Al Kattar Sahar

Directeur De Thèse

: Boukarim

Chawki Directeur De Thèse

Liagre Bertrand

Professeur M Chawki Boukarim

Professeur M Bertrand Liagre

M Philippe Billiald

Professeur M Asaad Eid

Assistant Professeur David-Yannick Leger

Mona Mme

Professeur Diab-Assaf

Professeur M Abdo Jurjus

Role of Metformin and Probiotics in The Crosstalk Between Inflammatory Bowel Disease, Colorectal Cancer and Diabetes

Keywords: Colorectal cancer, Inflammatory Bowel Diseases, Diabetes, Probiotics, Inflammation, Metformin

Background: the co-occurrence of colorectal cancer (CRC), Inflammatory Bowel diseases (IBD) and diabetes mellitus along with inflammation and dysmicrobism has been frequently reported. Several studies shed light on the anti-oncogenic potential of metformin in colorectal carcinogenesis as well as the beneficial effects of probiotics on inflammatory diseases.

Aims: this study aimed to demonstrate that metformin in association with probiotics act in a synergic effect in breaking the crosstalk, thus inhibiting CRC progression, improving diabetes and reducing inflammation.

Methodology: ninety-six male Balb/c mice, 6-8 weeks old, were divided into 16 control and experimental groups to assess the effect of the different treatments and combinations at the clinical, histological and molecular levels. Pro-inflammatory cytokines IL-6 and TNF- levels were assessed, as well as reactive oxygen and nitrogen species. Moreover, the proliferation index of colocytes was determined by Ki-67 immunohistochemistry.

Results: metformin and probiotics showed beneficial outcomes on CRC and diabetes, alone and most importantly in combination. Their effects were exerted by reversing the histopathological alterations and by inhibiting the inflammatory process whereby a downregulation of IL-6 and TNF-α as well as oxidative stress were depicted.

Conclusion:

the characterization of the effects of probiotics and metformin on CRC and diabetes sheds light on the role of inflammation and microbiota in this crosstalk. Deciphering more the downstream signaling pathways elicited by these compounds will help in developing new effective targeted treatment modalities.

Dedication

ُ ك ُ ن ُ ُ معُهللاُوالُت بايل ُ والُت ُ ن ُغريُخايلُالب ُ ال ُ نتُت ُأ ٍ فبنيُرمشة ُ رم ُ ش ه ا ُ ي غ ُ ّ ري ُ ىلُحال ُا ُهللاُمنُحالٍ
To the loving memory of my guardian angel, Weddo Pr. Bertrand Liagre, the lively, energetic and cheerful Director. You have made this journey much easier and my stay at Limoges memorable. I could have never imagined having a better advisor.

I owe my deepest gratitude to my thesis Director at the Lebanese University, Pr. Chawki Bou Karim, for his encouragement, insightful advices and guidance during the completion of this project. Thank you for placing your trust and confidence in my abilities.

Besides my advisors, I would like to thank my thesis committe members, Pr. Philippe Billiald, Pr. David-Yannick Leger, Pr. Asaad Eid, and Pr. Mona Diab-Assaf for generously offering their time, support, and assistance throughout the preparation and review of this thesis. I would like to thank two beautiful people, Dr. Rosalyn Jurjus and Mrs. Aline Pinon, I am grateful for your kindness, help and positive vibes. Special thanks go to my committee chair, Pr. Abdo Jurjus. I am grateful for being a part of your lab for the past 6 years, you have pushed me to sharpen my thinking and brought my work to a higher level. Without your guidance and persistent help, this dissertation would not have been possible. I would also like to thank my friends, for bringing so much fun, and laughter to my life. You are all part of this success story. I wish to express my love and my gratitude to my Parents, for their payers and sacrifices. Thank you for giving me strength to reach for the stars and chase my dreams. "Ma petite Nanou", you are the better version of me, I love you unconditionally and I will always be there for you.

Finally, I would like to express my gratitude for my Husband, who has been by my side throughout this PhD, living every single minute of it, and without whom, I wouldn't have had the courage to embark on this journey in the first place. You are the best thing that ever happened to me. Table 1: Enzymatic reactions that participate in ROS/NOS generation in the GI tract. .......... Table 2:

Table of contents

List of Figures

List of Tables

Introduction

Colorectal cancer (CRC) also referred to as bowel cancer, colon cancer, or rectal cancer is the development of cancer from the colon or rectum. According to the World Health Organization GLOBOCAN database, CRC is the third most commonly diagnosed cancer worldwide with 1.8 million new cases and almost 881,000 deaths in 2018 [1]. Its burden is predicted to increase by 60% in 2030 due to several factors, mainly the aging of the population and the accommodation to the sedentary lifestyle [2]. In Lebanon, CRC has a high prevalence as it accounts for 8.5% of all cancers [3].

The majority of CRC patients are diagnosed with a resectable localized disease. Surgery, followed by adjuvant therapy for high-risk patients, is considered as the optimum curative treatment approach in such cases. However, ultimately, approximately half of all diagnosed CRC patients will develop disseminated advanced disease, which requires medical management and in most cases will be fatal [4]. In parallel, diabetes mellitus affected 451 million people worldwide in 2017 and its prevalence keeps an increasing trend. Actually, this number is expected to rise to 693 million by 2045 [5]. The increasing trend of diabetes makes it imperative that research should focus on its prevention as well as its treatment. Several recent studies focused on the role of inflammation in the onset of diabetes, its poor prognosis and on understanding the mechanisms linking inflammation to diabetes and its related complications.

Such interrelationship has stimulated interest in targeting inflammatory pathways as part of the strategy to prevent or control diabetes and its complications, especially in preparing a favorable ground for malignant diseases [6].

Although there has been some debate regarding the effects of diabetes on CRC, meta-analyses consistently show that Diabetes Mellitus (DM) is an independent risk factor for CRC and that diabetic patients with CRC may have worse outcomes than their non-diabetic counterparts [7].

Moreover, these two disease entities share a panoply of common risk factors. The pathophysiological mechanisms of diabetes, including insulin resistance, hyperglycemia and resulting hyperinsulinemia, are all associated with the development and progression of cancer [8]. The co-occurrence of DM and CRC along with inflammation and dysbiosis has been frequently reported by our team [9,10] and by others [11].

In this study, a new therapeutic approach in treating colorectal cancer associated with diabetes was used. Hereby, metformin, an antidiabetic drug frequently and widely used, was tested for its anticancer properties along with probiotics, the microbiota modulating agents.

Metformin is known to inhibit hepatic glucose production and decreases insulin resistance in peripheral tissues, thereby reducing levels of circulating glucose levels and improving insulin sensitivity. Epidemiological studies have shown that patients with type 2 DM who are taking metformin have a lower risk of cancer and better outcomes compared with patients who do not take metformin. Although there has been substantial evidence from in-vivo and in-vitro studies supporting the potential efficacy of metformin as an anti-cancer agent, there have been no

clinical studies investigating the effect of metformin on CRC in presence of probiotics [12].

On the other hand, Probiotics have obtained increasing medical importance because of their beneficial effects upon the host health. In addition to the homeostasis regulation of the intestinal epithelial system and immune responses, probiotics have shown to possess antitumor activity using various mechanisms [13].

In the following chapters, the characteristics of colorectal cancer and diabetes at the clinical, molecular and histological levels will be discussed.

Chapter one will cover the anatomy and physiology of the gastrointestinal tract and the large intestine in particular, with a focus on the intestinal barrier, as well as an extensive definition of the microbiota and its importance in the context of inflammation and carcinogenesis.

Additionally, the key players in the crosstalk between CRC, diabetes and inflammation will be highlighted, mainly oxidative stress and inflammation.

Afterwards, metformin and probiotics' potential in treating and preventing colorectal cancer and ameliorating the diabetic phenotype will be discussed.

The subsequent chapters will outline the work done on the established in-vivo model while trying to decipher the mechanisms of action of our treatment combination, metformin and probiotics.

Résumé

Contexte: la concomitance du cancer colorectal (CCR), des maladies inflammatoires de l'intestin et du diabète sucré avec l'inflammation et le dismicrobisme a été fréquemment rapportée. Plusieurs études ont mis en lumière le potentiel anti-oncogénique de la metformine sur la carcinogenèse colorectale, ainsi que les effets bénéfiques des probiotiques sur les maladies inflammatoires.

Objectifs

Chapter 1: Literature review

A-Overview of the gastrointestinal tract

The portion of the alimentary canal extending from the proximal part of the esophagus to the distal part of the anal canal is a hallow tube of varying diameter. This tube has the same structural organization throughout its length. Its wall is formed by four distinctive layers from the lumen outward (Figure 1).

They are as follows:

 Mucosa, consisting of a lining epithelium, an underlying connective tissue called the lamina propria and the muscularis mucosa composed of smooth muscle. These structures differ between the different parts of the alimentary canal to adapt to tits specific functions. The main functions of the mucosa are protection, absorption and secretion.

 Submucosa: consisting of dense irregular connective tissue layer containing blood and lymphatic vessels, a nerve plexus and occasional glands.

 Muscularis externa, consisting in most parts of two thick concentric layers of smooth muscle. Contractions of the muscularis extern mix and propel the contents of the digestive tract.

 Serosa and adventitia: A serous membrane consisting of a simple squamous epithelium, the mesothelium and a small amount of underlying connective tissue. An adventitia consisting only of connective tissue is found where the wall of the tube is directly attached or fixed to adjoining structures (i.e, body wall and certain retroperitoneal organs). It is the outermost layer of the alimentary canal. This composite diagram shows the wall structure of the alimentary canal in four representative organs: esophagus, stomach, small intestine, and large intestine [14].

.

B-Morphology of the large intestine

The large intestine is a tubular structure about 30 to 40 cm in length at birth, reaching about 150 cm in the adult. Viewed as a whole, the various parts of the large intestine form a horseshoe-shaped arc (Figure 2). It extends from the cecum to the anal canal and consists of different segments: the cecum, the vermiform appendix, colon, rectum, and the anal canal. The colon is further subdivided on the basis of its anatomic location into ascending colon, descending colon and sigmoid colon [14,15].

 The cecum: a pouch connected to the ascending colon and the ileum; it prolongs below the ileocecal junction and is surrounded by a mesentery. The cecum has the greatest diameter (7.5 cm approximately), narrowing down to the sigmoid.

 Appendix: a narrow, fingerlike extension of the cecum. The appendix contains many lymphoid nodules; it is suspended by a mesentery (the mesoappendix).

 Ascending colon: It is located retroperitoneally, and ascends on the right flank to reach the liver, where it turns into the right colic flexure (or hepatic flexure).

 Transverse colon: It is attached by the transverse mesocolon, it runs from the right and bends on the left to form the splenic flexure.

 Descending colon: It descends retroperitoneally along the left flank to intersect with the sigmoid colon in the left groin area.

 Sigmoid colon: It is suspended by the sigmoid mesocolon and runs medially to join the midline rectum in the pelvis. The sigmoid colon has the smallest diameter of about 2.5 cm.

 Rectum and anal canal: They extend from the middle sacrum until the anus [14,15].

The large intestine has a greater luminal diameter than the small intestine, and is characterized by the presence of distinct features at the gross level:

 Teniae coli: three longitudinal equally spaced bands of muscularis externa, primarily visible in the cecum and colon. In addition, they are absent in the rectum, anal canal and vermiform appendix. Teniae coli contribute in peristalsis  Haustra: Pouches or sacculations of the colon formed by teniae coli's contractions.

-Omental appendices: small fatty accumulations covered by visceral peritoneum, suspending on the outer surface of the colon [14,15]. The colon, like the entire intestinal tract consists in general of four layers: (1) a mucosa, (2) a submucosa, (3) a double-layered musculosa, and (4) a serosa (Figure 3).

Mucosa

The mucosa of the large intestine has a smooth surface; neither plicae circulares nor villi are present. It contains the same cell types of the small intestine except Paneth cells, which are normally absent in humans. It consists of the epithelium and lamina propria.

The epithelium contains numerous straight intestinal glands (Crypts of Lieberkühn) that extend through the full thickness of the mucosae. The gland consists of simple columnar epithelium, as does the intestinal surface from which they invaginate.

The primary function of the columnar absorptive cells is reabsorption of water and electrolytes.

This reabsorption is accomplished by the Na+/K+ activated ATPase driven transport system.

Goblet cells are more numerous in the large intestine than the small intestine. They produce mucin that is secreted continuously to lubricate the bowel, facilitating the passage of the increasingly solid content. Goblet cells may mature deep in the intestinal gland. They secrete mucus continuously, even to the point where they reach the luminal surface.

Columnar absorptive cells predominate over goblet cells in most of the colon, the ratio decreases, however, approaching 1:1 near the rectum where the number of goblet cells increases.

-Epithelial cell renewal in the large intestine: All intestinal epithelial cells in the large intestine derive from a single stem cell population; these stem cells are located at the bottom of the intestinal gland. The lower third of the gland constitutes the intestinal stem cell niche, where newly generated cells undergo two to three more divisions as they begin their migration up to the luminal surface, where they are shed about 5 days later.

Lamina propria

Although the lamina propria of the large intestine contains the same basic components as the rest of the digestive tract, it demonstrates some additional features such as:

 Collagen table: a thick layer of collagen and proteoglycans that participates in the regulation of water and electrolyte transport.

 Pericryptal fibroblast sheath: a well-developed fibroblast population of regularly replicating cells. It is suggested that the macrophages of the lamina propria in the large intestine may arise as a terminal differentiation of the pericryptal fibroblasts.

 GALT: extensively developed, large lymphatic nodules that distort the systematic spacing of the intestinal glands and extend into the submucosa.

Muscularis externa

The outer layer of the muscularis externa of the colon and cecum is marked by the presence of distinct structures, the teniae coli, and haustra coli. The muscularis externa of the large intestine produces two major types of contraction: segmentation and peristalsis.

-Segmentation: a local contraction that does not induce the propulsion of bowel load.

-Peristalsis: contractions that engender the distal mass movement which occur typically once a day to empty the distal colon.

Submucosa and serosa

The submucosa of the large intestine corresponds to the layer of blood vessels, nerves and connective tissue surrounding the mucosa, the submucosa supports the other layers of the large intestine. The outer layer of the large intestine is typically a serosa; however, when the intestine is in direct contact with other structures, the external layer is an adventitia.

C-The intestinal barrier

The intestinal mucosa is a semi-permeable structure forming a physical and immunological defense barrier (Figure 4). This barrier has a dual role as it allows the selective uptake of substances including essential nutrients and electrolytes. It also restricts the passage of harmful intraluminal entities, including foreign antigens, bacteria, and their toxins.

Three main layers constitute the intestinal barrier: The outer mucus layer, the central single layer and the inner lamina propria [START_REF] Groschwitz | Intestinal barrier function: molecular regulation and disease pathogenesis[END_REF].

The anatomy of these layers is described in part A. Their specific roles in the activities of the barrier are listed below.

The outer mucus layer

The mucosal layer is a chemical barrier overlying the intestinal epithelium; it is considered as the first line of defense that limits the contact between the microbiome and epithelial cells.

In the colon, the mucus is organized as a bilayer composed of an adherent inner layer and a loose detached outer layer; however, the small intestine has only one single detached mucus layer.

The main components of the mucosal layer are mucins. Besides, it comprises the commensal gut microbiota, as well as immune-sensing and regulatory proteins such as antimicrobial peptides (AMPs), and secretory immunoglobulin A (sIgA) [START_REF] Baumgart | Intestinal barrier function[END_REF][START_REF] Chelakkot | Mechanisms regulating intestinal barrier integrity and its pathological implications[END_REF].

 Mucins

Mucins are highly glycosylated proteins produced and released by goblet cells, they are the main constituents of the mucus layer. Depletion of mucins leads to mucosal injury, diarrhea and inflammation affect the barrier and confer a predisposition to inflammatory bowel diseases and colorectal cancers [START_REF] Chelakkot | Mechanisms regulating intestinal barrier integrity and its pathological implications[END_REF][START_REF] France | The mucosal barrier at a glance[END_REF]. In humans there are five oligomerizing secreted mucins (MUC2, MUC5AC, MUC5B, MUC6, and MUC19); with MUC2 as the predominant type in the small and large intestine [START_REF] Wells | Homeostasis of the gut barrier and potential biomarkers[END_REF].

 Antimicrobial peptides (AMPs)

AMPs are small cationic peptides considered a fundamental component of the innate immunity.

The main AMPs in mammals are defensins and cathelicidins. These AMPs exhibit a broad spectrum of antimicrobial activity, their secretion could be continuous or inducible by proinflammatory cytokines or pathogens [START_REF] Diamond | The roles of antimicrobial peptides in innate host defense[END_REF].

 Secretory IgA (SIgA)

SIgA is the most abundant class of antibody found in the intestinal lumen of humans and most other mammals. It is known to be the first line of defense in protecting the intestinal epithelium from enteric pathogens and toxins, and maintaining homeostasis [START_REF] Mantis | Secretory IgA's complex roles in immunity and mucosal homeostasis in the gut[END_REF].

The central single cell layer

This is a continuous sheet formed by a monolayer of polarized columnar intestinal epithelial cells (IEC). These cells are tightly attached by apical junctional complexes: the tight junctions (TJs) and adherents junctions(AJs) [START_REF] Baumgart | Intestinal barrier function[END_REF].

Epithelial barrier function is mediated by a series of intercellular junctions that include an apical tight junction (TJ), subjacent adherens junction (AJ), and desmosomes. Given their close structural and functional proximity, the TJ and AJ are collectively referred to as the apical junctional complex (AJC). The backbone of intercellular junctions consists of transmembrane proteins that associate with cytoplasmic plaque proteins anchored to the cytoskeleton [START_REF] Luissint | Inflammation and the intestinal barrier: leukocyteepithelial cell interactions, cell junction remodeling, and mucosal repair[END_REF].

 Tight junctions

TJs are the adhesive junctional complexes located at the apical side of the cells, they are multiprotein complexes that function as a selective paracellular barrier facilitating the passage of ions and solutes, while preventing luminal antigens, microorganisms and their toxins [START_REF] Groschwitz | Intestinal barrier function: molecular regulation and disease pathogenesis[END_REF].

TJs are highly dynamic and their permeability is affected by external and intracellular stimuli and their alteration is implicated in several intestinal and systemic diseases [START_REF] Ma | Chapter 25 -Tight Junctions and the Intestinal Barrier[END_REF].

Under pathophysiological conditions , secreted cytokines such as TNF-α, IFN-γ, IL-1β, IL-4, IL-6, IL-12, IL-13, insulin, and insulin-like growth factor, mediate a dysfunction and a possible leak of the TJ barrier, resulting in immune activation and tissue inflammation [START_REF] Luissint | Inflammation and the intestinal barrier: leukocyteepithelial cell interactions, cell junction remodeling, and mucosal repair[END_REF][START_REF] Al-Sadi | Mechanism of cytokine modulation of epithelial tight junction barrier[END_REF].

Most importantly, TNF-α-induced alteration of intestinal TJ barrier has been proposed as a chief proinflammatory mechanism in IBD and CRC among others [START_REF] Ma | Chapter 25 -Tight Junctions and the Intestinal Barrier[END_REF].

 Adherens junctions

Adherens junctions (AJs) are protein complexes located on the lateral cellular membrane at points of cell to cell contact. They are formed by interactions between transmembrane proteins, intracellular adaptor proteins and the cytoskeleton [START_REF] Groschwitz | Intestinal barrier function: molecular regulation and disease pathogenesis[END_REF].The main function of adherens junctions is their stabilization of the tight junction which contributes to epithelial barrier function [START_REF] France | The mucosal barrier at a glance[END_REF].

The major element of the epithelial adherens junction is E-cadherin (or CDH1), a singlespanning transmembrane protein. Disruption of E-cadherin was shown to induce abnormal epithelial differentiation, aggravate inflammatory responses, crypt hyperproliferation and epithelial dysplasia. Moreover, E-cadherin polymorphisms are linked to inflammatory bowel disease and colonic adenocarcinoma [START_REF] France | The mucosal barrier at a glance[END_REF].

 Desmosomes

Desmosomes (DMs) provide mechanical strength to the epithelium. Their transmembrane cadherins include desmoglein and desmocollin proteins. 7 DM cadherins exist including 4 desmogleins and 3 desmocollins. In humans, desmoglein 2 and desmocollin 2 are expressed in human intestinal epithelial cells.

The role of DM proteins in regulating the intestinal epithelial barrier is not fully established yet, however, these junctions play an important role in regulating the mechanical stress generated by the intestinal epithelium [START_REF] Luissint | Inflammation and the intestinal barrier: leukocyteepithelial cell interactions, cell junction remodeling, and mucosal repair[END_REF].

The inner lamina propria

This layer, the lamina propria is a supportive layer of conjunctive tissue situated beneath the intestinal epithelium. Innate and adaptive immune cells such as T cells, B cells, dendritic cells and macrophages, reside in the lamina propria [START_REF] Baumgart | Intestinal barrier function[END_REF][START_REF] Vancamelbeke | The intestinal barrier: a fundamental role in health and disease[END_REF].

These cells play central roles in the immunomodulation and defense mechanisms. Their functions include phagocytosis, elimination of pathogenic substances, cytokine production, as well as the conservation of epithelial barrier function. In addition to their ability to produce prostaglandin E2, they produce also anti-inflammatory cytokines such as IL-10 among others [START_REF] Cardoso-Silva | Intestinal Barrier Function in Gluten-Related Disorders[END_REF].

D-Gut Microbiome

The human body comprises more than 100 trillion microbes, most of which are held in the gut, forming different communities living in a vast range of body niches. This population is called the microbiome and encompasses a wide selection of microorganisms, including archaea, viruses, fungi and anaerobic bacteria, the most studied group since they are the most abundant.

These microbial communities are achieved after birth and are essential for preserving body homeostasis [START_REF] Mármol | Colorectal Carcinoma: A General Overview and Future Perspectives in Colorectal Cancer[END_REF].It is estimated that a total of 3.3 million microbial genes form the collective microbial genome, this number is about 150 times greater than the human genome. Thus, the presence of this wide array of genes sheds light on the important impact of intestinal microorganisms on the human body [START_REF] Gerritsen | Intestinal microbiota in human health and disease: the impact of probiotics[END_REF].

The dynamic host-microbiome interaction is controlled by a wide range of factors, genetic and environmental, such as age, alcohol, diet, antibiotic use, mode of birth...among others [START_REF] Mármol | Colorectal Carcinoma: A General Overview and Future Perspectives in Colorectal Cancer[END_REF].

Intestinal microorganisms play a critical role in human physiology and metabolism. Given its wide spectrum of types and functions, it is not surprising that alterations in normal flora are on the basis of several systemic and intestinal diseases [START_REF] Cénit | Rapidly expanding knowledge on the role of the gut microbiome in health and disease[END_REF]. An alteration in the normal microbiota is defined as dysbiosis, a condition characterized by a disruption in the normal relationship between the host and the intestinal microbiota [START_REF] Carding | Dysbiosis of the gut microbiota in disease[END_REF].

Metabolic functions of the microbiota

Many reports consider the intestinal microbiota as a metabolic organ as it interacts with the host and upholds human health via various fundamental pathways. These pathways are involved in the metabolism of energy, amino acids, nucleotides, carbohydrates, cofactors and vitamins, as well as the biosynthesis of secondary metabolites. [START_REF] Gerritsen | Intestinal microbiota in human health and disease: the impact of probiotics[END_REF].

Importantly, the fermentation of complex carbohydrates by microbiota leads to short-chain fatty acids (SCFAs) production. These SCFAs constitute a vital energy source and essentially they possess immunomodulatory and anti-inflammatory properties [START_REF] Venegas | Short chain fatty acids (SCFAs)-mediated gut epithelial and immune regulation and its relevance for inflammatory bowel diseases[END_REF]. Acetate, propionate, and butyrate are the major SCFAs produced.

-Acetate is the most abundant SCFA, it is a crucial metabolite for bacterial growth , plays a role in cholesterol metabolism and lipogenesis, and in central appetite regulation.

-Propionate is transferred to the liver, where it regulates gluconeogenesis and satiety signaling through interaction with the gut fatty acid receptors.

-Butyrate is the main energy source for colonocytes, it has different functions ranging from induction of cancerous cells apoptosis, to intestinal gluconeogenesis activation, maintaining oxygen balance in the gut and preventing gut microbiota dysbiosis [START_REF] Valdes | Role of the gut microbiota in nutrition and health[END_REF].

Microbiome perturbations, immune dysfunction, and chronic disease

Several studies shed light on perturbations affecting the composition as well as the functions of gut microbiota in a wide spectrum of diseases. These include Inflammatory bowel diseases (IBD),clostridium difficile Infection, atopic asthma, behavioral disorders like autism spectrum disease (ASD) , celiac disease , colorectal cancer, obesity and type 2 diabetes mellitus (T2DM), cardiovascular disease, as well as autoimmune diseases like rheumatoid arthritis , multiple sclerosis, and type 1 diabetes mellitus [START_REF] Durack | The gut microbiome: Relationships with disease and opportunities for therapy[END_REF][START_REF] Wang | The Human Microbiota in Health and Disease[END_REF].

Dysbiosis and CRC

The microbiome is now considered one of the prime suspects responsible for the onset and evolution of gastrointestinal disorders and specifically colorectal carcinogenesis. Along this line, dysbiosis is now considered to be a key factor in the development of IBD and CRC [START_REF] Fong | Gut microbiota modulation: a novel strategy for prevention and treatment of colorectal cancer[END_REF][START_REF] Yu | Microbiota dysbiosis and barrier dysfunction in inflammatory bowel disease and colorectal cancers: exploring a common ground hypothesis[END_REF].

Although it is not yet clear how dysbiosis could induce colonic carcinogenesis, chronic inflammation appears to be the core mechanism. The first stages of both CRC and IBD diseases involve an alteration to the normal flora, which results in activation of the immune system, thus giving rise to a chronic inflammatory state characterized by an upregulation of proinflammatory cytokines and reactive oxygen species, thus creating a tumor-favorable microenvironment [START_REF] Koliarakis | Intestinal microbiota and colorectal cancer: a new aspect of research[END_REF].

E-Physiology of the large intestine

The large intestine has three major functions: secretory, digestive and absorptive (Figure 5).

Within 8-9 hours of ingestion, meals reach the large intestine. About 90% of the ingested water is absorbed by the small intestine, and the rest is absorbed by the large intestine, a process that converts liquid chyme residue into semi-solid stools or feces [START_REF] Azzouz | large intestine[END_REF].

The muscular anatomy of the colon is characterized by concentration of the longitudinal muscle into bands named teniae coli. Contraction of the teniae coli and the circular muscle results in haustrations. Segmenting colonic contractions help in water and electrolytes absorption, and contents pass slowly, usually taking days to pass through the colon augmenting the time for water and electrolytes absorption [START_REF] Nigam | Gastrointestinal tract 5: the anatomy and functions of the large intestine[END_REF][START_REF] Johnson | Gastrointestinal physiology[END_REF].

Closure of the anal canal is preserved by a tonic contraction of the internal anal sphincter.

Distention of the rectum stimulates the internal anal sphincter to relax and causes the need to defecate. Colonic movements are also controlled by activities of the intrinsic and extrinsic nerves and possibly by hormonal regulations [START_REF] Johnson | Gastrointestinal physiology[END_REF].

Feces are formed mostly of bacteria, old epithelial cells from the intestinal mucosa, inorganic waste, undigested food matter and fiber, small amounts fats and proteins. The brown color is due to hemoglobin breakdown products stercobilin and urobilin [START_REF] Nigam | Gastrointestinal tract 5: the anatomy and functions of the large intestine[END_REF].

Functional problems of defecation, vascular disorders of hemorrhoids, as well as several other gastrointestinal and systemic diseases are related to the anatomy of the large intestine [START_REF] Azzouz | large intestine[END_REF].

F-Pathologies of the intestines

A wide spectrum of pathologies affects the intestines, they include:

Diarrhea

Diarrhea, described as loose and watery feces is a frequent symptom of gastroenteritis, norovirus or food poisoning, as well as allergies, food intolerances and IBDs.

Constipation

Constipation is known as the intermittent and painful evacuation of slow moving hard feces.

Complications of constipation include abdominal distension and pain and may lead to gastro intestinal obstruction. Constipation is frequently caused by irregular bowel habits, a diet poor in fibers, and a sedentary lifestyle, however, it could also originate from more serious conditions such as diverticulitis, gastrointestinal tumor mass or paralytic ileus [START_REF] Nigam | Gastrointestinal tract 5: the anatomy and functions of the large intestine[END_REF].

Lactose intolerance

It is defined as the inability to digest dietary lactose leading to abdominal cramps, bloating and diarrhea [START_REF] Nigam | Gastrointestinal tract 5: the anatomy and functions of the large intestine[END_REF].

Celiac disease

Celiac disease is characterized by gluten intolerance which develops in genetically susceptible individuals. The exact etiology of this intolerance in unknown, it is characterized by a release of inflammatory mediators and an impairment of the intestinal mucosal lining [START_REF] Nigam | Gastrointestinal tract 5: the anatomy and functions of the large intestine[END_REF][START_REF] Lebwohl | Coeliac disease[END_REF].

Bowel obstruction

Obstruction of the large intestine is a serious medical problem requiring urgent attention and intervention. It is commonly caused by colorectal cancer and diverticular disease. Symptoms include abdominal pain, distension and constipation [START_REF] Wexner | Diseases of the colon[END_REF].

Diverticulitis

Colonic diverticula are pea-sized outpouchings from the colonic lumen caused by a mucosal herniation through the colonic wall at sites of vascular perforation a result of increased pressure; for instance, while straining during excretion [START_REF] Nigam | Gastrointestinal tract 5: the anatomy and functions of the large intestine[END_REF][START_REF] Tochigi | Management of complicated diverticulitis of the colon[END_REF].

Malabsorption syndrome

Malabsorption syndrome encompasses a number of disorders in which the small intestine is unable to absorb enough of certain nutrients like proteins, fats, minerals, vitamins and/or carbohydrates as well as fluids, resulting in deficiencies and malnutrition [START_REF] Clark | Malabsorption Syndromes[END_REF].

Appendicitis

This condition is defined as an inflammation of the appendix due to an obstruction of the lumen, resulting in ischaemic injury and bacterial infection. Appendicitis is considered a medical emergency, because a probable perforation will lead to peritonitis, possibly leading to death within hours [START_REF] Dixon | Acute appendicitis[END_REF].

Irritable Bowel Syndrome (IBS)

IBS is a common, chronic gastrointestinal disorder characterized by long-lasting abdominal pain, change in bowel habits, abdominal distension, bloating, and urgency. It is a disorder of gastrointestinal motility, which is influenced by stress and psychosocial dysfunction [START_REF] Wexner | Diseases of the colon[END_REF][START_REF] Distrutti | Gut microbiota role in irritable bowel syndrome: New therapeutic strategies[END_REF].

Inflammatory bowel disease (IBD)

Inflammatory bowel diseases (IBD) are a spectrum of immune-related conditions that include ulcerative colitis (UC) and Crohn's disease(CD)empirically defined by clinical, pathological, endoscopic and radiological features [START_REF] Xavier | Unravelling the pathogenesis of inflammatory bowel disease[END_REF]. The onset of IBD typically occurs in the second and third decades of life and a majority of affected individuals progress to relapsing and chronic disease. Family aggregation has long been recognized [START_REF] Xavier | Unravelling the pathogenesis of inflammatory bowel disease[END_REF].

Several genetic and environmental as well as life style related factors are involved in the onset of IBD and the exact etiology of this multifactorial disease is not well defined yet , However, an active inflammatory status characterized by an upregulation of a wide cytokine network remains the hallmark of this pathology [START_REF] Xavier | Unravelling the pathogenesis of inflammatory bowel disease[END_REF][START_REF] Friedrich | Cytokine Networks in the Pathophysiology of Inflammatory Bowel Disease[END_REF].

Some differences between UC and CD were described as UC is characterized by a diffuse mucosal inflammation that extends proximally from the rectum to a varying degree.

However, in CD, the involvement of the terminal ileum is most common and the earliest mucosal lesions appear over Peyer's patches. Unlike ulcerative colitis, Crohn's disease may be patchy and segmental, and inflammation typically transmural [START_REF] Xavier | Unravelling the pathogenesis of inflammatory bowel disease[END_REF].

It is well documented that IBD represents a major healthcare burden of significant global morbidity, with highest prevalence in Europe and North America and increasing incidence in Asia. [START_REF] Friedrich | Cytokine Networks in the Pathophysiology of Inflammatory Bowel Disease[END_REF]. Symptoms include abdominal cramps, bloating, gas, urgency, diarrhea and bleeding. The key feature of IBD is dysbiosis, defined as a shift in the composition of the gut microbiota; however, the specific role of dysbiosis in its pathogenesis remains poorly understood. Importantly, IBD is considered an key risk factor for colorectal carcinogenesis [START_REF] Caruso | Host-microbiota interactions in inflammatory bowel disease[END_REF].

G-Colorectal cancer 1. Epidemiology of CRC

Colorectal cancer (CRC) is the third most commonly diagnosed cancer worldwide (Figure 6) with 1.8 million new cases and almost 881,000 deaths in 2018, according to the World Health Organization GLOBOCAN database [1]. Its burden is predicted to increase by 60% in 2030 [2].

In Lebanon, CRC accounts for 8.5% of all cancers. It is the highest incidence rate in the MENA region, as 1463 cases of CRC were diagnosed in 2018 (Figure 8) in a population of almost 6 million [3].

More than 90% of CRCs are adenocarcinomas, a malignant neoplasm that develops from glandular epithelial cells of the colon and rectum; other rare types include squamous cell carcinoma, adenosquamous carcinoma, spindle cell carcinoma and undifferentiated carcinoma [START_REF] Fleming | Colorectal carcinoma: Pathologic aspects[END_REF] .

Classification of CRC

Colorectal cancer is basically due to the onset of mutations in specific genes such as oncogenes, tumor suppressor genes and genes related to DNA repair mechanisms, thus CRC is classified based on the origin of the mutation into 3 categories: sporadic, inherited and familial CRC (Figure 9).

 Sporadic cancers:

Seventy percent of CRC cases follow a specific succession of mutations converting an adenoma to a carcinoma. This sequence starts with a mutation of the APC (adenomatous polyposis coli) gene triggering polyp formation, followed by a chain of mutations in KRAS, TP53 and DCC (Deleted in colorectal carcinoma) genes, thus leading to the carcinoma state [START_REF] Armaghany | Genetic alterations in colorectal cancer[END_REF].

 Inherited cancers:

Inherited CRC account to only 5% of incidences. They are classified into 2 main categories: familial adenomatous polyposis (FAP) characterized by a large number of colorectal adenomas and hereditary non-polyposis colorectal cancer (HNPCC), which is related to mutations in DNA repair mechanisms. The main form HNPCC is Lynch syndrome, it is caused by inherited mutations in one of the alleles coding for DNA repair proteins such as MSH2, MLH1, MLH6, PMS1 and PMS2 [START_REF] Kuipers | Colorectal cancer[END_REF].

 Familial colorectal cancer:

This type accounts for approximately 25% of all cases, it is not included in the inherited type as it occurs in families without evidence for one of the known inherited syndromes. Nonsyndromic or familial CRC is a cluster of CRC that is distinguished from the hereditary syndromes. It is an heterogeneous disorder that includes patients with unrecognized hereditary syndromes and an unclear molecular mechanism. Possibly, a combination of environmental and inherited genetic factors play a role in the onset of CRC in these families [START_REF] Armelao | Familial colorectal cancer: a review[END_REF].

Risk factors

The etiology of CRC is multifactorial encompassing genetic, environmental and lifestyle related factors including westernized diet, alcohol consumption and obesity… among others.

However, chronic inflammation, in particular inflammatory bowel disease (IBD) and dysbiosis in enteric microbiota remain the key players in this process [10,[START_REF] Gao | Gut Microbe-Mediated Suppression of Inflammation-Associated Colon Carcinogenesis by Luminal Histamine Production[END_REF][START_REF] Jeon | Effect of vitamin C on azoxymethane (AOM)/dextran sulfate sodium (DSS)-induced colitis-associated early colon cancer in mice[END_REF][START_REF] Zitvogel | The microbiome in cancer immunotherapy: Diagnostic tools and therapeutic strategies[END_REF]. Even though CRC is influenced by hereditary components, most CRC cases are sporadic and slowly develop over several years following the adenoma-carcinoma sequence [START_REF] Vecchia | Metabolic pathways regulating colorectal cancer initiation and progression[END_REF].

Although much has yet to be learned about it, several factors are known to increase a person's chance to develop CRC:

 Age: the risk of developing CRC is markedly increased after 50 years of age.

 IBD: A personal history of inflammatory bowel disease (IBD) increases the chances of having CRC by 2.75 in patients with UC and 2.64% in patients with Crohn's [START_REF] Keller | Colorectal cancer in inflammatory bowel disease: review of the evidence[END_REF].

 Familial history of CRC: Having relatives with CRC raises the risk of CRC development.

 Lifestyle related factors: This group of factors plays an important role in the predisposition to CRC; it regroups sedentary lifestyle, obesity, bad nutritional habits, red and burned meat consumption, smoking and alcohol consumption…among others [START_REF] Haggar | Colorectal cancer epidemiology: incidence, mortality, survival, and risk factors[END_REF][START_REF] Ryan-Harshman | Diet and colorectal cancer: Review of the evidence[END_REF].

Dysbiosis and CRC

The microbiome comprised of the collective genome of microbes inhabiting the gut, is now considered one of the prime suspects responsible for the onset and evolution of gastrointestinal disorders and specifically colorectal carcinogenesis. Definitely, dysbiosis is now known to be a key factor in the development of IBD and CRC [START_REF] Fong | Gut microbiota modulation: a novel strategy for prevention and treatment of colorectal cancer[END_REF][START_REF] Yu | Microbiota dysbiosis and barrier dysfunction in inflammatory bowel disease and colorectal cancers: exploring a common ground hypothesis[END_REF][START_REF] Gagnière | Gut microbiota imbalance and colorectal cancer[END_REF].

Growing attention has been given to the role of microbial infection in carcinogenesis in recent decades, and microbes are suspected to be involved in approximately 20% of cancers, and especially CRC. Several observations led to the focus on microbiota as a key player in CRC:

Variability of the incidence of CRC highly suggested the involvement of certain environmental risk factors, such as high-fat diets, obesity or Western lifestyle. In addition, Knudson's two-hit hypothesis suggested that host factors play an important role in the predisposition to carcinogenesis. In this scenario, a second environmental hit can lead to uncontrolled cellular proliferation which could be a possible infectious cause [START_REF] Gagnière | Gut microbiota imbalance and colorectal cancer[END_REF].

Additionally, experimental studies have focused on the role of dysbiosis on CRC. In 1975, the first observation linking gut microbiota with CRC was reported in germ-free rats that developed less chemically induced colorectal tumor than conventional rats. These results have been reproduced in several in vitro and in-vivo CRC models [START_REF] Weisburger | Germ-free status and colon tumor induction by N-methyl-N'-nitro-N-nitrosoguanidine[END_REF].

The contribution of bacteria to CRC could be due to two different scenarios:

 A dysbiotic microbial community with pro-carcinogenic features that are capable of remodeling the microbiome as a whole to drive pro-inflammatory responses and epithelial cell transformation, leading to cancer;

 And the "driver-passenger" theory, wherein intestinal bacteria, termed "bacteria drivers", initiate CRC by inducing epithelial DNA damage and tumorigenesis, in turn promoting the proliferation of passenger bacteria that have a growth advantage in the tumoral microenvironment [START_REF] Gagnière | Gut microbiota imbalance and colorectal cancer[END_REF].

Although it is not yet clear how dysbiosis could induce colonic carcinogenesis, chronic inflammation appears to be the core mechanism. The first stages of both CRC and IBD diseases involve an alteration to the normal flora, which results in activation of the immune system, thus giving rise to a chronic inflammatory state characterized by an upregulation of proinflammatory cytokines and reactive oxygen species among others, creating a tumor-favorable microenvironment [START_REF] Koliarakis | Intestinal microbiota and colorectal cancer: a new aspect of research[END_REF].

Staging and prognosis of CRC

Pathologic staging for CRC is done by the tumor-node-metastasis (TNM) system as defined by the American Joint Committee on Cancer.

In TNM staging system, T describes the size of the tumor and any spread of cancer into nearby tissue, N describes spread of cancer to proximate lymph nodes; and M is for metastasis.

In colorectal carcinogenesis, TNM is based on depth of invasion of the bowel wall, extent of regional lymph node involvement, and presence of distant sites of disease. The classification for CRC is described below [START_REF] Micheau | Colorectal cancer staging-Radiological classifications commonly used in medical imaging[END_REF].

Primary Tumor (T)

 TX Primary tumor cannot be assessed Metastases in more than one organ/site or the peritoneum TNM staging is the most common staging system (Figure 10); it predicts the disease prognosis.

Molecular pathways of CRC

The mechanism of CRC development consists of four major stages: initiation, promotion, progression and metastasis, passing through a stepwise accumulation of multiple genetic and epigenetic aberrations, subsequently leading to invasive and metastatic tumors (Figure 11). These sequences implicate the progression of normal colon epithelial cells to aberrant crypt foci, followed by early and advanced polyps with subsequent progression to early cancer and then advanced cancer.

 The Chromosomal instability (CIN) pathway

The Chromosomal instability (CIN) is the classical pathway, it accounts for 80%-85% of all CRC cases. It is characterized by disparities in the number of chromosomes, thus leading to loss of heterozygosity (LOH) [START_REF] Pino | The chromosomal instability pathway in colon cancer[END_REF].

Several mechanisms are implicated in this process such as alterations in chromosome segregation, telomere dysfunction and DNA damage response, affecting crucial genes such as APC, KRAS, PI3K and TP53 … among others. Importantly, APC mutations lead to a nuclear translocation of β-catenin thus enhancing tumorigenesis and invasion. On the other hand, the KRAS and PI3K mutations activate MAP kinase pathway which leads to cell proliferation.

TP53 mutations also affects the p53 gene and leads to uncontrolled cell cycle progression [START_REF] Mármol | Colorectal Carcinoma: A General Overview and Future Perspectives in Colorectal Cancer[END_REF].

 The Microsatellite instability (MSI) pathway

The MSI pathway is caused by a loss of DNA repair mechanisms leading to mutations in oncogenes or tumor suppressor genes such as MLH1, MSH2, MSH6, PMS1 and PMS2 . Loss of expression of mismatch repair genes (MMR) can be caused by spontaneous events (promoter hypermethylation) or germinal mutations such as those found in Lynch syndrome. In general, MSI tumors present a better prognosis than sporadic tumors [START_REF] Mármol | Colorectal Carcinoma: A General Overview and Future Perspectives in Colorectal Cancer[END_REF].

 CpG island methylator phenotype (CIMP)

The CpG island methylator phenotype is due to epigenetic instability, a common feature in colorectal carcinogenesis. The main characteristic of CIMP tumors is the hypermethylation of oncogene promoters MINT clones, p16, THBS, and MLH1, which leads to genetic silencing and a loss of protein expression [START_REF] Mármol | Colorectal Carcinoma: A General Overview and Future Perspectives in Colorectal Cancer[END_REF][START_REF] Advani | Pathological, and Molecular Characteristics of CpG Island Methylator Phenotype in Colorectal Cancer: A Systematic Review and Meta-analysis[END_REF].

CIMP has a distinct phenotype , it is characterized by key clinical, pathologic, and molecular features, including female sex, old age, high MSI, BRAF mutations, and right-sided tumor location [START_REF] Advani | Pathological, and Molecular Characteristics of CpG Island Methylator Phenotype in Colorectal Cancer: A Systematic Review and Meta-analysis[END_REF].

Screening for CRC

Screening is considered a secondary prevention as it can reduce CRC incidence and mortality by enabling detection and, therefore, treatment of precancerous lesions before their malignant transformation. Such a screening can be performed by:

 Fecal Occult Blood (FOBT)
This is a commonly adopted technique, the fecal occult blood test detects small amount of blood in stool samples, supporting CRC diagnosis. The two main types of FOBT are the immunochemical FOBT (or fecal immunochemical test -FIT) and the guaiac-based FOBT (Figure 12).

The guaiac-based test (g-FOBT) is the most commonly used. This test is based on the oxidation of the paper-embedded guaiac by hydrogen peroxide (developer) in presence of the heme component of hemoglobin thus generating a blue-colored product (Figure 12-A).

The i-FOBT was approved by the United States Food and Drug Administration in 2001 has been added to recent American Cancer Society (ACS) and GI Consortium guidelines for colorectal cancer screening. This test is based on an antibody-antigen detection of human hemoglobin (see Fig. 12-B) [START_REF] Kafrouni | Fecal Occult Blood Test Availabe online[END_REF].

 Colonoscopy

Colonoscopy is known as the gold-standard screening method. It is a highly sensitive but costly test. The efficacy of colonoscopy in CRC screening is explored by several studies shedding light on the decreased CRC risk and mortality in patients undergoing colonoscopy compared to their non-screened counterparts.

Colonoscopy is a safe technique. Complications, including colonic perforations and intestinal bleeding, are relatively rare.

Regardless of one's lifestyle habits, screening and detection of polyps with subsequent polypectomy plays a crucial role in preventing CRC development in asymptomatic patients and is recommended to begin in average-risk patients who are 50 years old [START_REF] Reynolds | [END_REF]. However, for individuals with predisposing conditions, earlier and more frequent testing is recommended [START_REF] Reynolds | [END_REF][START_REF] Keum | Global burden of colorectal cancer: emerging trends, risk factors and prevention strategies[END_REF] .

CRC Treatments

The choice of first-line treatment for CRC patients currently involves a multimodal approach based on tumor and patient related characteristics such as the number and localization of metastases, tumor progression, and prognosis among others [START_REF] Wolpin | Systemic treatment of colorectal cancer[END_REF][START_REF] Gustavsson | A review of the evolution of systemic chemotherapy in the management of colorectal cancer[END_REF].

Besides surgical resection, several regimens of these chemotherapy drugs are used sometimes in combination:

a. Intravenous Fluorouracil:

Fluorouracil is considered the cornerstone of systemic treatment for colorectal cancer. It is a fluorinated pyrimidine that acts primarily through inhibition of thymidylate synthase and is commonly administered with leucovorin, a reduced folate that is thought to stabilize fluorouracil's interaction with this enzyme.

b. Irinotecan:

It is a semisynthetic derivative of the natural alkaloid camptothecin that acts by inhibiting topoisomerase I, an enzyme that catalyzes breakage and rejoining of DNA strands during DNA replication .

c. Oxaliplatin:

It is a diaminocyclohexane platinum compound that forms DNA adducts, leading to impaired DNA replication and cellular apoptosis.

d. Bevacizumab:

It is a monoclonal antibody working as an Angiogenesis Inhibitor in a strategy to control malignant proliferation and spread through the inhibition of neoangiogenesis.

e. Epidermal Growth Factor Receptor (EGFR) Inhibitors:

Such inhibition is mainly performed by cetuximab and panitumuma: these antibodies are directed against the extracellular domain of EGFR. EGFR plays a crucial role in tumorigenesis as it enhances cellular growth and proliferation [START_REF] Wolpin | Systemic treatment of colorectal cancer[END_REF][START_REF] Gustavsson | A review of the evolution of systemic chemotherapy in the management of colorectal cancer[END_REF].

In addition to traditional chemotherapy, adjunct treatments are emerging with the aim of inhibiting inflammation , increasing chemotherapy efficacy and minimizing its resistance and side effects as well as the risk of developing secondary tumors. These include the use of agarose tumor macrobeads, anti-inflammatory drugs, metformin, probiotics, and gold-based drugs…among others [START_REF] Mármol | Colorectal Carcinoma: A General Overview and Future Perspectives in Colorectal Cancer[END_REF].

H-Diabetes

Diabetes mellitus is a group of metabolic disorders characterized by chronic hyperglycemia and dysregulated metabolism of carbohydrates, lipids and proteins induced by insulin insufficiency [START_REF] Wu | Risk factors contributing to type 2 diabetes and recent advances in the treatment and prevention[END_REF]. According to the International Diabetes Federation, diabetes affected 451 million people worldwide in 2017; this number is expected to rise to 693 million by 2045. This rising trend has been promoted by a shift into urban lifestyle, the spread of western style diet and lack of physical activity [5].

Types of diabetes mellitus

According to the American Diabetes Association 2019, diabetes is classified into four main types:

Type I diabetes: It is Insulin dependent diabetes, it is immune-mediated and characterized by a destruction of the pancreatic beta-cells and complete insulin deficiency.

Type II diabetes: This type is non-Insulin dependent, and it is characterized by insulin resistance.

Gestational diabetes: It is described as an intolerance to glucose with onset in pregnancy.

Specific types of diabetes:

They are due to other causes, such as monogenic diabetes syndromes (neonatal diabetes and maturity-onset diabetes of the young), diseases of the exocrine pancreas (such as cystic fibrosis and pancreatitis), and drug-or chemical-induced diabetes (such as with glucocorticoid use, in the treatment of HIV/AIDS, or after organ transplantation) .

Complications of diabetes

Diabetic patients are more prone to life threatening complications when compared to nondiabetic counterparts, these complications could be categorized into two main types: Notably, diabetic complications constitute the major cause of hospital admissions, disability, and mortality in diabetic patients [START_REF] Halim | The effects of inflammation, aging and oxidative stress on the pathogenesis of diabetes mellitus (type 2 diabetes)[END_REF].



Risk factors

Several risk factors predispose an individual to diabetes mellitus, such as age, inactive lifestyle, high blood pressure, poor glycemic control, smoking, obesity, bad lipid profile and cardiovascular diseases….among others [START_REF] Halim | The effects of inflammation, aging and oxidative stress on the pathogenesis of diabetes mellitus (type 2 diabetes)[END_REF].

Treatment of diabetes

While lifestyle modifications and metformin are considered the cornerstone of the initial management of diabetes mellitus, an increasing array of second and third-line pharmacological agents is being used.

Different families of oral and injectable drugs are available for the treatment of diabetes including sulfonylureas, meglitinides, insulin, alpha-glucosidase inhibitors,

Thiazolidinediones as well as Glucagon-like peptide-1 (RA-GLP1) receptor agonists, Dipeptidyl peptidase-4 inhibitors, and Sodium glucose cotransporter 2 (SGLT2) inhibitor. In addition, insulin analogues that better simulate endogenous insulin secretion have been developed.

However, it is worth noting that metformin remains the first choice of treatment for most patients. Other alternative treatments should be individualized, taking into consideration patient characteristics such as the level of hyperglycemia and the presence of co-morbidities, and the treatment properties such as its durability of lowering blood glucose, risk of hypoglycemia, effect on diabetic complications , effect on body weight, as well as its side effects and contraindications [START_REF] Marín-Peñalver | Update on the treatment of type 2 diabetes mellitus[END_REF].

I-Oxidative stress

The term "oxidative stress" was first used in 1970, "cells were subjected to oxidative stress" to describe the addition of H2O2 to erythrocytes. Since 1985, the term was used to denote oxidative damage to cells and organs [78].

Free radicals

Free radicals are unstable, highly reactive molecules containing one or more unpaired electron(s) [START_REF] Asmat | Diabetes mellitus and oxidative stress-A concise review[END_REF]. Free radicals can be classified into three main types [78]: Reactive oxygen and nitrogen species (RONS) are produced by all aerobic cells and play chief roles in several systemic functions and disease. RONS generation could be endogenous or exogenous.


 Endogenous sources of RONS include nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, myeloperoxidase (MPO), lipoxygenase, and angiotensin II.

 Exogenous sources of RONS encompass air and water pollution, tobacco, alcohol, heavy or transition metals, drugs (e.g., cyclosporine, tacrolimus, gentamycin, and bleomycin), industrial solvents, cooked food (e.g., smoked meat, waste oil, and fat) and radiation, which are metabolized into free radicals inside the body.

Whether they are endogenous or exogenous, RONS cause oxidative modification to carbohydrates, lipids, proteins, and cellular DNA [START_REF] Liguori | Oxidative stress, aging, and diseases[END_REF].

Antioxidants

Natural antioxidants are considered as defense mechanisms against reactive species, these substances could be classified into different categories according to their properties:

 Endogenous antioxidants : glutathione, lipoic acid, coenzyme Q, ferritin, uric acid, bilirubin, l-carnitine, melatonin, superoxide dismutase (SOD), catalase (CAT), glutathione peroxidases (GPXs), thioredoxins (TRX) and peroxiredoxins (PRX), among others [START_REF] Aguilar | Endogenous antioxidants: a review of their role in oxidative stress. A master regulator of oxidative stress-the transcription factor nrf2[END_REF].

 Natural antioxidants: have a dietary origin, such as ascorbic acid (Vitamin C), tocopherol (Vitamin E), β-carotene (Vitamin A), lipoic acid, uric acid, glutathione and polyphenol metabolites  Synthetic antioxidants: N-acetyl cysteine (NAC), pyruvate, selenium, butylated hydroxytoluene, butylated hydroxyanisole, and propyl gallate… among others [START_REF] Sosa | Oxidative stress and cancer: An overview[END_REF].

Oxidative stress in colorectal cancer

In the last decade, association between oxidative stress and CRC has been broadly studied. In the gastrointestinal tract, ROS generation is obtained via different enzymatic reactions (Table 1). Additionally, in colorectal carcinogenesis, markers of oxidative stress were found to be upregulated. This is depicted by a multitude of studies on human colorectal tumors whereby increased levels of ROS, nitric oxide (NO), 8-oxodG in DNA, lipid peroxides, glutathione peroxidase (GPx), catalase (CAT) and decreased methylation of cytosine in DNA, in addition to lipid modification and increased leukocyte activation, were found, linking oxidative stress to inflammatory cells and inflammation and consequently colorectal cancer [START_REF] Perše | Oxidative Stress in the Pathogenesis of Colorectal Cancer: Cause or Consequence?[END_REF].

One other possible way by which oxidative stress exerts its tumorigenic effect is through cell cycle modification, alteration of p53 expression, thus influencing proliferation of cancerous cells [START_REF] Piechota-Polanczyk | Review article: the role of oxidative stress in pathogenesis and treatment of inflammatory bowel diseases[END_REF].

Several studies also explored ROS inhibition as a treatment modality in CRC. This inhibition was found to ameliorate CRC phenotype especially in colitis associated colorectal cancer [START_REF] Piechota-Polanczyk | Review article: the role of oxidative stress in pathogenesis and treatment of inflammatory bowel diseases[END_REF],

thus linking again oxidative stress to colorectal carcinogenesis.

Catalase (CAT) 2H2O2 → O2+ H2O

The cytoplasm and peroxisomes of epithelium and lamina propria; leukocytes.

Glutathione peroxidase (GPx)

H2O2 + 2GSH → GSSG + 2H2O GPx1-peroxisomes of colon lymphatic tissue and the lamina propria, submucosa, muscularis and serosa;

GPx2-peroxisomes of the luminal epithelium;

GPx3-secreted by the intestinal epithelial cells;

GPx4-peroxisomes of colonic and ileal tissues.

Endothelial nitric oxide synthase (eNOS)

l-arginine + O2 → l-citrulline + NO • Cell membrane of the endothelial cells

Inducible nitric oxide synthase (iNOS)

NO • + O2

Oxidative stress and diabetes

It is well established that oxidative stress is considered as the main cause behind diabetes complications especially cardiovascular events, however, the exact mechanism by which oxidative stress facilitates the development of diabetic complications is not fully understood.

In diabetes, an increased intracellular glucose leads to an increased RONS production, which surpasses the neutralizing antioxidant capability of the cell. Thus, the activation of different molecular pathways involved in hyperglycemia-induced oxidative tissue could lead to the production of growth factors and pro-inflammatory cytokines, linking again diabetes to oxidative stress and inflammation [START_REF] Liguori | Oxidative stress, aging, and diseases[END_REF].

J-Inflammation and CRC

Chronic inflammation is characterized by a continuously active inflammatory reaction and tissue destruction. Many of the immune cells contributing to the pathology of chronic inflammation (macrophages, neutrophils and eosinophils) are involved in the carcinogenic process, directly or through the production of inflammatory cytokines and other secretory factors [START_REF] Chen | Inflammatory responses and inflammation-associated diseases in organs[END_REF][START_REF] Kany | Cytokines in Inflammatory Disease[END_REF].

Cytokines that promote colorectal and colitis-associated tumor development include TNF-α, IL-6, and IL-1 among others. On the other hand a variety of cytokines showed protective effects on CRC such as IL-10 and TGF-β that inhibit colorectal tumorigenesis [START_REF] Terzić | Inflammation and Colon Cancer[END_REF]. Most tumor-promoting cytokines are produced by lamina propria macrophages and dendritic cells (DC) during early stages of CRC development or by T cells during late-stage tumor progression [START_REF] Grivennikov | IL-6 and Stat3 are required for survival of intestinal epithelial cells and development of colitis-associated cancer[END_REF].

It is well established that chronic inflammation promotes carcinogenesis via several pathways, mainly by inducing gene mutations, inhibiting apoptosis, or stimulating angiogenesis and cell proliferation. Moreover, this inflammatory microenvironment promotes the accumulation of additional mutations and epigenetic changes, whereby, activated inflammatory cells produce reactive oxygen species (ROS) and reactive nitrogen intermediates that can induce DNA damage and mutation [START_REF] Morgillo | Carcinogenesis as a Result of Multiple Inflammatory and Oxidative Hits: a Comprehensive Review from Tumor Microenvironment to Gut Microbiota[END_REF].

Importantly, two key genes in the inflammatory process, cyclooxygenase-2 (COX-2) and nuclear factor kappa B (NF-κB), provide a mechanistic link between inflammation and cancer and are targets for chemoprevention, particularly, in CRC [START_REF] Desai | Mechanisms of Phytonutrient Modulation of Cyclooxygenase-2 (COX-2) and Inflammation Related to Cancer[END_REF][START_REF] Dixon | Mechanistic aspects of COX-2 expression in colorectal neoplasia. Recent results in cancer research. Fortschritte der Krebsforschung[END_REF]. Most, but not all, tumorpromoting cytokines trigger receptors on intestinal epithelial cells that activate oncogenic transcription factors and other oncogenic signaling pathways, such as extracellular signalregulated kinase or Akt/mammalian target of rapamycin (mTOR) [START_REF] Samadi | A multi-targeted approach to suppress tumor-promoting inflammation[END_REF][START_REF] Yang | Targeting cancer stem cell pathways for cancer therapy[END_REF].

Nuclear factor-κB

Nuclear factor-κB (NF-κB) transcription factors play a crucial role in many physiological and pathological processes (Figure 14). Given their important role in mediating inflammatory signals, a lot of attention has been given to NF-κB and its upstream activator, IκB kinase (IKK), and their involvement in inflammation and carcinogenesis [START_REF] Liu | NF-κB signaling in inflammation[END_REF] NF-κB regulates the expression of genes, many of which play important roles in the regulation of inflammation and apoptosis and have been associated with tumor progression, whereby activated NF-κB was found in 40% of CRC tissues and 67% of CRC cell lines [START_REF] Park | Roles of NF-κB in Cancer and Inflammatory Diseases and Their Therapeutic Approaches[END_REF]. The upstream IκB kinases are also activated in several types of cancer. Additionally, defective IKKα was found in several solid tumors such as breast, colon, ovarian, pancreatic, bladder, prostate carcinomas and melanoma [START_REF] Xia | an active player in human cancers[END_REF].

NF-κB pathway Description

Nuclear factor-κB (NF-κB) proteins include NF-κB2 p52/p100, NF-κB1 p50/p105, c-Rel, RelA/p65, and RelB. These proteins function as transcription factors that regulate the expression of genes influencing a broad range of biological processes including innate and adaptive immunity, inflammation, stress responses, B-cell development…among others.

The two characterized molecular pathways of NF-κB are the classical (or canonical) pathway, and the alternative (or noncanonical) pathway.

In the classical pathway, NF-κB/Rel proteins are bound and inhibited by IκB proteins. These

IκB are phosphorylated thus activated by proinflammatory cytokines, Lipopolysaccharide (LPS), growth factors, and antigen receptors. The activation of IκB is followed by the activation of NF-κB/Rel complexes that translocate to the nucleus where they induce target gene expression.

On the other hand, in the noncanonical NF-κB pathway, the activation of the kinase NIK, leads to the activation of IKKα complexes, which leads to the phosphorylation of C-terminal residues in NF-κB2 p100. This leads to the translocation of NF-κB complexes to the nucleus thus inducing target gene expression [START_REF] Cellsignaling | NF-kB signaling[END_REF].

Cyclooxygenase-2

Many human cancers show elevated prostaglandin (PG) levels owing to upregulation of cyclooxygenase-2 (COX-2). The chief downstream mediator of COX-2 is PGE2 that promotes cellular proliferation and angiogenesis, inhibits apoptosis, enhances invasiveness, and modulates immunosuppression. COX-2 is found to be significantly overexpressed in a variety of tumors, including CRC. It is regularly expressed at low levels in colonic mucosa, however, its activity increases dramatically following mutation of the APC gene, thus linking COX-2 to the pathogenesis and progression of colorectal carcinogenesis [START_REF] Wang | The intriguing biology of the tumour necrosis factor/tumour necrosis factor receptor superfamily: players, rules and the games[END_REF]100].

Selective inhibitors of COX-2 (coxibs) have established a noticeable efficacy in the treatment of pain and inflammation comparable to that of non-selective Non-steroidal anti-inflammatory drugs (NSAIDs) with better gastrointestinal safety. Subsequently, additional pharmacologic activities have emerged outside of coxibs' analgesic activity, such as their ability to induce apoptosis and anti-neoplasic effects [100].

Interleukin-6

Interleukin-6 (IL-6) was found to stimulate proliferation of premalignant enterocytes.

Additionally, IL-6 is a potent stimulator of colon cancer cell proliferation and tumor growth. It has an important role in colitis as it mediates a pathogenic immune response, hence its inactivation completely blocks colitis in several animal models and in patients [START_REF] Terzić | Inflammation and Colon Cancer[END_REF].

Most of the effects of IL-6 in cancer cells are mediated by STAT3, a transcription factor that is activated by many growth factors and cytokines, including IL-11; IL-22; TGF-α, TGF-β and EGF…among others.

IL-6 also promotes Th17 cell differentiation, which can promote and sustain IBD, and regulates the survival of other proinflammatory T cells, such as Th1 cells, while inhibiting the function of Treg cells [START_REF] Terzić | Inflammation and Colon Cancer[END_REF].

IL-6 cascade description

IL-6 signaling is induced by the complex formed with IL-6 and a specific IL-6R leading to a series of signals through two main pathways: the JAK/STAT pathway and the Ras/MAPK (mitogen-activated protein kinase) pathway (Figure 15).

TNF-α

Tumor Necrosis Factor alpha (TNF-α) is a multifunctional proinflammatory cytokine, playing an important role in various physiological and pathological processes, including cellular proliferation, differentiation and apoptosis…among others. TNF-α is produced during the initial inflammatory response. It induces and maintains inflammation through the production of cytokines, chemokines, endothelial adhesion molecules, and the recruitment of activated leukocytes to the site of infection or injury. A large number of cells are able to produce TNFα, mainly monocytes and macrophages but also lymphocytes, neutrophils, NK cells …among others [102].

TNF-α acts through two distinct receptors: TNFR1 p55 (TNF Receptor-1) which is found in all human tissues and TNFR2 p75 (TNF Receptor-2) which is mainly expressed in immune cells. Collectively, TNF-α is considered as a promoter of inflammation, angiogenesis, and tumor promoting factor, specifically, TNF-α expression is shown to be upregulated in CRC [START_REF] Terzić | Inflammation and Colon Cancer[END_REF].

However, the binding of TNF-R1 and TNF-R2 results in distinct downstream pathways, which favor both cell survival and apoptosis. Importantly, it has been shown that the activation of JNK, Erk1/2, p38 MAPK, and NF-κB pathways promotes cell survival. On the other hand, the activation of caspase-8 induces apoptosis [103-106].

TGF-β

Despite a primary tumor suppressor role, there is increasing evidence suggesting that the transforming growth factor-beta (TGF-β) can promote tumor growth, invasion, epithelial-mesenchymal transition (EMT) and metastasis in advanced stages of CRC. In addition, TGF-β has been shown to attenuate an anti-tumor immune response through the induction of regulatory T cells in spontaneous and inflammation-associated cancer [107].

Importantly, TGF-β is a multifunctional growth factor that possesses dual roles in tumor progression, as it acts as both anti-tumorigenic and pro-tumorigenic agent depending on the stage and characteristics of the tumor [107,108].

Interleukin-10

Interleukin-10 (IL-10) is also considered as a cytokine with a double role; interestingly, discrepancies are found between animal and human observations. For example, human IL-10 serum levels increase over time during CRC progression and correlate with poor CRC prognosis, shedding light on the tumor promoting role of this cytokine [109].

In contrast, several studies showed that IL-10-deficient mice develop colitis and colitisassociated cancer within two to three weeks after birth, shedding light on the importance of this cytokine in colorectal inflammation and carcinogenesis and posing IL-10 as a tumor protective agent. Additionally, the experimental IL-10 knockout mouse model results in a disease similar to human IBD and, therefore, has been proven useful as an experimental model for developing new and effective therapies for IBD and its associated carcinogenesis [100,108].

K-Inflammation and diabetes mellitus

Inflammation plays a major role in the onset and progression of diabetes, importantly diabetes is now considered as an inflammatory disease. Moreover, increased levels of circulating inflammatory markers were found in serum of diabetic patients. It is an etiological factor which promotes the onset of insulin resistance leading to DM. It also contributes to the predicted diabetes complications.

One important factor linking diabetes and inflammation is obesity, it is closely associated with the development of insulin resistance in inflammatory state and peripheral tissues.

Furthermore, the activation of the immune system is also a crucial factor that when associated to chronic inflammation, contributes extensively to the pathogenesis of DM. All these mechanisms and others are illustrated in Figure 16. The occurrence of diabetes correlates with multiple health conditions originating due to inflammatory mechanisms such as IBD [10,[START_REF] Halim | The effects of inflammation, aging and oxidative stress on the pathogenesis of diabetes mellitus (type 2 diabetes)[END_REF].

L-Metformin

Metformin is one of the most extensively prescribed metabolic modulators. It was first synthesized in the 1920s and has been used worldwide for treating diabetes mellitus, metabolic syndrome and polycystic ovary syndrome among others. Metformin is a safe, inexpensive medicine suitable for daily use. It is also suitable to patients who need chemopreventive agents as a long term therapy. Multiple studies have elucidated the mechanism of action for metformin. Metformin is known to activate AMP-activated protein kinase (AMPK), which inhibits the mTOR pathway that plays an important role in cellular translational processes and progression [111][112][113]. These properties prompted interest in metformin as a potential anticancer agent. Subsequently, a number of observational studies and meta-analysis have associated lower cancer incidence with metformin use as well as a lower risk of nonspecific cancer-related mortality [9,114] . Several studies have also demonstrated that metformin has an anti-proliferative effect associated with cell cycle arrest and apoptosis by down regulation of anti-apoptotic proteins as well as AMPK [115,116], however, its mechanism of action is not fully elucidated, especially in the context of CRC.

M-Probiotics

Having demonstrated the role of microbiota in colorectal cancer onset and progression, it is reasonable to suppose that it could be switched to a "non-carcinogenic" microbiome, thereby preventing the tumorigenic process [START_REF] Mármol | Colorectal Carcinoma: A General Overview and Future Perspectives in Colorectal Cancer[END_REF].

Dietary interventions and food supplements, such as selected probiotics, have emerged as a valid alternative to manipulate microbiota and manage several diseases such as diabetes, inflammatory diseases and colon cancer among others [117,118].

The World Health Organization /Food and Agriculture Organization (2001) defined probiotics as "live microorganisms which when administered in adequate amounts confer a health benefit on the host" [119].

Multiple reports showed the relevance of probiotic oral supplementation and the scarcity of adverse effects. However, contradictory results still exist in literature about the anticarcinogenic activities of probiotics [117].

Probiotics were also shown to have beneficial effects on diabetes mellitus, with numerous In the last few years, a growing interest in studying and using probiotic microorganisms has been observed, not only for the treatment of gastrointestinal diseases but also for the overall improvement of human health [START_REF] Mármol | Colorectal Carcinoma: A General Overview and Future Perspectives in Colorectal Cancer[END_REF].

Indeed, several studies and reviews highlighting both the systemic activity of probiotics and their beneficial role in ameliorating diabetes and allergic diseases such as ectopic eczema management have been published [10,123].

Consequently, it is clear that the manipulation of intestinal microbiota composition by means of probiotics may be a promising approach to ensure the correct maintenance and improvement of human health, in general and colorectal cancer in particular.

Chapter 3: Materials and Methods

Animals

In this study, a total of 96, 6-weeks old, male Balb/c mice were grouped according to their body weights and housed in medium sized polysulfone cages at the animal care facility of the American university of Beirut. The animals were kept at a constant temperature (21°C ± 2°C)

with an alternating 12-hour light/dark cycle. Animal chow (provided from Teklad-Envigo) and water were provided ad libitum.

Personnel working with the animals were trained and certified by the Animal Care facility of the American University of Beirut. Moreover, the study was approved by the Institutional Animal Care and Use Committee of the American University of Beirut, Lebanon (IACUC#16-04-370).

All animal experiments and procedures followed strictly the institutional and international ethical guidelines of the care and use of laboratory animals.

Experimental design

The Balb/c mice were divided into 4 main categories: diabetics and non-diabetics , CRC and non-CRC as illustrated in table 2. Animals were then randomly arranged according to the different treatment combinations:

-Non-treated controls -Treated with metformin (M) alone, -Treated with probiotics (P) alone -Treated with the combination of the 2 drugs (MP)

As detailed in Table 2, there were 16 animal subgroups.

Mice were individually labeled by tail coloring to facilitate the tracking of each mouse and the average group weight was adjusted to eliminate any significant weight variance among the different animal groups. -DSS preparation:

DSS (purchased from Sigma-Aldrich, Thermo Fisher Scientific, Inc), is the pro-inflammatory agent. DSS powder was weighed and dissolved in autoclaved water and administered to the animals, at optimized concentrations, in their drinking water. A DSS cycle consists of one week DSS and the two following weeks of regular drinking water.

Since the colitogenic effect of DSS is influenced by environmental factors , the used animal batches and strain as well as other factors, pilot studies were performed to define the optimal concentration and needed number of DSS cycles [125].

AOM (Sigma-Aldrich, Thermo Fisher Scientific, Inc) is the carcinogenic agent, it was injected intraperitoneally at a Maximum Tolerated Dose (MTD) of 10 mg/kg body weight [124].

Induction of Diabetes

Streptozotocin (STZ) (Sigma Aldrich, Fisher Scientific, Inc) at a single 150 mg/kg dose has been used to induce diabetes mellitus. Preceding its administration, STZ was directly suspended in citrate buffer (pH 4.4-4.5) and injected intraperitoneally [126,127].

Administration of probiotics and metformin

The commercial probiotic (P) used regroups a combination of seven strains of lactic acidproducing bacteria: Lactobacillus rhamnosus, Saccharomyces boulardii, Bifidobacterium breve, Bifidobacterium lactis, Lactobacillus acidophilus, Lactobacillus reuteri and Lactobacillus plantarum. A daily dose of 10 8 CFU per animal was administered in their drinking water.

Metformin (Glucophage) treatment was administered via drinking water at a dosage of 150 mg/kg body weight. .

Clinical course assessment

During the experimental period, clinical parameters were recorded, animals were monitored for body weight, stool aspect and rectal bleeding. The clinical disease activity index (DAI) emanated from these parameters with a score ranging from zero to four. DAI is calculated based on the formula : DAI = (Stool consistency + Fecal bleeding + Weight loss)/3 considering the following values : stool consistency (0, normal; 2, loose; 4, diarrhea), gross bleeding (0, absence; 2, blood stained; 4, presence) and weight loss (0, none; 1, 1%-5%; 2, 5%-10%; 3, 10%-20%; 4, >20%) [128].

Determination of blood glucose levels

Tail vein blood was used to measure blood glucose levels by am Accu-Chek® Performa blood glucose meter system. The reading spectrum for the Accu-Chek is 10-500 mg/dl values exceeding 500 mg/dl were registered as "HI".

Glucose measurements were done before diabetes induction and weekly after STZ injection.

Diabetes was diagnosed with BGL > 250 mg/dl. Moreover, all blood glucose measurements were taken at the same time, in the fed state early in the morning to eliminate inconsistency in blood glucose values caused by feeding patterns of the mice [129].

Measurement of fecal occult blood

In order to collect feces, each mouse was placed for a couple of minutes in an empty cage with no bedding, using a clean forceps, feces were removed and occult blood was measured using Guaiac fecal occult blood test kit, as per the manufacturer instructions [130].

Blood and serum collection

Terminal bleeding was performed on the experimental time point by cardiocentesis in accordance with the approved institutional animal care protocols. The blood was then centrifuged at 2500 rpm for 10 min and the serum was separated and stored at -20°C for further analysis.

Dissection

At the experiment endpoint, animals were sacrificed by cervical dislocation and dissected in order to remove their colon. Isolated colon was quickly flushed on ice with cold phosphatebuffered saline (PBS) in order to clean the colon and remove its content.

A portion of this clean colon was fixed in 10% buffered formalin for histological processing and the other bigger portion was snap frozen and kept in a liquid nitrogen tank for further molecular studies.

Histological studies

After being fixed in the liquid fixing agent (10% Formalin) for more than 48 hours, colon biopsies went through a series of processing steps: dehydration with graded ethanol, clearing using xylol, wax infiltration and paraffin embedded. Blocks were then cut into 5 μm sections, placed on a glass slide and stained for general morphology with hematoxylin and eosin (H&E)

[131].

The different sections were examined under light microscopy and photographed at different magnifications using an Olympus CX41 microscope. A scale from Hussein et al. as illustrated in table 3, was adapted and used to do the histologic scoring of the H&E stained sections [132].

Determination of intestinal cellular proliferation by immunohistochemistry using

Ki-67 stain

Paraffin-embedded sections were used to check for ki-67 labelling, slides were deparaffinization using a series of xylol and graded alcohol (Table 4). Then, antigen retrieval was done by immersion in a citrate buffer at pH=6. After a TBST wash, the primary antibody (Ki-67 Encorbio) was incubated overnight at a dilution of 1/1000 at 4°C .The next day the sections were washed and incubated with goat anti-rabbit IgG highly cross-adsorbed Secondary Antibody, Alexa Fluor 594 (Thermo Fisher Scientific, Inc Scientific) at a dilution of 1/1000

for 2 hours at room temperature. The nuclei were counterstained with DAPI and the slides coversliped and stored at 4°C until analysis.

Ki-67 was evaluated by examining the crypts, which were photographed and the number of Ki-67 positive cells per HPF (40x objective) were counted [133,134].

Reactive oxygen species measurement by dihydroethidium (DHE)

Frozen slides were prepared from tissues, snap frozen in liquid nitrogen (-80°C), and a solvent resistant pen was used to demarcate colonic tissue. DHE solution (Thermo Fisher Scientific, Inc) was dispensed over the tissue at a 1/1000 dilution. The slides were then placed in an incubator at 37°C for 30 min, the DHE residues removed, and slides counterstained with a mounting medium containing DAPI. Slides were coversliped and stored at 4°C at dark until At first, Griess reagent was prepared by mixing an equal volume of reagents A and B.

A 96 well microtiter plates were used, whereby 100 µl of Griess reagent was added to 100 µl of serum sample. The plate was incubated in dark for 10 min at room temperature and absorbance was measured at 550 nm. A sodium nitrite standard curve was established using a serial dilution of a NaNO2 solution. This curve was used to calculate the nitrite concentration (µM) [136,137].

Assessment of cytokine levels

The levels of IL-6 and TNF-α were assessed in both plasma and colon tissue using ELISA assay (Figure 20) performed according to the manufacturer's instructions (Thermo Fisher Scientific, Inc). The optical density was measured using a microtiter plate reader on 450 nm (Multiskan Ascent 96/384 plate reader, Thermo Fisher Scientific, Inc). The final results were expressed in pg/ml with the limit of detection of IL-6 and TNF-α as 4-500 pg/ml and 8-1000 pg/ml, respectively.

Statistical analysis

Statistical analyses were done using GraphPad Prism 8.0.1 software and data were expressed as a mean±SEM. Significant differences were assessed using the one-way ANOVA followed by Tukey-Kramer multiple comparisons test. A value of P<0.05 was considered significant [140].

Chapter 4: Results

Effect of metformin and probiotics on glycemia

All the emanating clinical, histological, immunohistochemical and molecular data were analyzed with an emphasis on the effect of the combination therapy, M and P, in treating CRC in non-diabetic and diabetic animals.

First of all, diabetes induction in all intended animals was successful. Blood glucose level was measured on a weekly basis as a direct indicator of diabetic status.

As expected, one week after an IP injection of 150 mg/kg STZ, glycemia levels increased beyond 250 mg/dl. These elevated levels were effectively maintained in all diabetic untreated mice (group D and DCRC) during the experimental period (Figure 21b).

All animals in the non-diabetic groups (NC, CRC, CRC+M, CRC+P, CRC+MP, M, P and MP), had glycemia levels less than 250 mg/dl at all time points and the difference between these groups was not statistically significant (ns) as shown in Figure 21a.

Nevertheless, animals in the CRC group that were not exposed to diabetes induction (group CRC), showed elevated glycemia peaks specifically in weeks 3, 5, 8 and 11 in which DSS was administered. Importantly, at week 8, these fluctuations in blood sugar levels were statistically significant when comparing normal animals in group NC to CRC group. These peaks were significantly lowered in animals treated with the MP mixture (CRC+MP versus CRC, # p<0.05) as shown in Figure 21a.

Diabetic (DCRC and D) groups showed the highest blood glucose levels at all time points with no statistical difference (ns) between the two groups. This reveals that colorectal carcinogenesis did not affect the animals' glycemia levels. In diabetic mice, the treatment with P and M alone or in combination significantly reduced glucose levels throughout the experimental period.

When compared to the untreated diabetic mice, with CRC induction (DCRC group), M and P single drug administration in DCRC+M and DCRC+P groups reduced significantly blood glucose values (Figure 21b, *p<0.05). Remarkably, the M and P mixture showed a significant effect, superior than either drug alone, as showed when comparing DCRC+M versus DCRC+MP groups ( † p<0.05) and DCRC+P versus DCRC+MP groups ( † p<0.05). These observations indicated that P helped M in alleviating the hyperglycemic phenotype induced by STZ (Figure 21b).

Effect of metformin and probiotics on CRC

The optimal procedure adopted for CRC induction was successful in all mice. The concentration of Dextran sulfate sodium (DSS) and its number of cycles were defined by pilot studies in which using different concentrations of DSS ranging from 1 till 3% were tested.

Treatment of male Balb/c mice with 1.5% DSS in their drinking water for 4 cycles, in addition to an injection of 10 mg/kg Azoxymethane (AOM), resulted in clinical signs and symptoms, gross and histological changes characteristic of CRC.

Animals that underwent this optimized DSS/AOM protocol showed signs of sickness starting the first cycle and these signs were intensified by each consecutive DSS cycle.

Weight loss (Figure 22), loose stools, diarrhea and rectal bleeding were the main detected changes, as calculated by the DAI. Additionally, these animals presented various signs of sickness such as bad posture, hunched back, decreased grooming, reduced mobility and responsiveness (Figure 23).

A weight gain was seen in non-diabetic and diabetic animals without colorectal cancer (i.e. in groups NC, M, P, MP, D, D+M, D+P and D+MP). Conversely, groups with colorectal cancer presented a weight loss; whereby the lowest weight averages were noted in CRC and DCRC groups with no statistically significant difference between these 2 groups. Treatment with metformin or probiotics alone and most importantly in combination had a positive effect in preventing the weight loss in the non-diabetic and diabetic CRC animals with # p<0.05 and *p<0.05 respectively (Figure 22). When looking at fecal occult blood, negative values were recorded in all animals belonging to the groups that were not exposed to CRC induction (NC, D, M, D+M, P, D+P, MP and D+MP groups). On the other hand, animals that had the CRC induction, displayed rectal bleeding and had positive fecal blood to varied extents. 17% of CRC and DCRC animals had positive occult blood on the first DSS administration (at week 3), they reached 100% positive rates by the end of the experiment (Table 5).

Metformin and probiotics treatment alone or most importantly in combination reduced the frequency of blood in the stools and hindered their appearance until week 5.

CRC+MP and DCRC+MP groups showed the best scores whereby blood in stools was detected in only 67% of the animals at week 5 and the positive rates were 50% and 33%, respectively , at week 13. In addition, when paralleling animals treated with single drug in non-diabetic and diabetic CRC, i.e. groups (CRC+M, CRC+P, DCRC+M and DCRC+P) to the MP combination groups (CRC+MP and DCRC+MP), lower proportions of positive occult blood in the combination treatment, were encountered at all time points (Table 5). Treatments elicited a decrease of the blood in the stools, thus indicating a recovery in the mucosa. As for the DAI, the highest disease activity indices were obtained in animals belonging to the CRC and DCRC groups (Figure 24). Treatment of CRC animals with M alone or P alone improved their clinical profile and decreased DAI, but not significantly.

Importantly, the combined MP treatment induced a decrease in DAI levels in non-diabetic CRC and diabetic CRC ( # p<0.05 and *p<0.05, respectively), as these groups (CRC+MP and DCRC+MP) had the lowest DAI along with an amelioration in their clinical status.

The addition of probiotics to the metformin treatment was beneficial as this combination decreased DAI in non-diabetics as seen when comparing animals treated with metformin alone and animals treated with the MP combination in CRC and DCRC groups (Figure 24). normal controls and in all of the other groups that were not subject to AOM/DSS CRC induction (i.e. M, P, MP, D, D+M, D+P and D+MP groups) had normal DAI close to zero.. Additionally, it is worth noting that when comparing diabetic animals to their non-diabetic counterparts, diabetics were shwon to present more signs of discomfort and sickness without significant alteration of the DAI score (Figure 24).

Effect of metformin and probiotics on the survival rates

A fluctuation in survival rates was detected between the different groups based on their experimental condition and administered treatment. The lowest survival rate was obtained in in diabetic CRC (DCRC) animals whereby 50% of the animals were dead by the experimental endpoint (week 13). Animals receiving the M and P treatments had survival rates of 67% and 83% in groups DCRC+M and DCRC+P, respectively. Importantly DCRC+MP group receiving the combined therapy had 100% survival rates (Figure 25). CRC non-diabetic animals had a survival rate of 67%, the survival was ameliorated when animals were treated with M and P alone or in combination in CRC+M, CRC+P and CRC+MP groups, as their survival rates reached 100% similarly to normal animals in groups NC, M, P and MP (Figure 25).

Metformin and probiotics effect on colon length

It is well known that a shortening of the colon reflects the extent of colorectal inflammation.

In this study, measurement of the colon showed differences between the various groups: the shortest colons were seen in mice exposed to AOM/DSS (CRC and DCRC groups) with 6.75±0.78 cm and 6.67±0.17 cm respectively, (Figures 26 and27). In non-diabetics, animals that were subject to CRC induction (CRC group), showed significantly shorter colons than the normal controls (NC) ( # p<0.05). Metformin administration in (CRC+M) group augmented the colon length, but this was not statistically significant.

However, administration of P alone in (CRC+P) group or in combination with metformin (CRC+MP) significantly increased colon length to reach 10.58±0.83 cm and 10.83±0.31 cm, respectively, with # p<0.05 (Figure 27), close to normal control (NC) group.

Significantly shorter colon were obtained when comparing DCRC group and D group with *p<0.05. Treatment with single drug, M or P ameliorated the colon length but not significantly in DCRC+M and DCRC+P groups. However, when the MP combination is administered, the colon length was significantly ameliorated, even that it reached the normal values (11±0.63 cm in DCRC+MP group) (*p<0.05) (Figure 27). Average colon length (cm) was recorded on the day of sacrifice at week 13, bar graphs represents the mean±SEM for each group (n=6). Significant differences between the groups were determined by one-way ANOVA followed by Tukey's Multiple Comparison Test. Non-diabetic groups were compared to their experimental CRC control, significance was expressed by # p<0.05. On the other hand, diabetic groups were compared to their experimental diabetic CRC control, significance was expressed by *p<0.05 Polyps were counted on the day of sacrifice, bar graphs represents the mean±SEM for each group (n=6). Significant differences between the groups were determined by one-way ANOVA followed by Tukey's Multiple Comparison Test. Non-diabetic groups were compared to their experimental CRC control , significance was expressed by #p<0.05. On the other hand, diabetic groups were compared to their experimental diabetic CRC control, significance was expressed by *p<0.05. Moreover, when comparing only 2 groups, connecting lines were used to indicate the compared groups with †p<0.05, (ns) stands for non-significant.

Histological alterations of the colon due to metformin and probiotics treatment

No alterations were depicted when examining colon sections of the normal mice in groups NC, and mice receiving metformin and probiotic alone or in combination in groups M, P and MP.

These groups showed low histological scores, they had normal colon histology characterized by the straight unbranched crypts reaching the muscularis mucosa, the intact columnar epithelium, with enterocytes lining the crypts, presence of numerous goblet cells , a thin smooth muscularis mucosae , as well as normal presence of inflammatory cells aggregates (Panel 26Aa).

In contrast, in CRC and DCRC groups, animals that were exposed to the AOM/DSS protocol showed significant histological alteration upon histological inspection of their respective colon tissues.

These animals in these groups had the highest histological alterations with 19.0±1.35 and 20.0±0.58, respectively (Figure 29C). Several changes were noted indicating the different levels of inflammation and dysplasia occurring in the colon. Mainly, an epithelial ulceration, an inflammation of the crypts, formation of crypt abscesses, as well as a dysregulation in the normal crypt architecture were noted. In addition, huge inflammatory cells infiltration in the mucosa and submucosa were present, along with a discontinued muscularis mucosa and goblet cells depletion ….as represented in Panels 26A-e and 26B-m for CRC and DCRC, respectively.

The comparison of the histological scores in the different non-diabetic and diabetic groups shed light on the beneficial effect of the combination therapy on the colonic tissue.

An improvement in colon crypts was noted in groups CRC+M and CRC+P, as seen in panels 28A-f and g, and this single drug treatment reduced the histological score to reach 14±1.0 and 11.83±0.31, respectively, with # p<0.05 when compared to untreated CRC (Figure 29C). The administration of MP combination, had an improved effect better than either drug alone and presented the lowest histological score (8.67±1.17 in CRC+MP group with # p<0.05) (Figure 29).

On the other hand, diabetic animals (D group), that were not exposed to CRC induction, showed a histological alteration score of 7.50±1.54, significantly higher than non-diabetic animals (NC group) with † p<0.05 (Figure 29C), thus emphasizing on the damage caused by diabetes alone on the colonic tissue illustrated by the increase in inflammatory cells infiltrates as seen in Panel 29B-i.

Additionally, single drug treatment in groups DCRC+M and DCRC+P was not able to prominently reverse the pathological damage caused by inflammation and dysplasia (Panel 29B-n and 29B-o). However, when probiotics and metformin were administered in combination, the improvement of the histological damage and the reduction in the histological score were statistically significant as noted in group DCRC+MP (Panel 29B-p) with *p<0.05 (Figure 29C).

Besides, the structural improvement between treatment with metformin alone (CRC+M, DCRC+M) and metformin with probiotics in CRC+MP and DCRC+MP groups was statistically significant, † p<0.05 (Figure 29C). One probable explanation could be that the dysregulated microbiota in CRC could stop metformin from exerting its protective effects on the colonic tissues. Adjustment of this dysbiosis with probiotics showed to be critical in modulating the anti-inflammatory and anticarcinogenic mechanism of action of metformin. Note the presence of large inflammatory cells infiltrates (circle), inflammatory cells invading the edematous submucosa (bracket), crypt abcess (black triangle) and cryptitis (black arrow) as well as crypt architecture disarray (star). Significant improvements in the combination treated mice in groups CRC+MP (7A-h) and DCRC+MP (7B-p) were noted. Original magnification 4X; scale bars 100 µm. Photos were adjusted for white balance using Adobe Photoshop®; (C) Histological alterations score. Data is expressed as Average ± SEM (n=6). Significant differences between the groups were determined by one-way ANOVA followed by Tukey's Multiple Comparison Test. Non-diabetic groups (CRC, CRC+M, CRC+P, CRC+MP, M, P and MP) were compared to their experimental CRC control (CRC), significance was expressed by #p<0.05. On the other hand, diabetic groups (DCRC, DCRC+M, DCRC+P, DCRC+MP, D+M, D+P and D+MP groups) were compared to their experimental diabetic CRC control (DCRC ), significance was expressed by *p<0.05. Moreover, when comparing only 2 groups, connecting lines were used to indicate the compared groups with †p<0.05, (ns) stands for non-significant.

Lower magnifications of each slide are presented in the figures 30 till 37. Non-diabetic groups were compared to their experimental CRC control , significance was expressed by # p<0.05. On the other hand, diabetic groups were compared to their experimental diabetic CRC control, significance was expressed by *p<0.05. Moreover, when comparing only 2 groups, connecting lines were used to indicate the compared groups with † p<0.05, (ns) stands for non-significant.

Modulation of reactive oxygen species production

Frozen colon sections were used to evaluate ROS activity, using dihydroethidium (DHE), a ROS responsive stain.

CRC and DCRC groups had the highest ROS levels, Moreover, diabetic animals in group D also had high ROS production with † p<0.05 when compared to NC group (Figure 39),thus, demonstrating the increased ROS generation in diabetes and shedding light on the shared oxidative stress increase between CRC, inflammation and diabetes.

In non-diabetics, single drug treatment reduced significantly ROS production, with the lowest DHE/DAPI ratios obtained in CRC+MP group # p<0.05 (Figure 39 ).

Concerning the diabetic animals, single drug treatment in DCRC animals did not affect the colonic ROS production. However, when the two drugs were combined, a significant reduction in DHE/DAPI ratios was noted in DCRC+MP group (Figure 39).

(Figure 40). Moreover, diabetic animals in D group, showed elevated levels of nitrite when compared to their non-diabetic counterparts with, however, no statistically significant difference. Serum nitrite production was measured by Griess assay. Results are expressed as mean±SEM (n=4). Significant differences were determined by one-way ANOVA followed by Tukey's Multiple Comparison Test. Non-diabetic groups were compared to their experimental CRC control, significance was expressed by # p<0.05. On the other hand, diabetic groups were compared to their experimental diabetic CRC control, significance was expressed by *p<0.05, (ns) stands for non-significant.

Effects of metformin and probiotics on IL-6 and TNF-α production

The analysis of the two cytokines, IL-6 and TNF-α, in the serum and colon extracts of the different animal groups showed interesting variations.

Normal animals in the NC group showed low levels of cytokines, on the other hand, mice in CRC and DCRC groups had significantly higher IL-6 and TNF-α levels in their colons (Figure 41-a, c) and sera (Figure 41 In addition, TNF-α levels were also significantly reduced in both colon and serum of CRC+MP group with , # p<0.05 and only in the serum of DCRC+MP group with *p<0.05.

Moreover, elevated IL-6 and TNF-α levels were noted in group D, despite the fact that animals in this group were not subjected to CRC induction, shedding light on the inflammatory environment created by diabetes in the colon, which has a major impact on carcinogenesis.

Collectively, metformin and probiotics were shown to work in synergy in the colon and serum of diabetic and non-diabetic CRC groups, whereby the MP treatment reduced the production of IL-6

and TNF-α, two key players in inflammation and tumorigenesis. Results are expressed as mean±SEM (n=4). Error bars represent SEM. Significant differences were determined by one-way ANOVA followed by Tukey's Multiple Comparison Test. Non-diabetic groups were compared to their experimental CRC control , significance was expressed by #p<0.05. On the other hand, diabetic groups were compared to their experimental diabetic CRC control, significance was expressed by *p<0.05, (ns) stands for non-significant. As for AOM, its hydroxylation results in the formation of the reactive metabolite MAM, a DNA-Alkylating agent that acts through the addition of methyl groups at the O6 or N7 position of guanine (O6-methyl-deoxyguanosine and N7-methyl-deoxyguanosine) of the DNA molecule.

The main advantages of AOM are its low cost, high potency and reproducibility, as well as its simple mode of administration, excellent stability in solution, and low price. As for diabetes mellitus, it is a group of metabolic disorders considered as one of the most prevalent and rapidly increasing comorbid disorders.

Medical literature in the past 10 years focused on the relationship between diabetes and CRC, whereby a higher risk on colorectal cancer onset was observed in diabetic patients when compared to their non-diabetic counterparts, Moreover, poor CRC outcomes has been associated with the onset of diabetes [11,144]. These observations parallel our results where cancer was worsened by diabetes; higher histological score and a poorer clinical profile were obtained in diabetic CRC non-treated animals when compared to their non-diabetic counterparts. Moreover, survival rates reflected this observation as the rates in diabetic CRC mice were significantly lower than the non-diabetic CRC animals.

It is well established that hyperglycemia coupled with dysbiosis and an increase in oxidative stress and chronic intestinal inflammation create a favorable environment for the development of the three medical conditions DM, IBD and CRC. Notably, a dysregulated gut microbiota is being recognized as a key player in this crosstalk [10,11]. In this study, probiotics use has been considered an attractive therapeutic target in the management of inflammatory diseases, carcinogenesis as well as diabetes after it has proven in a previous study to reverse the carcinogenic process in solid tumors [9].

In the past years, a large number of experimental studies were conducted, focusing on the protective role of probiotics on a wide spectrum of human disorders, especially colorectal These observations were in line with our results whereby metformin administration to CRC animals ameliorated their clinical profile, their DAI scores and their survival rates.

Furthermore, it reduced histological alterations scores and lowered significantly the oxidative stress in diabetic and non-diabetic animals treated with metformin alone, in comparison with the untreated CRC and DCRC animals.

Collectively, our results showed that treatment with metformin alone or probiotics alone had beneficial effects on diabetes and CRC to variable degrees. These variations might be ascribed to the inter-individual difference in the composition of gut microbiota especially with the inflamed microenvironment created by the induction of CRC and diabetes.

Several recent studies have focused on the gut microbiota as a key site of action for metformin.

This was supported by old data indicating that the efficacy of metformin is affected by antibiotics [160]. Additionally, the glucose lowering effects were found to be stronger following an intraduodenal versus intravenous administration of metformin [161].

A meta-analysis conducted recently showed a reduction in HbA1c, FBG and insulin resistance level in T2DM patients treated with probiotics [162]. Moreover, Experimental and clinical evidence showed that that the modulation of gut microbiota through probiotic administration has also a preventive effect against gestational diabetes mellitus [163].

In this study, the addition of probiotics to metformin, helped metformin in excreting its anticancerous and anti-inflammatory properties. Possibly, probiotics acted through the correction of dysbiosis which enhanced the activities of metformin. In fact, CRC and DCRC animals that were treated with the combination therapy showed a significant improvement in their diabetic and cancer status, when compared to groups treated with a single drug and to untreated groups.

It is well-established that cellular proliferation is a key factor influencing carcinogenesis onset The beneficial effects granted by metformin and probiotics were also substantiated by the histological analysis of the colonic mucosa. Whereby, the analysis of H&E stained colon tissues showed that the multiple histopathological alterations recorded during the course of the disease were reversed to significant extents. The main alterations observed include surface erosion, inflammatory cells infiltration, submucosal edema, polyp formation and dysplasia.

The combination (M + P) attenuated colorectal inflammatory severity, ameliorated colorectal crypt structure and significantly reduced the severity of inflammation as assessed in the histological score. It looks like that the combination treatment (M + P) could alleviate alterations in the intestinal wall through a distinct mechanism that each drug alone. It is very probable that the integrity of the mucosa needed a different mechanism. Thus, through the balanced microbiota, the junctional complexes of the epithelial cells were maintained; the secretory part of the balanced microbiota could have provided anti-inflammatory elements and restored the eubiosis state.

Moreover, gut barrier dysregulations and pathologies promote the production of proinflammatory cytokines mainly TNF-α and IL-6, which in turn activate inflammation and insulin resistance, shedding the light again on the inflammation crosstalk between CRC and diabetes [10]. This work showed an increase in IL-6 and TNF-α in CRC animals with or without diabetes induction in CRC and DCRC groups. The increase in cytokine production was not limited to colon tissue, as the levels of these cytokines were also elevated in the serum of the animals, thus emphasizing on the systemic inflammation occurring in diabetes and CRC.

A strong inhibition of these cytokines was detected in the colon of cancerous animals when probiotics and metformin were combined.

In addition, it is clear that a state of chronic inflammation accompanied with inflammatory alterations in the colonic mucosa are characteristic features of CRC. They consist of inflammatory cells aggregates and infiltration and enhanced production of a panel of cytokines [166]. Following recruitment, neutrophils get activated and secrete large amounts of proinflammatory mediators mainly IL-1β and IL-6 [167]. These changes, paralleled with elevated levels of reactive oxygen and nitrogen species (RONS) generated in the colon tissue, contribute to destructive mucosal damage, which creates a leaking mucosal barrier that could allow multiple bacteria, including toxic strains to infiltrate and grow leading to a chronic inflammation, an optimal environment for developing colitis associated CRC [START_REF] Yu | Microbiota dysbiosis and barrier dysfunction in inflammatory bowel disease and colorectal cancers: exploring a common ground hypothesis[END_REF].

In this study, CRC and diabetes induced an upregulation of free radicals production. This was indicated by the increased levels of RONS in diabetic and CRC animals. These levels were restored to normal by the administration of the combination therapy, metformin and probiotics.

The increased NO production correlates with the increase in the levels of pro-inflammatory mediators like TNF-α and IL-6, thus leading to exacerbation of the inflammatory chronic reaction, which is at the core of the IBD-CRC etiology [168]. Treatment with metformin in combination with probiotics was able to diminish the production of inflammatory mediators, of RONS, and to reestablish the colonic structure and function of the gut barrier.

This study had potential limitations; first, as with the majority of studies, the design of the current study was subject to limitations as the number of animals per group could be increased, moreover the experiment could have been repeated to validate the results. Second, the study was only conducted on male mice, both genders could be used to eliminate gender biases and explore the possible different responses between males and females. Third, additional molecular testing could have been done to further explore additional cytokines and molecular pathways involved in the crosstalk between CRC and diabetes. A dysregulated microbiota in a state of dysbiosis, impairs the functions of the gut barrier by affecting the tight junctions and the mucus layer, thus facilitates the translocation of pathogens and toxins towards lamina propria. This invasion leads to recruitment of inflammatory cells, their activation and the secretion of pro-inflammatory cytokines including IL-6 and TNF-. In parallel, an increase in RONS production induces a chronic inflammatory state, DNA damage and increased cell proliferation, known as key players in CRC progression. Moreover, inflammation promotes insulin resistance and disturbance in glucose homeostasis thus exacerbating diabetes and enhancing CRC. Probiotics and metformin administration, however, inhibited CRC progression, reduced inflammation and ameliorated diabetes. These beneficial effects are potentially due to a restoration of the gut barrier, production of SCFA, anti-microbial peptides, regulation of hepatic glucose production and modulating the balance between proliferation and apoptosis.

The result of such a balanced microenvironment is the preservation of a dynamic intestinal barrier, which controls and maintains homeostasis.

Figure 1 :Figure 2 :Figure 3 :

 123 Figure 1: Diagram of general organization of the alimentary canal. ....................................... Figure 2: Features of the musculature of the large intestine. ................................................... Figure 3: Histology of the colon (H&E). .................................................................................Figure 4: Schematic figure of the intestinal barrier and its components. ................................Figure 5: Functions of the large intestine ................................................................................Figure 6: Bar chart of Region-specific incidence age standardized rates by sex for cancers of the (A) colon and (B) Rectum in 2018.....................................................................................Figure 7: Estimated number of new cancer cases in 2018, worldwide, both sexes, all ages ...Figure 8: Number of new cancer cases in 2018 in Lebanon, both sexes all ages. ...................Figure 9: Proportion of colorectal cancer cases associated with sporadic and hereditary factors.. ..................................................................................................................................... Figure 10: TNM classification of colorectal cancer ................................................................Figure 11: Pathways of colorectal carcinogenesis. .................................................................. Figure 12: Schematic representation of the g-FOBT (A) and i-FOBT (B) blood detection reactions. ..................................................................................................................................Figure 13: Commonly encountered reactive oxygen species.. ................................................Figure 14: The NF-κB signaling pathway. ..............................................................................Figure 15: IL-6 signaling cascade. ...........................................................................................Figure 16: The relationship between inflammation and diabetes. ...........................................Figure 17: Blood collection by cardiac puncture (cardiocentesis).......................................... Figure 18: The principle of DHE protocol for ROS detection and quantification...................Figure 19: Griess reaction principle. ........................................................................................Figure 20: Principle of sandwich Enzyme-linked Immunosorbent assay ELISA. ..................Figure 21: Effects of probiotics and metformin on glycemia levels in non-diabetic(a) and diabetic (b) mice. ..................................................................................................................... Figure 22: Weight changes in the different animal groups during the experimental period. ..Figure 23: Hunched back animals with unhealthy clinical profile (left side), a mouse with diarrhea and rectal bleeding (right side). .................................................................................Figure 24: Disease activity index variations in the different groups. ......................................Figure 25: Kaplan-Meier survival curves of animals in the different groups. .........................Figure 26: Gross macroscopic images of the colon from non-diabetic groups (a) and diabetic groups (b). ................................................................................................................................Figure 27: Colon length variation. ...........................................................................................Figure 28: Polyp count variation in the different groups. ........................................................ Figure 29 Effect of probiotics and metformin on colon histology. .........................................Figure 30: Histology of CRC and DCRC animals ...................................................................Figure 31: Histology of CRC and DCRC animals treated with metformin alone. .................. Figure 32: Histology of CRC and DCRC animals treated with probiotics. .............................Figure 33: Histology of CRC and DCRC animals treated with the combination therapy. ......Figure 34: Histology of normal controls and Diabetic animals. ..............................................Figure 35: Histology of normal controls and diabetic animals treated with metformin alone.96

Figure 4 :

 4 Figure 1: Diagram of general organization of the alimentary canal. ....................................... Figure 2: Features of the musculature of the large intestine. ................................................... Figure 3: Histology of the colon (H&E). .................................................................................Figure 4: Schematic figure of the intestinal barrier and its components. ................................Figure 5: Functions of the large intestine ................................................................................Figure 6: Bar chart of Region-specific incidence age standardized rates by sex for cancers of the (A) colon and (B) Rectum in 2018.....................................................................................Figure 7: Estimated number of new cancer cases in 2018, worldwide, both sexes, all ages ...Figure 8: Number of new cancer cases in 2018 in Lebanon, both sexes all ages. ...................Figure 9: Proportion of colorectal cancer cases associated with sporadic and hereditary factors.. ..................................................................................................................................... Figure 10: TNM classification of colorectal cancer ................................................................Figure 11: Pathways of colorectal carcinogenesis. .................................................................. Figure 12: Schematic representation of the g-FOBT (A) and i-FOBT (B) blood detection reactions. ..................................................................................................................................Figure 13: Commonly encountered reactive oxygen species.. ................................................Figure 14: The NF-κB signaling pathway. ..............................................................................Figure 15: IL-6 signaling cascade. ...........................................................................................Figure 16: The relationship between inflammation and diabetes. ...........................................Figure 17: Blood collection by cardiac puncture (cardiocentesis).......................................... Figure 18: The principle of DHE protocol for ROS detection and quantification...................Figure 19: Griess reaction principle. ........................................................................................Figure 20: Principle of sandwich Enzyme-linked Immunosorbent assay ELISA. ..................Figure 21: Effects of probiotics and metformin on glycemia levels in non-diabetic(a) and diabetic (b) mice. ..................................................................................................................... Figure 22: Weight changes in the different animal groups during the experimental period. ..Figure 23: Hunched back animals with unhealthy clinical profile (left side), a mouse with diarrhea and rectal bleeding (right side). .................................................................................Figure 24: Disease activity index variations in the different groups. ......................................Figure 25: Kaplan-Meier survival curves of animals in the different groups. .........................Figure 26: Gross macroscopic images of the colon from non-diabetic groups (a) and diabetic groups (b). ................................................................................................................................Figure 27: Colon length variation. ...........................................................................................Figure 28: Polyp count variation in the different groups. ........................................................ Figure 29 Effect of probiotics and metformin on colon histology. .........................................Figure 30: Histology of CRC and DCRC animals ...................................................................Figure 31: Histology of CRC and DCRC animals treated with metformin alone. .................. Figure 32: Histology of CRC and DCRC animals treated with probiotics. .............................Figure 33: Histology of CRC and DCRC animals treated with the combination therapy. ......Figure 34: Histology of normal controls and Diabetic animals. ..............................................Figure 35: Histology of normal controls and diabetic animals treated with metformin alone.96

Figure 5 :

 5 Figure 1: Diagram of general organization of the alimentary canal. ....................................... Figure 2: Features of the musculature of the large intestine. ................................................... Figure 3: Histology of the colon (H&E). .................................................................................Figure 4: Schematic figure of the intestinal barrier and its components. ................................Figure 5: Functions of the large intestine ................................................................................Figure 6: Bar chart of Region-specific incidence age standardized rates by sex for cancers of the (A) colon and (B) Rectum in 2018.....................................................................................Figure 7: Estimated number of new cancer cases in 2018, worldwide, both sexes, all ages ...Figure 8: Number of new cancer cases in 2018 in Lebanon, both sexes all ages. ...................Figure 9: Proportion of colorectal cancer cases associated with sporadic and hereditary factors.. ..................................................................................................................................... Figure 10: TNM classification of colorectal cancer ................................................................Figure 11: Pathways of colorectal carcinogenesis. .................................................................. Figure 12: Schematic representation of the g-FOBT (A) and i-FOBT (B) blood detection reactions. ..................................................................................................................................Figure 13: Commonly encountered reactive oxygen species.. ................................................Figure 14: The NF-κB signaling pathway. ..............................................................................Figure 15: IL-6 signaling cascade. ...........................................................................................Figure 16: The relationship between inflammation and diabetes. ...........................................Figure 17: Blood collection by cardiac puncture (cardiocentesis).......................................... Figure 18: The principle of DHE protocol for ROS detection and quantification...................Figure 19: Griess reaction principle. ........................................................................................Figure 20: Principle of sandwich Enzyme-linked Immunosorbent assay ELISA. ..................Figure 21: Effects of probiotics and metformin on glycemia levels in non-diabetic(a) and diabetic (b) mice. ..................................................................................................................... Figure 22: Weight changes in the different animal groups during the experimental period. ..Figure 23: Hunched back animals with unhealthy clinical profile (left side), a mouse with diarrhea and rectal bleeding (right side). .................................................................................Figure 24: Disease activity index variations in the different groups. ......................................Figure 25: Kaplan-Meier survival curves of animals in the different groups. .........................Figure 26: Gross macroscopic images of the colon from non-diabetic groups (a) and diabetic groups (b). ................................................................................................................................Figure 27: Colon length variation. ...........................................................................................Figure 28: Polyp count variation in the different groups. ........................................................ Figure 29 Effect of probiotics and metformin on colon histology. .........................................Figure 30: Histology of CRC and DCRC animals ...................................................................Figure 31: Histology of CRC and DCRC animals treated with metformin alone. .................. Figure 32: Histology of CRC and DCRC animals treated with probiotics. .............................Figure 33: Histology of CRC and DCRC animals treated with the combination therapy. ......Figure 34: Histology of normal controls and Diabetic animals. ..............................................Figure 35: Histology of normal controls and diabetic animals treated with metformin alone.96

Figure 6 :

 6 Figure 1: Diagram of general organization of the alimentary canal. ....................................... Figure 2: Features of the musculature of the large intestine. ................................................... Figure 3: Histology of the colon (H&E). .................................................................................Figure 4: Schematic figure of the intestinal barrier and its components. ................................Figure 5: Functions of the large intestine ................................................................................Figure 6: Bar chart of Region-specific incidence age standardized rates by sex for cancers of the (A) colon and (B) Rectum in 2018.....................................................................................Figure 7: Estimated number of new cancer cases in 2018, worldwide, both sexes, all ages ...Figure 8: Number of new cancer cases in 2018 in Lebanon, both sexes all ages. ...................Figure 9: Proportion of colorectal cancer cases associated with sporadic and hereditary factors.. ..................................................................................................................................... Figure 10: TNM classification of colorectal cancer ................................................................Figure 11: Pathways of colorectal carcinogenesis. .................................................................. Figure 12: Schematic representation of the g-FOBT (A) and i-FOBT (B) blood detection reactions. ..................................................................................................................................Figure 13: Commonly encountered reactive oxygen species.. ................................................Figure 14: The NF-κB signaling pathway. ..............................................................................Figure 15: IL-6 signaling cascade. ...........................................................................................Figure 16: The relationship between inflammation and diabetes. ...........................................Figure 17: Blood collection by cardiac puncture (cardiocentesis).......................................... Figure 18: The principle of DHE protocol for ROS detection and quantification...................Figure 19: Griess reaction principle. ........................................................................................Figure 20: Principle of sandwich Enzyme-linked Immunosorbent assay ELISA. ..................Figure 21: Effects of probiotics and metformin on glycemia levels in non-diabetic(a) and diabetic (b) mice. ..................................................................................................................... Figure 22: Weight changes in the different animal groups during the experimental period. ..Figure 23: Hunched back animals with unhealthy clinical profile (left side), a mouse with diarrhea and rectal bleeding (right side). .................................................................................Figure 24: Disease activity index variations in the different groups. ......................................Figure 25: Kaplan-Meier survival curves of animals in the different groups. .........................Figure 26: Gross macroscopic images of the colon from non-diabetic groups (a) and diabetic groups (b). ................................................................................................................................Figure 27: Colon length variation. ...........................................................................................Figure 28: Polyp count variation in the different groups. ........................................................ Figure 29 Effect of probiotics and metformin on colon histology. .........................................Figure 30: Histology of CRC and DCRC animals ...................................................................Figure 31: Histology of CRC and DCRC animals treated with metformin alone. .................. Figure 32: Histology of CRC and DCRC animals treated with probiotics. .............................Figure 33: Histology of CRC and DCRC animals treated with the combination therapy. ......Figure 34: Histology of normal controls and Diabetic animals. ..............................................Figure 35: Histology of normal controls and diabetic animals treated with metformin alone.96

Figure 7 :

 7 Figure 1: Diagram of general organization of the alimentary canal. ....................................... Figure 2: Features of the musculature of the large intestine. ................................................... Figure 3: Histology of the colon (H&E). .................................................................................Figure 4: Schematic figure of the intestinal barrier and its components. ................................Figure 5: Functions of the large intestine ................................................................................Figure 6: Bar chart of Region-specific incidence age standardized rates by sex for cancers of the (A) colon and (B) Rectum in 2018.....................................................................................Figure 7: Estimated number of new cancer cases in 2018, worldwide, both sexes, all ages ...Figure 8: Number of new cancer cases in 2018 in Lebanon, both sexes all ages. ...................Figure 9: Proportion of colorectal cancer cases associated with sporadic and hereditary factors.. ..................................................................................................................................... Figure 10: TNM classification of colorectal cancer ................................................................Figure 11: Pathways of colorectal carcinogenesis. .................................................................. Figure 12: Schematic representation of the g-FOBT (A) and i-FOBT (B) blood detection reactions. ..................................................................................................................................Figure 13: Commonly encountered reactive oxygen species.. ................................................Figure 14: The NF-κB signaling pathway. ..............................................................................Figure 15: IL-6 signaling cascade. ...........................................................................................Figure 16: The relationship between inflammation and diabetes. ...........................................Figure 17: Blood collection by cardiac puncture (cardiocentesis).......................................... Figure 18: The principle of DHE protocol for ROS detection and quantification...................Figure 19: Griess reaction principle. ........................................................................................Figure 20: Principle of sandwich Enzyme-linked Immunosorbent assay ELISA. ..................Figure 21: Effects of probiotics and metformin on glycemia levels in non-diabetic(a) and diabetic (b) mice. ..................................................................................................................... Figure 22: Weight changes in the different animal groups during the experimental period. ..Figure 23: Hunched back animals with unhealthy clinical profile (left side), a mouse with diarrhea and rectal bleeding (right side). .................................................................................Figure 24: Disease activity index variations in the different groups. ......................................Figure 25: Kaplan-Meier survival curves of animals in the different groups. .........................Figure 26: Gross macroscopic images of the colon from non-diabetic groups (a) and diabetic groups (b). ................................................................................................................................Figure 27: Colon length variation. ...........................................................................................Figure 28: Polyp count variation in the different groups. ........................................................ Figure 29 Effect of probiotics and metformin on colon histology. .........................................Figure 30: Histology of CRC and DCRC animals ...................................................................Figure 31: Histology of CRC and DCRC animals treated with metformin alone. .................. Figure 32: Histology of CRC and DCRC animals treated with probiotics. .............................Figure 33: Histology of CRC and DCRC animals treated with the combination therapy. ......Figure 34: Histology of normal controls and Diabetic animals. ..............................................Figure 35: Histology of normal controls and diabetic animals treated with metformin alone.96

Figure 8 :

 8 Figure 1: Diagram of general organization of the alimentary canal. ....................................... Figure 2: Features of the musculature of the large intestine. ................................................... Figure 3: Histology of the colon (H&E). .................................................................................Figure 4: Schematic figure of the intestinal barrier and its components. ................................Figure 5: Functions of the large intestine ................................................................................Figure 6: Bar chart of Region-specific incidence age standardized rates by sex for cancers of the (A) colon and (B) Rectum in 2018.....................................................................................Figure 7: Estimated number of new cancer cases in 2018, worldwide, both sexes, all ages ...Figure 8: Number of new cancer cases in 2018 in Lebanon, both sexes all ages. ...................Figure 9: Proportion of colorectal cancer cases associated with sporadic and hereditary factors.. ..................................................................................................................................... Figure 10: TNM classification of colorectal cancer ................................................................Figure 11: Pathways of colorectal carcinogenesis. .................................................................. Figure 12: Schematic representation of the g-FOBT (A) and i-FOBT (B) blood detection reactions. ..................................................................................................................................Figure 13: Commonly encountered reactive oxygen species.. ................................................Figure 14: The NF-κB signaling pathway. ..............................................................................Figure 15: IL-6 signaling cascade. ...........................................................................................Figure 16: The relationship between inflammation and diabetes. ...........................................Figure 17: Blood collection by cardiac puncture (cardiocentesis).......................................... Figure 18: The principle of DHE protocol for ROS detection and quantification...................Figure 19: Griess reaction principle. ........................................................................................Figure 20: Principle of sandwich Enzyme-linked Immunosorbent assay ELISA. ..................Figure 21: Effects of probiotics and metformin on glycemia levels in non-diabetic(a) and diabetic (b) mice. ..................................................................................................................... Figure 22: Weight changes in the different animal groups during the experimental period. ..Figure 23: Hunched back animals with unhealthy clinical profile (left side), a mouse with diarrhea and rectal bleeding (right side). .................................................................................Figure 24: Disease activity index variations in the different groups. ......................................Figure 25: Kaplan-Meier survival curves of animals in the different groups. .........................Figure 26: Gross macroscopic images of the colon from non-diabetic groups (a) and diabetic groups (b). ................................................................................................................................Figure 27: Colon length variation. ...........................................................................................Figure 28: Polyp count variation in the different groups. ........................................................ Figure 29 Effect of probiotics and metformin on colon histology. .........................................Figure 30: Histology of CRC and DCRC animals ...................................................................Figure 31: Histology of CRC and DCRC animals treated with metformin alone. .................. Figure 32: Histology of CRC and DCRC animals treated with probiotics. .............................Figure 33: Histology of CRC and DCRC animals treated with the combination therapy. ......Figure 34: Histology of normal controls and Diabetic animals. ..............................................Figure 35: Histology of normal controls and diabetic animals treated with metformin alone.96

Figure 9 :

 9 Figure 1: Diagram of general organization of the alimentary canal. ....................................... Figure 2: Features of the musculature of the large intestine. ................................................... Figure 3: Histology of the colon (H&E). .................................................................................Figure 4: Schematic figure of the intestinal barrier and its components. ................................Figure 5: Functions of the large intestine ................................................................................Figure 6: Bar chart of Region-specific incidence age standardized rates by sex for cancers of the (A) colon and (B) Rectum in 2018.....................................................................................Figure 7: Estimated number of new cancer cases in 2018, worldwide, both sexes, all ages ...Figure 8: Number of new cancer cases in 2018 in Lebanon, both sexes all ages. ...................Figure 9: Proportion of colorectal cancer cases associated with sporadic and hereditary factors.. ..................................................................................................................................... Figure 10: TNM classification of colorectal cancer ................................................................Figure 11: Pathways of colorectal carcinogenesis. .................................................................. Figure 12: Schematic representation of the g-FOBT (A) and i-FOBT (B) blood detection reactions. ..................................................................................................................................Figure 13: Commonly encountered reactive oxygen species.. ................................................Figure 14: The NF-κB signaling pathway. ..............................................................................Figure 15: IL-6 signaling cascade. ...........................................................................................Figure 16: The relationship between inflammation and diabetes. ...........................................Figure 17: Blood collection by cardiac puncture (cardiocentesis).......................................... Figure 18: The principle of DHE protocol for ROS detection and quantification...................Figure 19: Griess reaction principle. ........................................................................................Figure 20: Principle of sandwich Enzyme-linked Immunosorbent assay ELISA. ..................Figure 21: Effects of probiotics and metformin on glycemia levels in non-diabetic(a) and diabetic (b) mice. ..................................................................................................................... Figure 22: Weight changes in the different animal groups during the experimental period. ..Figure 23: Hunched back animals with unhealthy clinical profile (left side), a mouse with diarrhea and rectal bleeding (right side). .................................................................................Figure 24: Disease activity index variations in the different groups. ......................................Figure 25: Kaplan-Meier survival curves of animals in the different groups. .........................Figure 26: Gross macroscopic images of the colon from non-diabetic groups (a) and diabetic groups (b). ................................................................................................................................Figure 27: Colon length variation. ...........................................................................................Figure 28: Polyp count variation in the different groups. ........................................................ Figure 29 Effect of probiotics and metformin on colon histology. .........................................Figure 30: Histology of CRC and DCRC animals ...................................................................Figure 31: Histology of CRC and DCRC animals treated with metformin alone. .................. Figure 32: Histology of CRC and DCRC animals treated with probiotics. .............................Figure 33: Histology of CRC and DCRC animals treated with the combination therapy. ......Figure 34: Histology of normal controls and Diabetic animals. ..............................................Figure 35: Histology of normal controls and diabetic animals treated with metformin alone.96

Figure 10 :

 10 Figure 1: Diagram of general organization of the alimentary canal. ....................................... Figure 2: Features of the musculature of the large intestine. ................................................... Figure 3: Histology of the colon (H&E). .................................................................................Figure 4: Schematic figure of the intestinal barrier and its components. ................................Figure 5: Functions of the large intestine ................................................................................Figure 6: Bar chart of Region-specific incidence age standardized rates by sex for cancers of the (A) colon and (B) Rectum in 2018.....................................................................................Figure 7: Estimated number of new cancer cases in 2018, worldwide, both sexes, all ages ...Figure 8: Number of new cancer cases in 2018 in Lebanon, both sexes all ages. ...................Figure 9: Proportion of colorectal cancer cases associated with sporadic and hereditary factors.. ..................................................................................................................................... Figure 10: TNM classification of colorectal cancer ................................................................Figure 11: Pathways of colorectal carcinogenesis. .................................................................. Figure 12: Schematic representation of the g-FOBT (A) and i-FOBT (B) blood detection reactions. ..................................................................................................................................Figure 13: Commonly encountered reactive oxygen species.. ................................................Figure 14: The NF-κB signaling pathway. ..............................................................................Figure 15: IL-6 signaling cascade. ...........................................................................................Figure 16: The relationship between inflammation and diabetes. ...........................................Figure 17: Blood collection by cardiac puncture (cardiocentesis).......................................... Figure 18: The principle of DHE protocol for ROS detection and quantification...................Figure 19: Griess reaction principle. ........................................................................................Figure 20: Principle of sandwich Enzyme-linked Immunosorbent assay ELISA. ..................Figure 21: Effects of probiotics and metformin on glycemia levels in non-diabetic(a) and diabetic (b) mice. ..................................................................................................................... Figure 22: Weight changes in the different animal groups during the experimental period. ..Figure 23: Hunched back animals with unhealthy clinical profile (left side), a mouse with diarrhea and rectal bleeding (right side). .................................................................................Figure 24: Disease activity index variations in the different groups. ......................................Figure 25: Kaplan-Meier survival curves of animals in the different groups. .........................Figure 26: Gross macroscopic images of the colon from non-diabetic groups (a) and diabetic groups (b). ................................................................................................................................Figure 27: Colon length variation. ...........................................................................................Figure 28: Polyp count variation in the different groups. ........................................................ Figure 29 Effect of probiotics and metformin on colon histology. .........................................Figure 30: Histology of CRC and DCRC animals ...................................................................Figure 31: Histology of CRC and DCRC animals treated with metformin alone. .................. Figure 32: Histology of CRC and DCRC animals treated with probiotics. .............................Figure 33: Histology of CRC and DCRC animals treated with the combination therapy. ......Figure 34: Histology of normal controls and Diabetic animals. ..............................................Figure 35: Histology of normal controls and diabetic animals treated with metformin alone.96

Figure 11 :

 11 Figure 1: Diagram of general organization of the alimentary canal. ....................................... Figure 2: Features of the musculature of the large intestine. ................................................... Figure 3: Histology of the colon (H&E). .................................................................................Figure 4: Schematic figure of the intestinal barrier and its components. ................................Figure 5: Functions of the large intestine ................................................................................Figure 6: Bar chart of Region-specific incidence age standardized rates by sex for cancers of the (A) colon and (B) Rectum in 2018.....................................................................................Figure 7: Estimated number of new cancer cases in 2018, worldwide, both sexes, all ages ...Figure 8: Number of new cancer cases in 2018 in Lebanon, both sexes all ages. ...................Figure 9: Proportion of colorectal cancer cases associated with sporadic and hereditary factors.. ..................................................................................................................................... Figure 10: TNM classification of colorectal cancer ................................................................Figure 11: Pathways of colorectal carcinogenesis. .................................................................. Figure 12: Schematic representation of the g-FOBT (A) and i-FOBT (B) blood detection reactions. ..................................................................................................................................Figure 13: Commonly encountered reactive oxygen species.. ................................................Figure 14: The NF-κB signaling pathway. ..............................................................................Figure 15: IL-6 signaling cascade. ...........................................................................................Figure 16: The relationship between inflammation and diabetes. ...........................................Figure 17: Blood collection by cardiac puncture (cardiocentesis).......................................... Figure 18: The principle of DHE protocol for ROS detection and quantification...................Figure 19: Griess reaction principle. ........................................................................................Figure 20: Principle of sandwich Enzyme-linked Immunosorbent assay ELISA. ..................Figure 21: Effects of probiotics and metformin on glycemia levels in non-diabetic(a) and diabetic (b) mice. ..................................................................................................................... Figure 22: Weight changes in the different animal groups during the experimental period. ..Figure 23: Hunched back animals with unhealthy clinical profile (left side), a mouse with diarrhea and rectal bleeding (right side). .................................................................................Figure 24: Disease activity index variations in the different groups. ......................................Figure 25: Kaplan-Meier survival curves of animals in the different groups. .........................Figure 26: Gross macroscopic images of the colon from non-diabetic groups (a) and diabetic groups (b). ................................................................................................................................Figure 27: Colon length variation. ...........................................................................................Figure 28: Polyp count variation in the different groups. ........................................................ Figure 29 Effect of probiotics and metformin on colon histology. .........................................Figure 30: Histology of CRC and DCRC animals ...................................................................Figure 31: Histology of CRC and DCRC animals treated with metformin alone. .................. Figure 32: Histology of CRC and DCRC animals treated with probiotics. .............................Figure 33: Histology of CRC and DCRC animals treated with the combination therapy. ......Figure 34: Histology of normal controls and Diabetic animals. ..............................................Figure 35: Histology of normal controls and diabetic animals treated with metformin alone.96

Figure 12 :

 12 Figure 1: Diagram of general organization of the alimentary canal. ....................................... Figure 2: Features of the musculature of the large intestine. ................................................... Figure 3: Histology of the colon (H&E). .................................................................................Figure 4: Schematic figure of the intestinal barrier and its components. ................................Figure 5: Functions of the large intestine ................................................................................Figure 6: Bar chart of Region-specific incidence age standardized rates by sex for cancers of the (A) colon and (B) Rectum in 2018.....................................................................................Figure 7: Estimated number of new cancer cases in 2018, worldwide, both sexes, all ages ...Figure 8: Number of new cancer cases in 2018 in Lebanon, both sexes all ages. ...................Figure 9: Proportion of colorectal cancer cases associated with sporadic and hereditary factors.. ..................................................................................................................................... Figure 10: TNM classification of colorectal cancer ................................................................Figure 11: Pathways of colorectal carcinogenesis. .................................................................. Figure 12: Schematic representation of the g-FOBT (A) and i-FOBT (B) blood detection reactions. ..................................................................................................................................Figure 13: Commonly encountered reactive oxygen species.. ................................................Figure 14: The NF-κB signaling pathway. ..............................................................................Figure 15: IL-6 signaling cascade. ...........................................................................................Figure 16: The relationship between inflammation and diabetes. ...........................................Figure 17: Blood collection by cardiac puncture (cardiocentesis).......................................... Figure 18: The principle of DHE protocol for ROS detection and quantification...................Figure 19: Griess reaction principle. ........................................................................................Figure 20: Principle of sandwich Enzyme-linked Immunosorbent assay ELISA. ..................Figure 21: Effects of probiotics and metformin on glycemia levels in non-diabetic(a) and diabetic (b) mice. ..................................................................................................................... Figure 22: Weight changes in the different animal groups during the experimental period. ..Figure 23: Hunched back animals with unhealthy clinical profile (left side), a mouse with diarrhea and rectal bleeding (right side). .................................................................................Figure 24: Disease activity index variations in the different groups. ......................................Figure 25: Kaplan-Meier survival curves of animals in the different groups. .........................Figure 26: Gross macroscopic images of the colon from non-diabetic groups (a) and diabetic groups (b). ................................................................................................................................Figure 27: Colon length variation. ...........................................................................................Figure 28: Polyp count variation in the different groups. ........................................................ Figure 29 Effect of probiotics and metformin on colon histology. .........................................Figure 30: Histology of CRC and DCRC animals ...................................................................Figure 31: Histology of CRC and DCRC animals treated with metformin alone. .................. Figure 32: Histology of CRC and DCRC animals treated with probiotics. .............................Figure 33: Histology of CRC and DCRC animals treated with the combination therapy. ......Figure 34: Histology of normal controls and Diabetic animals. ..............................................Figure 35: Histology of normal controls and diabetic animals treated with metformin alone.96

Figure 13 :

 13 Figure 1: Diagram of general organization of the alimentary canal. ....................................... Figure 2: Features of the musculature of the large intestine. ................................................... Figure 3: Histology of the colon (H&E). .................................................................................Figure 4: Schematic figure of the intestinal barrier and its components. ................................Figure 5: Functions of the large intestine ................................................................................Figure 6: Bar chart of Region-specific incidence age standardized rates by sex for cancers of the (A) colon and (B) Rectum in 2018.....................................................................................Figure 7: Estimated number of new cancer cases in 2018, worldwide, both sexes, all ages ...Figure 8: Number of new cancer cases in 2018 in Lebanon, both sexes all ages. ...................Figure 9: Proportion of colorectal cancer cases associated with sporadic and hereditary factors.. ..................................................................................................................................... Figure 10: TNM classification of colorectal cancer ................................................................Figure 11: Pathways of colorectal carcinogenesis. .................................................................. Figure 12: Schematic representation of the g-FOBT (A) and i-FOBT (B) blood detection reactions. ..................................................................................................................................Figure 13: Commonly encountered reactive oxygen species.. ................................................Figure 14: The NF-κB signaling pathway. ..............................................................................Figure 15: IL-6 signaling cascade. ...........................................................................................Figure 16: The relationship between inflammation and diabetes. ...........................................Figure 17: Blood collection by cardiac puncture (cardiocentesis).......................................... Figure 18: The principle of DHE protocol for ROS detection and quantification...................Figure 19: Griess reaction principle. ........................................................................................Figure 20: Principle of sandwich Enzyme-linked Immunosorbent assay ELISA. ..................Figure 21: Effects of probiotics and metformin on glycemia levels in non-diabetic(a) and diabetic (b) mice. ..................................................................................................................... Figure 22: Weight changes in the different animal groups during the experimental period. ..Figure 23: Hunched back animals with unhealthy clinical profile (left side), a mouse with diarrhea and rectal bleeding (right side). .................................................................................Figure 24: Disease activity index variations in the different groups. ......................................Figure 25: Kaplan-Meier survival curves of animals in the different groups. .........................Figure 26: Gross macroscopic images of the colon from non-diabetic groups (a) and diabetic groups (b). ................................................................................................................................Figure 27: Colon length variation. ...........................................................................................Figure 28: Polyp count variation in the different groups. ........................................................ Figure 29 Effect of probiotics and metformin on colon histology. .........................................Figure 30: Histology of CRC and DCRC animals ...................................................................Figure 31: Histology of CRC and DCRC animals treated with metformin alone. .................. Figure 32: Histology of CRC and DCRC animals treated with probiotics. .............................Figure 33: Histology of CRC and DCRC animals treated with the combination therapy. ......Figure 34: Histology of normal controls and Diabetic animals. ..............................................Figure 35: Histology of normal controls and diabetic animals treated with metformin alone.96

Figure 14 :

 14 Figure 1: Diagram of general organization of the alimentary canal. ....................................... Figure 2: Features of the musculature of the large intestine. ................................................... Figure 3: Histology of the colon (H&E). .................................................................................Figure 4: Schematic figure of the intestinal barrier and its components. ................................Figure 5: Functions of the large intestine ................................................................................Figure 6: Bar chart of Region-specific incidence age standardized rates by sex for cancers of the (A) colon and (B) Rectum in 2018.....................................................................................Figure 7: Estimated number of new cancer cases in 2018, worldwide, both sexes, all ages ...Figure 8: Number of new cancer cases in 2018 in Lebanon, both sexes all ages. ...................Figure 9: Proportion of colorectal cancer cases associated with sporadic and hereditary factors.. ..................................................................................................................................... Figure 10: TNM classification of colorectal cancer ................................................................Figure 11: Pathways of colorectal carcinogenesis. .................................................................. Figure 12: Schematic representation of the g-FOBT (A) and i-FOBT (B) blood detection reactions. ..................................................................................................................................Figure 13: Commonly encountered reactive oxygen species.. ................................................Figure 14: The NF-κB signaling pathway. ..............................................................................Figure 15: IL-6 signaling cascade. ...........................................................................................Figure 16: The relationship between inflammation and diabetes. ...........................................Figure 17: Blood collection by cardiac puncture (cardiocentesis).......................................... Figure 18: The principle of DHE protocol for ROS detection and quantification...................Figure 19: Griess reaction principle. ........................................................................................Figure 20: Principle of sandwich Enzyme-linked Immunosorbent assay ELISA. ..................Figure 21: Effects of probiotics and metformin on glycemia levels in non-diabetic(a) and diabetic (b) mice. ..................................................................................................................... Figure 22: Weight changes in the different animal groups during the experimental period. ..Figure 23: Hunched back animals with unhealthy clinical profile (left side), a mouse with diarrhea and rectal bleeding (right side). .................................................................................Figure 24: Disease activity index variations in the different groups. ......................................Figure 25: Kaplan-Meier survival curves of animals in the different groups. .........................Figure 26: Gross macroscopic images of the colon from non-diabetic groups (a) and diabetic groups (b). ................................................................................................................................Figure 27: Colon length variation. ...........................................................................................Figure 28: Polyp count variation in the different groups. ........................................................ Figure 29 Effect of probiotics and metformin on colon histology. .........................................Figure 30: Histology of CRC and DCRC animals ...................................................................Figure 31: Histology of CRC and DCRC animals treated with metformin alone. .................. Figure 32: Histology of CRC and DCRC animals treated with probiotics. .............................Figure 33: Histology of CRC and DCRC animals treated with the combination therapy. ......Figure 34: Histology of normal controls and Diabetic animals. ..............................................Figure 35: Histology of normal controls and diabetic animals treated with metformin alone.96

Figure 15 :

 15 Figure 1: Diagram of general organization of the alimentary canal. ....................................... Figure 2: Features of the musculature of the large intestine. ................................................... Figure 3: Histology of the colon (H&E). .................................................................................Figure 4: Schematic figure of the intestinal barrier and its components. ................................Figure 5: Functions of the large intestine ................................................................................Figure 6: Bar chart of Region-specific incidence age standardized rates by sex for cancers of the (A) colon and (B) Rectum in 2018.....................................................................................Figure 7: Estimated number of new cancer cases in 2018, worldwide, both sexes, all ages ...Figure 8: Number of new cancer cases in 2018 in Lebanon, both sexes all ages. ...................Figure 9: Proportion of colorectal cancer cases associated with sporadic and hereditary factors.. ..................................................................................................................................... Figure 10: TNM classification of colorectal cancer ................................................................Figure 11: Pathways of colorectal carcinogenesis. .................................................................. Figure 12: Schematic representation of the g-FOBT (A) and i-FOBT (B) blood detection reactions. ..................................................................................................................................Figure 13: Commonly encountered reactive oxygen species.. ................................................Figure 14: The NF-κB signaling pathway. ..............................................................................Figure 15: IL-6 signaling cascade. ...........................................................................................Figure 16: The relationship between inflammation and diabetes. ...........................................Figure 17: Blood collection by cardiac puncture (cardiocentesis).......................................... Figure 18: The principle of DHE protocol for ROS detection and quantification...................Figure 19: Griess reaction principle. ........................................................................................Figure 20: Principle of sandwich Enzyme-linked Immunosorbent assay ELISA. ..................Figure 21: Effects of probiotics and metformin on glycemia levels in non-diabetic(a) and diabetic (b) mice. ..................................................................................................................... Figure 22: Weight changes in the different animal groups during the experimental period. ..Figure 23: Hunched back animals with unhealthy clinical profile (left side), a mouse with diarrhea and rectal bleeding (right side). .................................................................................Figure 24: Disease activity index variations in the different groups. ......................................Figure 25: Kaplan-Meier survival curves of animals in the different groups. .........................Figure 26: Gross macroscopic images of the colon from non-diabetic groups (a) and diabetic groups (b). ................................................................................................................................Figure 27: Colon length variation. ...........................................................................................Figure 28: Polyp count variation in the different groups. ........................................................ Figure 29 Effect of probiotics and metformin on colon histology. .........................................Figure 30: Histology of CRC and DCRC animals ...................................................................Figure 31: Histology of CRC and DCRC animals treated with metformin alone. .................. Figure 32: Histology of CRC and DCRC animals treated with probiotics. .............................Figure 33: Histology of CRC and DCRC animals treated with the combination therapy. ......Figure 34: Histology of normal controls and Diabetic animals. ..............................................Figure 35: Histology of normal controls and diabetic animals treated with metformin alone.96

Figure 16 :

 16 Figure 1: Diagram of general organization of the alimentary canal. ....................................... Figure 2: Features of the musculature of the large intestine. ................................................... Figure 3: Histology of the colon (H&E). .................................................................................Figure 4: Schematic figure of the intestinal barrier and its components. ................................Figure 5: Functions of the large intestine ................................................................................Figure 6: Bar chart of Region-specific incidence age standardized rates by sex for cancers of the (A) colon and (B) Rectum in 2018.....................................................................................Figure 7: Estimated number of new cancer cases in 2018, worldwide, both sexes, all ages ...Figure 8: Number of new cancer cases in 2018 in Lebanon, both sexes all ages. ...................Figure 9: Proportion of colorectal cancer cases associated with sporadic and hereditary factors.. ..................................................................................................................................... Figure 10: TNM classification of colorectal cancer ................................................................Figure 11: Pathways of colorectal carcinogenesis. .................................................................. Figure 12: Schematic representation of the g-FOBT (A) and i-FOBT (B) blood detection reactions. ..................................................................................................................................Figure 13: Commonly encountered reactive oxygen species.. ................................................Figure 14: The NF-κB signaling pathway. ..............................................................................Figure 15: IL-6 signaling cascade. ...........................................................................................Figure 16: The relationship between inflammation and diabetes. ...........................................Figure 17: Blood collection by cardiac puncture (cardiocentesis).......................................... Figure 18: The principle of DHE protocol for ROS detection and quantification...................Figure 19: Griess reaction principle. ........................................................................................Figure 20: Principle of sandwich Enzyme-linked Immunosorbent assay ELISA. ..................Figure 21: Effects of probiotics and metformin on glycemia levels in non-diabetic(a) and diabetic (b) mice. ..................................................................................................................... Figure 22: Weight changes in the different animal groups during the experimental period. ..Figure 23: Hunched back animals with unhealthy clinical profile (left side), a mouse with diarrhea and rectal bleeding (right side). .................................................................................Figure 24: Disease activity index variations in the different groups. ......................................Figure 25: Kaplan-Meier survival curves of animals in the different groups. .........................Figure 26: Gross macroscopic images of the colon from non-diabetic groups (a) and diabetic groups (b). ................................................................................................................................Figure 27: Colon length variation. ...........................................................................................Figure 28: Polyp count variation in the different groups. ........................................................ Figure 29 Effect of probiotics and metformin on colon histology. .........................................Figure 30: Histology of CRC and DCRC animals ...................................................................Figure 31: Histology of CRC and DCRC animals treated with metformin alone. .................. Figure 32: Histology of CRC and DCRC animals treated with probiotics. .............................Figure 33: Histology of CRC and DCRC animals treated with the combination therapy. ......Figure 34: Histology of normal controls and Diabetic animals. ..............................................Figure 35: Histology of normal controls and diabetic animals treated with metformin alone.96

Figure 17 :

 17 Figure 1: Diagram of general organization of the alimentary canal. ....................................... Figure 2: Features of the musculature of the large intestine. ................................................... Figure 3: Histology of the colon (H&E). .................................................................................Figure 4: Schematic figure of the intestinal barrier and its components. ................................Figure 5: Functions of the large intestine ................................................................................Figure 6: Bar chart of Region-specific incidence age standardized rates by sex for cancers of the (A) colon and (B) Rectum in 2018.....................................................................................Figure 7: Estimated number of new cancer cases in 2018, worldwide, both sexes, all ages ...Figure 8: Number of new cancer cases in 2018 in Lebanon, both sexes all ages. ...................Figure 9: Proportion of colorectal cancer cases associated with sporadic and hereditary factors.. ..................................................................................................................................... Figure 10: TNM classification of colorectal cancer ................................................................Figure 11: Pathways of colorectal carcinogenesis. .................................................................. Figure 12: Schematic representation of the g-FOBT (A) and i-FOBT (B) blood detection reactions. ..................................................................................................................................Figure 13: Commonly encountered reactive oxygen species.. ................................................Figure 14: The NF-κB signaling pathway. ..............................................................................Figure 15: IL-6 signaling cascade. ...........................................................................................Figure 16: The relationship between inflammation and diabetes. ...........................................Figure 17: Blood collection by cardiac puncture (cardiocentesis).......................................... Figure 18: The principle of DHE protocol for ROS detection and quantification...................Figure 19: Griess reaction principle. ........................................................................................Figure 20: Principle of sandwich Enzyme-linked Immunosorbent assay ELISA. ..................Figure 21: Effects of probiotics and metformin on glycemia levels in non-diabetic(a) and diabetic (b) mice. ..................................................................................................................... Figure 22: Weight changes in the different animal groups during the experimental period. ..Figure 23: Hunched back animals with unhealthy clinical profile (left side), a mouse with diarrhea and rectal bleeding (right side). .................................................................................Figure 24: Disease activity index variations in the different groups. ......................................Figure 25: Kaplan-Meier survival curves of animals in the different groups. .........................Figure 26: Gross macroscopic images of the colon from non-diabetic groups (a) and diabetic groups (b). ................................................................................................................................Figure 27: Colon length variation. ...........................................................................................Figure 28: Polyp count variation in the different groups. ........................................................ Figure 29 Effect of probiotics and metformin on colon histology. .........................................Figure 30: Histology of CRC and DCRC animals ...................................................................Figure 31: Histology of CRC and DCRC animals treated with metformin alone. .................. Figure 32: Histology of CRC and DCRC animals treated with probiotics. .............................Figure 33: Histology of CRC and DCRC animals treated with the combination therapy. ......Figure 34: Histology of normal controls and Diabetic animals. ..............................................Figure 35: Histology of normal controls and diabetic animals treated with metformin alone.96

Figure 19 :

 19 Figure 1: Diagram of general organization of the alimentary canal. ....................................... Figure 2: Features of the musculature of the large intestine. ................................................... Figure 3: Histology of the colon (H&E). .................................................................................Figure 4: Schematic figure of the intestinal barrier and its components. ................................Figure 5: Functions of the large intestine ................................................................................Figure 6: Bar chart of Region-specific incidence age standardized rates by sex for cancers of the (A) colon and (B) Rectum in 2018.....................................................................................Figure 7: Estimated number of new cancer cases in 2018, worldwide, both sexes, all ages ...Figure 8: Number of new cancer cases in 2018 in Lebanon, both sexes all ages. ...................Figure 9: Proportion of colorectal cancer cases associated with sporadic and hereditary factors.. ..................................................................................................................................... Figure 10: TNM classification of colorectal cancer ................................................................Figure 11: Pathways of colorectal carcinogenesis. .................................................................. Figure 12: Schematic representation of the g-FOBT (A) and i-FOBT (B) blood detection reactions. ..................................................................................................................................Figure 13: Commonly encountered reactive oxygen species.. ................................................Figure 14: The NF-κB signaling pathway. ..............................................................................Figure 15: IL-6 signaling cascade. ...........................................................................................Figure 16: The relationship between inflammation and diabetes. ...........................................Figure 17: Blood collection by cardiac puncture (cardiocentesis).......................................... Figure 18: The principle of DHE protocol for ROS detection and quantification...................Figure 19: Griess reaction principle. ........................................................................................Figure 20: Principle of sandwich Enzyme-linked Immunosorbent assay ELISA. ..................Figure 21: Effects of probiotics and metformin on glycemia levels in non-diabetic(a) and diabetic (b) mice. ..................................................................................................................... Figure 22: Weight changes in the different animal groups during the experimental period. ..Figure 23: Hunched back animals with unhealthy clinical profile (left side), a mouse with diarrhea and rectal bleeding (right side). .................................................................................Figure 24: Disease activity index variations in the different groups. ......................................Figure 25: Kaplan-Meier survival curves of animals in the different groups. .........................Figure 26: Gross macroscopic images of the colon from non-diabetic groups (a) and diabetic groups (b). ................................................................................................................................Figure 27: Colon length variation. ...........................................................................................Figure 28: Polyp count variation in the different groups. ........................................................ Figure 29 Effect of probiotics and metformin on colon histology. .........................................Figure 30: Histology of CRC and DCRC animals ...................................................................Figure 31: Histology of CRC and DCRC animals treated with metformin alone. .................. Figure 32: Histology of CRC and DCRC animals treated with probiotics. .............................Figure 33: Histology of CRC and DCRC animals treated with the combination therapy. ......Figure 34: Histology of normal controls and Diabetic animals. ..............................................Figure 35: Histology of normal controls and diabetic animals treated with metformin alone.96

Figure 20 :

 20 Figure 1: Diagram of general organization of the alimentary canal. ....................................... Figure 2: Features of the musculature of the large intestine. ................................................... Figure 3: Histology of the colon (H&E). .................................................................................Figure 4: Schematic figure of the intestinal barrier and its components. ................................Figure 5: Functions of the large intestine ................................................................................Figure 6: Bar chart of Region-specific incidence age standardized rates by sex for cancers of the (A) colon and (B) Rectum in 2018.....................................................................................Figure 7: Estimated number of new cancer cases in 2018, worldwide, both sexes, all ages ...Figure 8: Number of new cancer cases in 2018 in Lebanon, both sexes all ages. ...................Figure 9: Proportion of colorectal cancer cases associated with sporadic and hereditary factors.. ..................................................................................................................................... Figure 10: TNM classification of colorectal cancer ................................................................Figure 11: Pathways of colorectal carcinogenesis. .................................................................. Figure 12: Schematic representation of the g-FOBT (A) and i-FOBT (B) blood detection reactions. ..................................................................................................................................Figure 13: Commonly encountered reactive oxygen species.. ................................................Figure 14: The NF-κB signaling pathway. ..............................................................................Figure 15: IL-6 signaling cascade. ...........................................................................................Figure 16: The relationship between inflammation and diabetes. ...........................................Figure 17: Blood collection by cardiac puncture (cardiocentesis).......................................... Figure 18: The principle of DHE protocol for ROS detection and quantification...................Figure 19: Griess reaction principle. ........................................................................................Figure 20: Principle of sandwich Enzyme-linked Immunosorbent assay ELISA. ..................Figure 21: Effects of probiotics and metformin on glycemia levels in non-diabetic(a) and diabetic (b) mice. ..................................................................................................................... Figure 22: Weight changes in the different animal groups during the experimental period. ..Figure 23: Hunched back animals with unhealthy clinical profile (left side), a mouse with diarrhea and rectal bleeding (right side). .................................................................................Figure 24: Disease activity index variations in the different groups. ......................................Figure 25: Kaplan-Meier survival curves of animals in the different groups. .........................Figure 26: Gross macroscopic images of the colon from non-diabetic groups (a) and diabetic groups (b). ................................................................................................................................Figure 27: Colon length variation. ...........................................................................................Figure 28: Polyp count variation in the different groups. ........................................................ Figure 29 Effect of probiotics and metformin on colon histology. .........................................Figure 30: Histology of CRC and DCRC animals ...................................................................Figure 31: Histology of CRC and DCRC animals treated with metformin alone. .................. Figure 32: Histology of CRC and DCRC animals treated with probiotics. .............................Figure 33: Histology of CRC and DCRC animals treated with the combination therapy. ......Figure 34: Histology of normal controls and Diabetic animals. ..............................................Figure 35: Histology of normal controls and diabetic animals treated with metformin alone.96

Figure 21 :

 21 Figure 1: Diagram of general organization of the alimentary canal. ....................................... Figure 2: Features of the musculature of the large intestine. ................................................... Figure 3: Histology of the colon (H&E). .................................................................................Figure 4: Schematic figure of the intestinal barrier and its components. ................................Figure 5: Functions of the large intestine ................................................................................Figure 6: Bar chart of Region-specific incidence age standardized rates by sex for cancers of the (A) colon and (B) Rectum in 2018.....................................................................................Figure 7: Estimated number of new cancer cases in 2018, worldwide, both sexes, all ages ...Figure 8: Number of new cancer cases in 2018 in Lebanon, both sexes all ages. ...................Figure 9: Proportion of colorectal cancer cases associated with sporadic and hereditary factors.. ..................................................................................................................................... Figure 10: TNM classification of colorectal cancer ................................................................Figure 11: Pathways of colorectal carcinogenesis. .................................................................. Figure 12: Schematic representation of the g-FOBT (A) and i-FOBT (B) blood detection reactions. ..................................................................................................................................Figure 13: Commonly encountered reactive oxygen species.. ................................................Figure 14: The NF-κB signaling pathway. ..............................................................................Figure 15: IL-6 signaling cascade. ...........................................................................................Figure 16: The relationship between inflammation and diabetes. ...........................................Figure 17: Blood collection by cardiac puncture (cardiocentesis).......................................... Figure 18: The principle of DHE protocol for ROS detection and quantification...................Figure 19: Griess reaction principle. ........................................................................................Figure 20: Principle of sandwich Enzyme-linked Immunosorbent assay ELISA. ..................Figure 21: Effects of probiotics and metformin on glycemia levels in non-diabetic(a) and diabetic (b) mice. ..................................................................................................................... Figure 22: Weight changes in the different animal groups during the experimental period. ..Figure 23: Hunched back animals with unhealthy clinical profile (left side), a mouse with diarrhea and rectal bleeding (right side). .................................................................................Figure 24: Disease activity index variations in the different groups. ......................................Figure 25: Kaplan-Meier survival curves of animals in the different groups. .........................Figure 26: Gross macroscopic images of the colon from non-diabetic groups (a) and diabetic groups (b). ................................................................................................................................Figure 27: Colon length variation. ...........................................................................................Figure 28: Polyp count variation in the different groups. ........................................................ Figure 29 Effect of probiotics and metformin on colon histology. .........................................Figure 30: Histology of CRC and DCRC animals ...................................................................Figure 31: Histology of CRC and DCRC animals treated with metformin alone. .................. Figure 32: Histology of CRC and DCRC animals treated with probiotics. .............................Figure 33: Histology of CRC and DCRC animals treated with the combination therapy. ......Figure 34: Histology of normal controls and Diabetic animals. ..............................................Figure 35: Histology of normal controls and diabetic animals treated with metformin alone.96

Figure 22 :

 22 Figure 1: Diagram of general organization of the alimentary canal. ....................................... Figure 2: Features of the musculature of the large intestine. ................................................... Figure 3: Histology of the colon (H&E). .................................................................................Figure 4: Schematic figure of the intestinal barrier and its components. ................................Figure 5: Functions of the large intestine ................................................................................Figure 6: Bar chart of Region-specific incidence age standardized rates by sex for cancers of the (A) colon and (B) Rectum in 2018.....................................................................................Figure 7: Estimated number of new cancer cases in 2018, worldwide, both sexes, all ages ...Figure 8: Number of new cancer cases in 2018 in Lebanon, both sexes all ages. ...................Figure 9: Proportion of colorectal cancer cases associated with sporadic and hereditary factors.. ..................................................................................................................................... Figure 10: TNM classification of colorectal cancer ................................................................Figure 11: Pathways of colorectal carcinogenesis. .................................................................. Figure 12: Schematic representation of the g-FOBT (A) and i-FOBT (B) blood detection reactions. ..................................................................................................................................Figure 13: Commonly encountered reactive oxygen species.. ................................................Figure 14: The NF-κB signaling pathway. ..............................................................................Figure 15: IL-6 signaling cascade. ...........................................................................................Figure 16: The relationship between inflammation and diabetes. ...........................................Figure 17: Blood collection by cardiac puncture (cardiocentesis).......................................... Figure 18: The principle of DHE protocol for ROS detection and quantification...................Figure 19: Griess reaction principle. ........................................................................................Figure 20: Principle of sandwich Enzyme-linked Immunosorbent assay ELISA. ..................Figure 21: Effects of probiotics and metformin on glycemia levels in non-diabetic(a) and diabetic (b) mice. ..................................................................................................................... Figure 22: Weight changes in the different animal groups during the experimental period. ..Figure 23: Hunched back animals with unhealthy clinical profile (left side), a mouse with diarrhea and rectal bleeding (right side). .................................................................................Figure 24: Disease activity index variations in the different groups. ......................................Figure 25: Kaplan-Meier survival curves of animals in the different groups. .........................Figure 26: Gross macroscopic images of the colon from non-diabetic groups (a) and diabetic groups (b). ................................................................................................................................Figure 27: Colon length variation. ...........................................................................................Figure 28: Polyp count variation in the different groups. ........................................................ Figure 29 Effect of probiotics and metformin on colon histology. .........................................Figure 30: Histology of CRC and DCRC animals ...................................................................Figure 31: Histology of CRC and DCRC animals treated with metformin alone. .................. Figure 32: Histology of CRC and DCRC animals treated with probiotics. .............................Figure 33: Histology of CRC and DCRC animals treated with the combination therapy. ......Figure 34: Histology of normal controls and Diabetic animals. ..............................................Figure 35: Histology of normal controls and diabetic animals treated with metformin alone.96

Figure 23 :

 23 Figure 1: Diagram of general organization of the alimentary canal. ....................................... Figure 2: Features of the musculature of the large intestine. ................................................... Figure 3: Histology of the colon (H&E). .................................................................................Figure 4: Schematic figure of the intestinal barrier and its components. ................................Figure 5: Functions of the large intestine ................................................................................Figure 6: Bar chart of Region-specific incidence age standardized rates by sex for cancers of the (A) colon and (B) Rectum in 2018.....................................................................................Figure 7: Estimated number of new cancer cases in 2018, worldwide, both sexes, all ages ...Figure 8: Number of new cancer cases in 2018 in Lebanon, both sexes all ages. ...................Figure 9: Proportion of colorectal cancer cases associated with sporadic and hereditary factors.. ..................................................................................................................................... Figure 10: TNM classification of colorectal cancer ................................................................Figure 11: Pathways of colorectal carcinogenesis. .................................................................. Figure 12: Schematic representation of the g-FOBT (A) and i-FOBT (B) blood detection reactions. ..................................................................................................................................Figure 13: Commonly encountered reactive oxygen species.. ................................................Figure 14: The NF-κB signaling pathway. ..............................................................................Figure 15: IL-6 signaling cascade. ...........................................................................................Figure 16: The relationship between inflammation and diabetes. ...........................................Figure 17: Blood collection by cardiac puncture (cardiocentesis).......................................... Figure 18: The principle of DHE protocol for ROS detection and quantification...................Figure 19: Griess reaction principle. ........................................................................................Figure 20: Principle of sandwich Enzyme-linked Immunosorbent assay ELISA. ..................Figure 21: Effects of probiotics and metformin on glycemia levels in non-diabetic(a) and diabetic (b) mice. ..................................................................................................................... Figure 22: Weight changes in the different animal groups during the experimental period. ..Figure 23: Hunched back animals with unhealthy clinical profile (left side), a mouse with diarrhea and rectal bleeding (right side). .................................................................................Figure 24: Disease activity index variations in the different groups. ......................................Figure 25: Kaplan-Meier survival curves of animals in the different groups. .........................Figure 26: Gross macroscopic images of the colon from non-diabetic groups (a) and diabetic groups (b). ................................................................................................................................Figure 27: Colon length variation. ...........................................................................................Figure 28: Polyp count variation in the different groups. ........................................................ Figure 29 Effect of probiotics and metformin on colon histology. .........................................Figure 30: Histology of CRC and DCRC animals ...................................................................Figure 31: Histology of CRC and DCRC animals treated with metformin alone. .................. Figure 32: Histology of CRC and DCRC animals treated with probiotics. .............................Figure 33: Histology of CRC and DCRC animals treated with the combination therapy. ......Figure 34: Histology of normal controls and Diabetic animals. ..............................................Figure 35: Histology of normal controls and diabetic animals treated with metformin alone.96

Figure 24 :

 24 Figure 1: Diagram of general organization of the alimentary canal. ....................................... Figure 2: Features of the musculature of the large intestine. ................................................... Figure 3: Histology of the colon (H&E). .................................................................................Figure 4: Schematic figure of the intestinal barrier and its components. ................................Figure 5: Functions of the large intestine ................................................................................Figure 6: Bar chart of Region-specific incidence age standardized rates by sex for cancers of the (A) colon and (B) Rectum in 2018.....................................................................................Figure 7: Estimated number of new cancer cases in 2018, worldwide, both sexes, all ages ...Figure 8: Number of new cancer cases in 2018 in Lebanon, both sexes all ages. ...................Figure 9: Proportion of colorectal cancer cases associated with sporadic and hereditary factors.. ..................................................................................................................................... Figure 10: TNM classification of colorectal cancer ................................................................Figure 11: Pathways of colorectal carcinogenesis. .................................................................. Figure 12: Schematic representation of the g-FOBT (A) and i-FOBT (B) blood detection reactions. ..................................................................................................................................Figure 13: Commonly encountered reactive oxygen species.. ................................................Figure 14: The NF-κB signaling pathway. ..............................................................................Figure 15: IL-6 signaling cascade. ...........................................................................................Figure 16: The relationship between inflammation and diabetes. ...........................................Figure 17: Blood collection by cardiac puncture (cardiocentesis).......................................... Figure 18: The principle of DHE protocol for ROS detection and quantification...................Figure 19: Griess reaction principle. ........................................................................................Figure 20: Principle of sandwich Enzyme-linked Immunosorbent assay ELISA. ..................Figure 21: Effects of probiotics and metformin on glycemia levels in non-diabetic(a) and diabetic (b) mice. ..................................................................................................................... Figure 22: Weight changes in the different animal groups during the experimental period. ..Figure 23: Hunched back animals with unhealthy clinical profile (left side), a mouse with diarrhea and rectal bleeding (right side). .................................................................................Figure 24: Disease activity index variations in the different groups. ......................................Figure 25: Kaplan-Meier survival curves of animals in the different groups. .........................Figure 26: Gross macroscopic images of the colon from non-diabetic groups (a) and diabetic groups (b). ................................................................................................................................Figure 27: Colon length variation. ...........................................................................................Figure 28: Polyp count variation in the different groups. ........................................................ Figure 29 Effect of probiotics and metformin on colon histology. .........................................Figure 30: Histology of CRC and DCRC animals ...................................................................Figure 31: Histology of CRC and DCRC animals treated with metformin alone. .................. Figure 32: Histology of CRC and DCRC animals treated with probiotics. .............................Figure 33: Histology of CRC and DCRC animals treated with the combination therapy. ......Figure 34: Histology of normal controls and Diabetic animals. ..............................................Figure 35: Histology of normal controls and diabetic animals treated with metformin alone.96

Figure 25 :

 25 Figure 1: Diagram of general organization of the alimentary canal. ....................................... Figure 2: Features of the musculature of the large intestine. ................................................... Figure 3: Histology of the colon (H&E). .................................................................................Figure 4: Schematic figure of the intestinal barrier and its components. ................................Figure 5: Functions of the large intestine ................................................................................Figure 6: Bar chart of Region-specific incidence age standardized rates by sex for cancers of the (A) colon and (B) Rectum in 2018.....................................................................................Figure 7: Estimated number of new cancer cases in 2018, worldwide, both sexes, all ages ...Figure 8: Number of new cancer cases in 2018 in Lebanon, both sexes all ages. ...................Figure 9: Proportion of colorectal cancer cases associated with sporadic and hereditary factors.. ..................................................................................................................................... Figure 10: TNM classification of colorectal cancer ................................................................Figure 11: Pathways of colorectal carcinogenesis. .................................................................. Figure 12: Schematic representation of the g-FOBT (A) and i-FOBT (B) blood detection reactions. ..................................................................................................................................Figure 13: Commonly encountered reactive oxygen species.. ................................................Figure 14: The NF-κB signaling pathway. ..............................................................................Figure 15: IL-6 signaling cascade. ...........................................................................................Figure 16: The relationship between inflammation and diabetes. ...........................................Figure 17: Blood collection by cardiac puncture (cardiocentesis).......................................... Figure 18: The principle of DHE protocol for ROS detection and quantification...................Figure 19: Griess reaction principle. ........................................................................................Figure 20: Principle of sandwich Enzyme-linked Immunosorbent assay ELISA. ..................Figure 21: Effects of probiotics and metformin on glycemia levels in non-diabetic(a) and diabetic (b) mice. ..................................................................................................................... Figure 22: Weight changes in the different animal groups during the experimental period. ..Figure 23: Hunched back animals with unhealthy clinical profile (left side), a mouse with diarrhea and rectal bleeding (right side). .................................................................................Figure 24: Disease activity index variations in the different groups. ......................................Figure 25: Kaplan-Meier survival curves of animals in the different groups. .........................Figure 26: Gross macroscopic images of the colon from non-diabetic groups (a) and diabetic groups (b). ................................................................................................................................Figure 27: Colon length variation. ...........................................................................................Figure 28: Polyp count variation in the different groups. ........................................................ Figure 29 Effect of probiotics and metformin on colon histology. .........................................Figure 30: Histology of CRC and DCRC animals ...................................................................Figure 31: Histology of CRC and DCRC animals treated with metformin alone. .................. Figure 32: Histology of CRC and DCRC animals treated with probiotics. .............................Figure 33: Histology of CRC and DCRC animals treated with the combination therapy. ......Figure 34: Histology of normal controls and Diabetic animals. ..............................................Figure 35: Histology of normal controls and diabetic animals treated with metformin alone.96

Figure 26 :

 26 Figure 1: Diagram of general organization of the alimentary canal. ....................................... Figure 2: Features of the musculature of the large intestine. ................................................... Figure 3: Histology of the colon (H&E). .................................................................................Figure 4: Schematic figure of the intestinal barrier and its components. ................................Figure 5: Functions of the large intestine ................................................................................Figure 6: Bar chart of Region-specific incidence age standardized rates by sex for cancers of the (A) colon and (B) Rectum in 2018.....................................................................................Figure 7: Estimated number of new cancer cases in 2018, worldwide, both sexes, all ages ...Figure 8: Number of new cancer cases in 2018 in Lebanon, both sexes all ages. ...................Figure 9: Proportion of colorectal cancer cases associated with sporadic and hereditary factors.. ..................................................................................................................................... Figure 10: TNM classification of colorectal cancer ................................................................Figure 11: Pathways of colorectal carcinogenesis. .................................................................. Figure 12: Schematic representation of the g-FOBT (A) and i-FOBT (B) blood detection reactions. ..................................................................................................................................Figure 13: Commonly encountered reactive oxygen species.. ................................................Figure 14: The NF-κB signaling pathway. ..............................................................................Figure 15: IL-6 signaling cascade. ...........................................................................................Figure 16: The relationship between inflammation and diabetes. ...........................................Figure 17: Blood collection by cardiac puncture (cardiocentesis).......................................... Figure 18: The principle of DHE protocol for ROS detection and quantification...................Figure 19: Griess reaction principle. ........................................................................................Figure 20: Principle of sandwich Enzyme-linked Immunosorbent assay ELISA. ..................Figure 21: Effects of probiotics and metformin on glycemia levels in non-diabetic(a) and diabetic (b) mice. ..................................................................................................................... Figure 22: Weight changes in the different animal groups during the experimental period. ..Figure 23: Hunched back animals with unhealthy clinical profile (left side), a mouse with diarrhea and rectal bleeding (right side). .................................................................................Figure 24: Disease activity index variations in the different groups. ......................................Figure 25: Kaplan-Meier survival curves of animals in the different groups. .........................Figure 26: Gross macroscopic images of the colon from non-diabetic groups (a) and diabetic groups (b). ................................................................................................................................Figure 27: Colon length variation. ...........................................................................................Figure 28: Polyp count variation in the different groups. ........................................................ Figure 29 Effect of probiotics and metformin on colon histology. .........................................Figure 30: Histology of CRC and DCRC animals ...................................................................Figure 31: Histology of CRC and DCRC animals treated with metformin alone. .................. Figure 32: Histology of CRC and DCRC animals treated with probiotics. .............................Figure 33: Histology of CRC and DCRC animals treated with the combination therapy. ......Figure 34: Histology of normal controls and Diabetic animals. ..............................................Figure 35: Histology of normal controls and diabetic animals treated with metformin alone.96

Figure 27 :

 27 Figure 1: Diagram of general organization of the alimentary canal. ....................................... Figure 2: Features of the musculature of the large intestine. ................................................... Figure 3: Histology of the colon (H&E). .................................................................................Figure 4: Schematic figure of the intestinal barrier and its components. ................................Figure 5: Functions of the large intestine ................................................................................Figure 6: Bar chart of Region-specific incidence age standardized rates by sex for cancers of the (A) colon and (B) Rectum in 2018.....................................................................................Figure 7: Estimated number of new cancer cases in 2018, worldwide, both sexes, all ages ...Figure 8: Number of new cancer cases in 2018 in Lebanon, both sexes all ages. ...................Figure 9: Proportion of colorectal cancer cases associated with sporadic and hereditary factors.. ..................................................................................................................................... Figure 10: TNM classification of colorectal cancer ................................................................Figure 11: Pathways of colorectal carcinogenesis. .................................................................. Figure 12: Schematic representation of the g-FOBT (A) and i-FOBT (B) blood detection reactions. ..................................................................................................................................Figure 13: Commonly encountered reactive oxygen species.. ................................................Figure 14: The NF-κB signaling pathway. ..............................................................................Figure 15: IL-6 signaling cascade. ...........................................................................................Figure 16: The relationship between inflammation and diabetes. ...........................................Figure 17: Blood collection by cardiac puncture (cardiocentesis).......................................... Figure 18: The principle of DHE protocol for ROS detection and quantification...................Figure 19: Griess reaction principle. ........................................................................................Figure 20: Principle of sandwich Enzyme-linked Immunosorbent assay ELISA. ..................Figure 21: Effects of probiotics and metformin on glycemia levels in non-diabetic(a) and diabetic (b) mice. ..................................................................................................................... Figure 22: Weight changes in the different animal groups during the experimental period. ..Figure 23: Hunched back animals with unhealthy clinical profile (left side), a mouse with diarrhea and rectal bleeding (right side). .................................................................................Figure 24: Disease activity index variations in the different groups. ......................................Figure 25: Kaplan-Meier survival curves of animals in the different groups. .........................Figure 26: Gross macroscopic images of the colon from non-diabetic groups (a) and diabetic groups (b). ................................................................................................................................Figure 27: Colon length variation. ...........................................................................................Figure 28: Polyp count variation in the different groups. ........................................................ Figure 29 Effect of probiotics and metformin on colon histology. .........................................Figure 30: Histology of CRC and DCRC animals ...................................................................Figure 31: Histology of CRC and DCRC animals treated with metformin alone. .................. Figure 32: Histology of CRC and DCRC animals treated with probiotics. .............................Figure 33: Histology of CRC and DCRC animals treated with the combination therapy. ......Figure 34: Histology of normal controls and Diabetic animals. ..............................................Figure 35: Histology of normal controls and diabetic animals treated with metformin alone.96

Figure 28 :

 28 Figure 1: Diagram of general organization of the alimentary canal. ....................................... Figure 2: Features of the musculature of the large intestine. ................................................... Figure 3: Histology of the colon (H&E). .................................................................................Figure 4: Schematic figure of the intestinal barrier and its components. ................................Figure 5: Functions of the large intestine ................................................................................Figure 6: Bar chart of Region-specific incidence age standardized rates by sex for cancers of the (A) colon and (B) Rectum in 2018.....................................................................................Figure 7: Estimated number of new cancer cases in 2018, worldwide, both sexes, all ages ...Figure 8: Number of new cancer cases in 2018 in Lebanon, both sexes all ages. ...................Figure 9: Proportion of colorectal cancer cases associated with sporadic and hereditary factors.. ..................................................................................................................................... Figure 10: TNM classification of colorectal cancer ................................................................Figure 11: Pathways of colorectal carcinogenesis. .................................................................. Figure 12: Schematic representation of the g-FOBT (A) and i-FOBT (B) blood detection reactions. ..................................................................................................................................Figure 13: Commonly encountered reactive oxygen species.. ................................................Figure 14: The NF-κB signaling pathway. ..............................................................................Figure 15: IL-6 signaling cascade. ...........................................................................................Figure 16: The relationship between inflammation and diabetes. ...........................................Figure 17: Blood collection by cardiac puncture (cardiocentesis).......................................... Figure 18: The principle of DHE protocol for ROS detection and quantification...................Figure 19: Griess reaction principle. ........................................................................................Figure 20: Principle of sandwich Enzyme-linked Immunosorbent assay ELISA. ..................Figure 21: Effects of probiotics and metformin on glycemia levels in non-diabetic(a) and diabetic (b) mice. ..................................................................................................................... Figure 22: Weight changes in the different animal groups during the experimental period. ..Figure 23: Hunched back animals with unhealthy clinical profile (left side), a mouse with diarrhea and rectal bleeding (right side). .................................................................................Figure 24: Disease activity index variations in the different groups. ......................................Figure 25: Kaplan-Meier survival curves of animals in the different groups. .........................Figure 26: Gross macroscopic images of the colon from non-diabetic groups (a) and diabetic groups (b). ................................................................................................................................Figure 27: Colon length variation. ...........................................................................................Figure 28: Polyp count variation in the different groups. ........................................................ Figure 29 Effect of probiotics and metformin on colon histology. .........................................Figure 30: Histology of CRC and DCRC animals ...................................................................Figure 31: Histology of CRC and DCRC animals treated with metformin alone. .................. Figure 32: Histology of CRC and DCRC animals treated with probiotics. .............................Figure 33: Histology of CRC and DCRC animals treated with the combination therapy. ......Figure 34: Histology of normal controls and Diabetic animals. ..............................................Figure 35: Histology of normal controls and diabetic animals treated with metformin alone.96

Figure 30 :

 30 Figure 1: Diagram of general organization of the alimentary canal. ....................................... Figure 2: Features of the musculature of the large intestine. ................................................... Figure 3: Histology of the colon (H&E). .................................................................................Figure 4: Schematic figure of the intestinal barrier and its components. ................................Figure 5: Functions of the large intestine ................................................................................Figure 6: Bar chart of Region-specific incidence age standardized rates by sex for cancers of the (A) colon and (B) Rectum in 2018.....................................................................................Figure 7: Estimated number of new cancer cases in 2018, worldwide, both sexes, all ages ...Figure 8: Number of new cancer cases in 2018 in Lebanon, both sexes all ages. ...................Figure 9: Proportion of colorectal cancer cases associated with sporadic and hereditary factors.. ..................................................................................................................................... Figure 10: TNM classification of colorectal cancer ................................................................Figure 11: Pathways of colorectal carcinogenesis. .................................................................. Figure 12: Schematic representation of the g-FOBT (A) and i-FOBT (B) blood detection reactions. ..................................................................................................................................Figure 13: Commonly encountered reactive oxygen species.. ................................................Figure 14: The NF-κB signaling pathway. ..............................................................................Figure 15: IL-6 signaling cascade. ...........................................................................................Figure 16: The relationship between inflammation and diabetes. ...........................................Figure 17: Blood collection by cardiac puncture (cardiocentesis).......................................... Figure 18: The principle of DHE protocol for ROS detection and quantification...................Figure 19: Griess reaction principle. ........................................................................................Figure 20: Principle of sandwich Enzyme-linked Immunosorbent assay ELISA. ..................Figure 21: Effects of probiotics and metformin on glycemia levels in non-diabetic(a) and diabetic (b) mice. ..................................................................................................................... Figure 22: Weight changes in the different animal groups during the experimental period. ..Figure 23: Hunched back animals with unhealthy clinical profile (left side), a mouse with diarrhea and rectal bleeding (right side). .................................................................................Figure 24: Disease activity index variations in the different groups. ......................................Figure 25: Kaplan-Meier survival curves of animals in the different groups. .........................Figure 26: Gross macroscopic images of the colon from non-diabetic groups (a) and diabetic groups (b). ................................................................................................................................Figure 27: Colon length variation. ...........................................................................................Figure 28: Polyp count variation in the different groups. ........................................................ Figure 29 Effect of probiotics and metformin on colon histology. .........................................Figure 30: Histology of CRC and DCRC animals ...................................................................Figure 31: Histology of CRC and DCRC animals treated with metformin alone. .................. Figure 32: Histology of CRC and DCRC animals treated with probiotics. .............................Figure 33: Histology of CRC and DCRC animals treated with the combination therapy. ......Figure 34: Histology of normal controls and Diabetic animals. ..............................................Figure 35: Histology of normal controls and diabetic animals treated with metformin alone.96

Figure 31 :

 31 Figure 1: Diagram of general organization of the alimentary canal. ....................................... Figure 2: Features of the musculature of the large intestine. ................................................... Figure 3: Histology of the colon (H&E). .................................................................................Figure 4: Schematic figure of the intestinal barrier and its components. ................................Figure 5: Functions of the large intestine ................................................................................Figure 6: Bar chart of Region-specific incidence age standardized rates by sex for cancers of the (A) colon and (B) Rectum in 2018.....................................................................................Figure 7: Estimated number of new cancer cases in 2018, worldwide, both sexes, all ages ...Figure 8: Number of new cancer cases in 2018 in Lebanon, both sexes all ages. ...................Figure 9: Proportion of colorectal cancer cases associated with sporadic and hereditary factors.. ..................................................................................................................................... Figure 10: TNM classification of colorectal cancer ................................................................Figure 11: Pathways of colorectal carcinogenesis. .................................................................. Figure 12: Schematic representation of the g-FOBT (A) and i-FOBT (B) blood detection reactions. ..................................................................................................................................Figure 13: Commonly encountered reactive oxygen species.. ................................................Figure 14: The NF-κB signaling pathway. ..............................................................................Figure 15: IL-6 signaling cascade. ...........................................................................................Figure 16: The relationship between inflammation and diabetes. ...........................................Figure 17: Blood collection by cardiac puncture (cardiocentesis).......................................... Figure 18: The principle of DHE protocol for ROS detection and quantification...................Figure 19: Griess reaction principle. ........................................................................................Figure 20: Principle of sandwich Enzyme-linked Immunosorbent assay ELISA. ..................Figure 21: Effects of probiotics and metformin on glycemia levels in non-diabetic(a) and diabetic (b) mice. ..................................................................................................................... Figure 22: Weight changes in the different animal groups during the experimental period. ..Figure 23: Hunched back animals with unhealthy clinical profile (left side), a mouse with diarrhea and rectal bleeding (right side). .................................................................................Figure 24: Disease activity index variations in the different groups. ......................................Figure 25: Kaplan-Meier survival curves of animals in the different groups. .........................Figure 26: Gross macroscopic images of the colon from non-diabetic groups (a) and diabetic groups (b). ................................................................................................................................Figure 27: Colon length variation. ...........................................................................................Figure 28: Polyp count variation in the different groups. ........................................................ Figure 29 Effect of probiotics and metformin on colon histology. .........................................Figure 30: Histology of CRC and DCRC animals ...................................................................Figure 31: Histology of CRC and DCRC animals treated with metformin alone. .................. Figure 32: Histology of CRC and DCRC animals treated with probiotics. .............................Figure 33: Histology of CRC and DCRC animals treated with the combination therapy. ......Figure 34: Histology of normal controls and Diabetic animals. ..............................................Figure 35: Histology of normal controls and diabetic animals treated with metformin alone.96

Figure 33 :

 33 Figure 1: Diagram of general organization of the alimentary canal. ....................................... Figure 2: Features of the musculature of the large intestine. ................................................... Figure 3: Histology of the colon (H&E). .................................................................................Figure 4: Schematic figure of the intestinal barrier and its components. ................................Figure 5: Functions of the large intestine ................................................................................Figure 6: Bar chart of Region-specific incidence age standardized rates by sex for cancers of the (A) colon and (B) Rectum in 2018.....................................................................................Figure 7: Estimated number of new cancer cases in 2018, worldwide, both sexes, all ages ...Figure 8: Number of new cancer cases in 2018 in Lebanon, both sexes all ages. ...................Figure 9: Proportion of colorectal cancer cases associated with sporadic and hereditary factors.. ..................................................................................................................................... Figure 10: TNM classification of colorectal cancer ................................................................Figure 11: Pathways of colorectal carcinogenesis. .................................................................. Figure 12: Schematic representation of the g-FOBT (A) and i-FOBT (B) blood detection reactions. ..................................................................................................................................Figure 13: Commonly encountered reactive oxygen species.. ................................................Figure 14: The NF-κB signaling pathway. ..............................................................................Figure 15: IL-6 signaling cascade. ...........................................................................................Figure 16: The relationship between inflammation and diabetes. ...........................................Figure 17: Blood collection by cardiac puncture (cardiocentesis).......................................... Figure 18: The principle of DHE protocol for ROS detection and quantification...................Figure 19: Griess reaction principle. ........................................................................................Figure 20: Principle of sandwich Enzyme-linked Immunosorbent assay ELISA. ..................Figure 21: Effects of probiotics and metformin on glycemia levels in non-diabetic(a) and diabetic (b) mice. ..................................................................................................................... Figure 22: Weight changes in the different animal groups during the experimental period. ..Figure 23: Hunched back animals with unhealthy clinical profile (left side), a mouse with diarrhea and rectal bleeding (right side). .................................................................................Figure 24: Disease activity index variations in the different groups. ......................................Figure 25: Kaplan-Meier survival curves of animals in the different groups. .........................Figure 26: Gross macroscopic images of the colon from non-diabetic groups (a) and diabetic groups (b). ................................................................................................................................Figure 27: Colon length variation. ...........................................................................................Figure 28: Polyp count variation in the different groups. ........................................................ Figure 29 Effect of probiotics and metformin on colon histology. .........................................Figure 30: Histology of CRC and DCRC animals ...................................................................Figure 31: Histology of CRC and DCRC animals treated with metformin alone. .................. Figure 32: Histology of CRC and DCRC animals treated with probiotics. .............................Figure 33: Histology of CRC and DCRC animals treated with the combination therapy. ......Figure 34: Histology of normal controls and Diabetic animals. ..............................................Figure 35: Histology of normal controls and diabetic animals treated with metformin alone.96

Figure 34 :

 34 Figure 1: Diagram of general organization of the alimentary canal. ....................................... Figure 2: Features of the musculature of the large intestine. ................................................... Figure 3: Histology of the colon (H&E). .................................................................................Figure 4: Schematic figure of the intestinal barrier and its components. ................................Figure 5: Functions of the large intestine ................................................................................Figure 6: Bar chart of Region-specific incidence age standardized rates by sex for cancers of the (A) colon and (B) Rectum in 2018.....................................................................................Figure 7: Estimated number of new cancer cases in 2018, worldwide, both sexes, all ages ...Figure 8: Number of new cancer cases in 2018 in Lebanon, both sexes all ages. ...................Figure 9: Proportion of colorectal cancer cases associated with sporadic and hereditary factors.. ..................................................................................................................................... Figure 10: TNM classification of colorectal cancer ................................................................Figure 11: Pathways of colorectal carcinogenesis. .................................................................. Figure 12: Schematic representation of the g-FOBT (A) and i-FOBT (B) blood detection reactions. ..................................................................................................................................Figure 13: Commonly encountered reactive oxygen species.. ................................................Figure 14: The NF-κB signaling pathway. ..............................................................................Figure 15: IL-6 signaling cascade. ...........................................................................................Figure 16: The relationship between inflammation and diabetes. ...........................................Figure 17: Blood collection by cardiac puncture (cardiocentesis).......................................... Figure 18: The principle of DHE protocol for ROS detection and quantification...................Figure 19: Griess reaction principle. ........................................................................................Figure 20: Principle of sandwich Enzyme-linked Immunosorbent assay ELISA. ..................Figure 21: Effects of probiotics and metformin on glycemia levels in non-diabetic(a) and diabetic (b) mice. ..................................................................................................................... Figure 22: Weight changes in the different animal groups during the experimental period. ..Figure 23: Hunched back animals with unhealthy clinical profile (left side), a mouse with diarrhea and rectal bleeding (right side). .................................................................................Figure 24: Disease activity index variations in the different groups. ......................................Figure 25: Kaplan-Meier survival curves of animals in the different groups. .........................Figure 26: Gross macroscopic images of the colon from non-diabetic groups (a) and diabetic groups (b). ................................................................................................................................Figure 27: Colon length variation. ...........................................................................................Figure 28: Polyp count variation in the different groups. ........................................................ Figure 29 Effect of probiotics and metformin on colon histology. .........................................Figure 30: Histology of CRC and DCRC animals ...................................................................Figure 31: Histology of CRC and DCRC animals treated with metformin alone. .................. Figure 32: Histology of CRC and DCRC animals treated with probiotics. .............................Figure 33: Histology of CRC and DCRC animals treated with the combination therapy. ......Figure 34: Histology of normal controls and Diabetic animals. ..............................................Figure 35: Histology of normal controls and diabetic animals treated with metformin alone.96

Figure 35 :

 35 Figure 1: Diagram of general organization of the alimentary canal. ....................................... Figure 2: Features of the musculature of the large intestine. ................................................... Figure 3: Histology of the colon (H&E). .................................................................................Figure 4: Schematic figure of the intestinal barrier and its components. ................................Figure 5: Functions of the large intestine ................................................................................Figure 6: Bar chart of Region-specific incidence age standardized rates by sex for cancers of the (A) colon and (B) Rectum in 2018.....................................................................................Figure 7: Estimated number of new cancer cases in 2018, worldwide, both sexes, all ages ...Figure 8: Number of new cancer cases in 2018 in Lebanon, both sexes all ages. ...................Figure 9: Proportion of colorectal cancer cases associated with sporadic and hereditary factors.. ..................................................................................................................................... Figure 10: TNM classification of colorectal cancer ................................................................Figure 11: Pathways of colorectal carcinogenesis. .................................................................. Figure 12: Schematic representation of the g-FOBT (A) and i-FOBT (B) blood detection reactions. ..................................................................................................................................Figure 13: Commonly encountered reactive oxygen species.. ................................................Figure 14: The NF-κB signaling pathway. ..............................................................................Figure 15: IL-6 signaling cascade. ...........................................................................................Figure 16: The relationship between inflammation and diabetes. ...........................................Figure 17: Blood collection by cardiac puncture (cardiocentesis).......................................... Figure 18: The principle of DHE protocol for ROS detection and quantification...................Figure 19: Griess reaction principle. ........................................................................................Figure 20: Principle of sandwich Enzyme-linked Immunosorbent assay ELISA. ..................Figure 21: Effects of probiotics and metformin on glycemia levels in non-diabetic(a) and diabetic (b) mice. ..................................................................................................................... Figure 22: Weight changes in the different animal groups during the experimental period. ..Figure 23: Hunched back animals with unhealthy clinical profile (left side), a mouse with diarrhea and rectal bleeding (right side). .................................................................................Figure 24: Disease activity index variations in the different groups. ......................................Figure 25: Kaplan-Meier survival curves of animals in the different groups. .........................Figure 26: Gross macroscopic images of the colon from non-diabetic groups (a) and diabetic groups (b). ................................................................................................................................Figure 27: Colon length variation. ...........................................................................................Figure 28: Polyp count variation in the different groups. ........................................................ Figure 29 Effect of probiotics and metformin on colon histology. .........................................Figure 30: Histology of CRC and DCRC animals ...................................................................Figure 31: Histology of CRC and DCRC animals treated with metformin alone. .................. Figure 32: Histology of CRC and DCRC animals treated with probiotics. .............................Figure 33: Histology of CRC and DCRC animals treated with the combination therapy. ......Figure 34: Histology of normal controls and Diabetic animals. ..............................................Figure 35: Histology of normal controls and diabetic animals treated with metformin alone.96

Figure 36 :

 36 Figure 36: Histology of normal and diabetic animals treated with probiotics alone. .............. Figure 37: Histology of normal and diabetic animals treated with the MP combination. .......Figure 38: Effect of probiotics and metformin on colon proliferation. ................................. Figure 39: Reactive oxygen species modulation by metformin and probiotics. .................... Figure 40: Nitrite modulation by metformin and probiotics. ................................................. Figure 41: Variation of IL-6 and TNF-α levels from colon extraction (a,c) and serum (b,d) of the different groups. ...............................................................................................................Figure 42: Proposed mechanisms of action elicited by metformin and probiotics in CRC and diabetes. .................................................................................................................................

Figure 42 :

 42 Figure 36: Histology of normal and diabetic animals treated with probiotics alone. .............. Figure 37: Histology of normal and diabetic animals treated with the MP combination. .......Figure 38: Effect of probiotics and metformin on colon proliferation. ................................. Figure 39: Reactive oxygen species modulation by metformin and probiotics. .................... Figure 40: Nitrite modulation by metformin and probiotics. ................................................. Figure 41: Variation of IL-6 and TNF-α levels from colon extraction (a,c) and serum (b,d) of the different groups. ...............................................................................................................Figure 42: Proposed mechanisms of action elicited by metformin and probiotics in CRC and diabetes. .................................................................................................................................

  Experimental design. ................................................................................................. Table 3: Histological changes. ................................................................................................. Table 4: Deparafinization protocol adapted from Abcam IHC. .............................................. Table 5: Effects of metformin and probiotics on fecal occult blood (FOB). ...........................

:

  cette étude vise à démontrer que la metformine en association avec des probiotiques agit en synergie en brisant la diaphonie, inhibant ainsi la progression du CCR, améliorant le diabète et réduisant l'inflammation. Méthodologie: quatre-vingt-seize souris mâles balb/c, âgées de 6 à 8 semaines, ont été divisées en 16 groupes témoins et expérimentaux pour évaluer l'effet des différents traitements et combinaisons aux niveaux clinique, histologique et moléculaire. Les niveaux de cytokines proinflammatoires IL-6 et TNF-α ont été évalués, ainsi que les espèces réactives de l'oxygène et de l'azote. De plus, l'indice de prolifération a été déterminé par immunohistochimie Ki-67. Résultats: la metformine et les probiotiques ont montré des résultats bénéfiques sur le CCR et le diabète, seuls et surtout en association. Les traitements ont réduit le processus inflammatoire en diminuant la production d'IL-6 et de TNF-α ainsi que le stress oxydatif. Conclusion: la caractérisation des effets des probiotiques et de la metformine sur le CCR et le diabète révèle le rôle de l'inflammation et du microbiote dans cette diaphonie. Décrypter les voies de signalisation en aval induites par ces composés aidera à développer de nouvelles thérapies ciblées efficaces. Mots-Clés: Cancer Colorectal, Maladies inflammatoires de l'intestin, Diabète, Inflammation, Probiotiques, Metformine.

Figure 1 :

 1 Figure 1: Diagram of general organization of the alimentary canal.

Figure 2 :

 2 Figure 2: Features of the musculature of the large intestine [16].

Figure 3 :

 3 Figure 3 : Histology of the colon (H&E). A: A cross section through the large intestine is shown at low magnification. Note the four layers that the colon wall: the mucosa (Muc), the submucosa (SubM), the muscularis externa (ME), and the serosa (S). B: Lamina propria: This figure reveals the muscularis mucosae (MM) and the cells in the lamina propria (LP), many of which can be recognized as lymphocytes and plasma cells. C: Intestinal glands: The cells that line the surface of the colon and the glands are principally absorptive cells (AC) and goblet cells (GC). Arrows show the opening of the glands. Note the following abbreviations: A: adipose tissue, BV: blood vessels, GI: intestinal glands M: mitotic figures, ME(c):circular layer of muscularis externa,ME(l): longitudinal layer of muscularis externa, TC: tenia coli, arrowheads: smooth muscle cells showing rounded nuclei, arrows: opening of intestinal glands [14].

Figure 4 :

 4 Figure 4: Schematic figure of the intestinal barrier and its components [29].

Figure 5 :

 5 Figure 5: Functions of the large intestine [44].

Figure 6 :

 6 Figure 6: Bar chart of Region-specific incidence age standardized rates by sex for cancers of the (A) colon and (B) Rectum in 2018 [1].

Figure 8 :

 8 Figure 8: Number of new cancer cases in 2018 in Lebanon, both sexes all ages [1].

Figure 9 :

 9 Figure 9: Proportion of colorectal cancer cases associated with sporadic and hereditary factors [58].

Figure 10 :

 10 Figure 10: TNM classification of colorectal cancer [68].

Figure 11 :

 11 Figure 11: Pathways of colorectal carcinogenesis [58].

Figure 12 :

 12 Figure 12: Schematic representation of the g-FOBT (A) and i-FOBT (B) blood detection reactions. g-FOBT, guaiac-based fecal occult blood test; i-FOBT, immunochemical fecal occult blood test [71].

  Acute metabolic complications: These complications are short term and comprise hypoglycemia, diabetic ketoacidosis and hyperosmolar non-ketotic coma.  Late systemic complications: These are long term chronic complications, further classified into two other sub-categories: macrovascular and microvascular. o Macrovascular complications comprising coronary heart disease, peripheral vascular disease and stroke. o Microvascular complications including neuropathy, retinopathy and nephropathy o Diabetic foot is considered a micro-and macrovascular complication. Other complications exist such as depression and erectile dysfunction [75].

  Reactive oxygen species (ROS)Reactive Nitrogen species (RNS)Reactive Chlorine species (RCS) Among the most important ROS are the hydroxyl radical (OH), the superoxide radical anion (O2-), nitric oxide (NO ), and peroxyl radicals (ROO), as well as non-radical species such as hydrogen peroxide (H2O2), singlet oxygen (1O2), hypochlorous acid (HOCl), and peroxynitrite (ONOO-) as depicted in Figure 13[START_REF] Poprac | Targeting Free Radicals in Oxidative Stress-Related Human Diseases[END_REF].

Figure 13 :

 13 Figure 13: Commonly encountered reactive oxygen species [80].

Figure 14 :

 14 Figure 14: The NF-κB signaling pathway [98].

Figure 15 :

 15 Figure 15: IL-6 signaling cascade [101].

  The JAK/STAT pathway (Blue rectangle): it is a member of a tyrosine protein-activated kinases family. The main transcription factors involved are STAT3 and STAT1. They can be released after tyrosine phosphorylation, thus forming a homologous or heterodimer, transferred to the nucleus, thereby activating specific DNA binding sequences to promote gene expression. The Ras/MAPK pathway (Red rectangle): Activation of the membrane-bound guanosine triphosphatase or Ras protein usually induces activation of downstream signaling proteins such as MAPK cascade phosphorylation. The main substrates for phosphorylation of MAPK are c-Myc, c-Jun, c-Fos [101].

Figure 16 :

 16 Figure 16: The relationship between inflammation and diabetes [110].

  studies reporting their ability to reduce insulin resistance,HbA1C levels and improve glucose tolerance [120]. In brief, probiotics possess a wide spectrum of mechanisms through which they exert their protective effects [119,121,122]:  Shift in gut microbial composition  Preservation of epithelial barrier function  Defense against harmful gut flora  Competitive elimination of pathogens  Enhancement of host immunity against pathogens by producing anti-microbial peptides that result in the suppression of specific microorganisms.  Anti-carcinogenic effects  Anti-inflammatory actions  Improvement of lactose intolerance symptoms  Amelioration of glucose metabolism  Decrease in insulin resistance  Lipid profile amelioration.

Figure 17 :

 17 Figure 17: Blood collection by cardiac puncture (cardiocentesis).

Figure 19 :

 19 Figure 19: Griess reaction principle [138].

Figure 20 :

 20 Figure 20: Principle of sandwich Enzyme-linked Immunosorbent assay ELISA [139].

Figure 21 :

 21 Figure 21: Effects of probiotics and metformin on glycemia levels in non-diabetic (a) and diabetic (b) mice.

Figure 22 :

 22 Figure 22: Weight changes in the different animal groups during the experimental period.

Figure 23 :

 23 Figure 23: Hunched back animals with unhealthy clinical profile (left side), a mouse with diarrhea and rectal bleeding (right side).

Figure 24 :

 24 Figure 24: Disease activity index variations in the different groups.

Figure 25 :

 25 Figure 25: Kaplan-Meier survival curves of animals in the different groups.

Figure 26 :

 26 Figure 26: Gross macroscopic images of the colon from non-diabetic groups (a) and diabetic groups (b).

ab

  Diabetes alone did not affect colon length, as no statistically significant difference was obtained when comparing non-diabetic and diabetic animals in all subgroups.

Figure 27 :

 27 Figure 27: Colon length variation.

Figure 28 :

 28 Figure 28: Polyp count variation in the different groups.

37 Figure 29

 3729 Figure 29 Effect of probiotics and metformin on colon histology. (A-B) Representative images of H&E-stained colon sections illustrating the histological changes in the nondiabetic (Panel A) and diabetic (Panel B) groups.

Figure 37 :

 37 Figure 37: Histology of normal and diabetic animals treated with the MP combination.

Figure 38 :

 38 Figure 38: Effect of probiotics and metformin on colon proliferation.

Figure 40 :

 40 Figure 40: Nitrite modulation by metformin and probiotics.

  -b, d) with # p<0.05 and *p<0.05, respectively. Single drug treatment in diabetic and non-diabetic animals decreased the levels of IL-6 (Figure 41-a,b) and TNF-α (Figure 41-c,d) in both colonic tissue and serum of CRC+M, CRC+P , DCRC+M and DCRC+P animals, with no statistical significance. However, when the MP combination is administered, a significantly lower level of IL-6 was detected in the colon of non-diabetics (CRC+MP, # p<0.05) and the serum of diabetics (DCRC+MP, *p<0.05) as seen in Figures 41-a and 41-b.

Figure 41 :

 41 Figure 41: Variation of IL-6 and TNF-α levels from colon extraction (a,c) and serum (b,d) of the different groups.

Chapter 5 :

 5 DiscussionThis work explored further the association between diabetes mellitus and CRC in particular the molecular mechanisms underlying these two disease entities using a well-established experimental model with distinct clinical characteristics. Protocols using AOM and DSS, in combination or alone, have been frequently used in different mice strains, demonstrating the relevance of the AOM/DSS mouse model in the study of the mechanisms of human colorectal carcinogenesis and its therapeutic targets. Due to the synergic effects of the tumor-inducing agent (AOM) and the tumor-promoting agent (DSS), the AOM/DSS model replicates colorectal carcinogenesis promoted by an initial acute inflammation phase, showing a shorter latency period than models based on AOM or DSS administration alone and not in combination. Even that they act in synergy, AOM and DSS have divergent mechanisms of action. DSS has a toxic effect on the epithelial lining of the colon and creates a severe colitis, which is characterized by loss of body weight, rectal bleeding and diarrhea [124,125]. This state of chronic inflammation triggered and maintained by DSS plays a major role in colorectal carcinogenesis through different mechanisms such as the alteration of the epithelial cells, the disruption of the gut barrier, as well as through the excessive production of oxidative species such as ROS, cytokines and pro-inflammatory molecules [141].

  cancer and diabetes. Several possible mechanisms of action of probiotics have been proposedincluding: improvement of the gastrointestinal mucosa, modifications in the intestinal microbiota and in its metabolic activity, immunomodulation, improvement of glycemia and HbA1c, decrease of cellular proliferation, induction of apoptosis and suppression of inflammatory reactions…among others[145,146].In our study, a treatment combining a probiotics mixture and metformin was administered to diabetic and non-diabetic Balb/c mice that were exposed to CRC induction. A thorough analysis of the emanating clinical, histological and molecular data resulted in the beneficial effects of metformin and probiotics on our colitis associated colorectal cancer mouse model.A large number of previous reports focused on probiotics and metformin and their beneficial effects on diabetes and CRC[111,143,147]. The findings of our work parallel these previous studies as our treatment protocol showed beneficial effects on diabetes and CRC to different extents; whereby treatment with probiotics along with metformin helped in inhibiting the damage caused by the administration of AOM/DSS to Balb/c mice. Less weight loss, a better disease activity index, a lower number of colon polyps and a better colonic architecture were detected in treated animals. Moreover, the inflammatory reaction was suppresses as manifested by a reduction in the secretion of TNF-α and IL-6.AOM/DSS induced colorectal cancer, is also characterized by a dysregulation of the intestinal barrier, therefore dysbiosis was considered a hallmark in our CRC mouse model which can lead to alterations in the luminal microbiota and consequently to the alteration of the host physiology, thus promoting the development and progression of CRC by different processes.Such processes include the creation of a chronic inflammatory state or hyperactive immune response, altering stem cell dynamics, the biosynthesis of toxic and genotoxic metabolites and dysregulation of the host metabolism[148].It was documented that dysbiosis in the gut acts as a driving force during the progression from inflammation to carcinogenesis[149,150], thus, probiotics have the ability to inhibit tumor progression mainly through the manipulation of the intestinal microbiota leading probably to homeostatic state or equilibrium. In this study, probiotics reduced glycemia when compared to the untreated diabetic mice, with or without CRC induction, metformin and probiotics single drug treatment significantly reduced blood glucose levels. These results go in parallel with preliminary interventions in humans suggesting that probiotics may ameliorate glucose metabolism, insulin resistance, and HbA1c levels[151,152]. Moreover, our findings suggest a possible chemopreventive effect of probiotics supplementation on CRC, whereby probiotics promoted intestinal homeostasis and regulated the inflammatory response. Non-diabetic and diabetic animals treated with probiotics alone in groups CRC+P and DCRC+P, had a better clinical profile when compared to the untreated DCRC and CRC groups and their DAI scores were improved as well as their survival rates. In addition, occult blood appearance was decreased and delayed; polyp formation, inflammatory cells infiltration and RONS secretions were significantly decreased with probiotics treatment. These results parallel several previous studies, for instance Mendes et al. showed that probiotics supplementation reduced inflammatory cell infiltration and lowered chemokine expression[150], Another study by Chen et al, described the protective effect of two strains C. butyricum and B. subtilis on CRC, these probiotics inhibited the proliferation of cancerous cells , induced cell cycle arrest and apoptosis. Their effect was also verified in-vivo as they inhibited tumor development in a DMH-induced CRC mouse model[153]. A recent study , conducted in 2020 showed that Probiotics therapy is beneficial on high fat induced carcinogenesis, as it was able to reduce obesity and reverse the microbial imbalance caused by the HFD administration , along with a reduction tumor incidence, thus linking metabolic disorders to carcinogenesis with microbial alterations as a common denominator[154].Concerning our second drug, metformin, it is widely and commonly used for the treatment of diabetes mellitus. It main advantages are its good safety profile and low cost. Besides its antidiabetic effects, several properties are being recently associated with metformin, mainly its antitumor, antiaging, cardiovascular and neuroprotective properties[144,155,156].The efficacy of metformin in decreasing inflammation and oxidative stress as well as in preventing tumorigenesis has been shown to be mediated mainly through the inhibition of various pro-inflammatory mediators and oxidative stress[157].Moreover, a recent study showed the beneficial effects of Metformin on metastatic colorectal cancer specifically KRAS-mutation mCRC patients that usually have limited therapeutic options [158]. Additionally , Wang et al showed that Metformin administration reduced the overall mortality of CRC patients with T2DM[159].

  and development. In the present work, cellular proliferation was evaluated using Ki-67 labeling of colon tissue. High levels of Ki-67 positive cells were detected in the crypts of CRC and DCRC animals with extended labelling to most of the crypt surface. This is in accordance with other studies showing that in CRC, a reversal in the distribution of proliferating cells from the bottom of the crypt into the upper crypt and luminal surface occurs[164]. However, in normal conditions proliferating cells are concentrated at the bottom half of the crypt, and the upper half of the crypt usually consists of non-dividing migrating cells[165]. An inhibition of proliferating colonocytes was induced by metformin and probiotics especially when combined, shedding light again on the protective effect of these 2 compounds on colorectal carcinogenesis.

Figure 42 :

 42 Figure 42: Proposed mechanisms of action elicited by metformin and probiotics in CRC and diabetes.

Table 1 :

 1 Enzymatic reactions that participate in ROS/NOS generation in the GI tract[START_REF] Piechota-Polanczyk | Review article: the role of oxidative stress in pathogenesis and treatment of inflammatory bowel diseases[END_REF].

	Enzyme	Reaction	Site of action

Complex I and III/ubiquinone of the mitochondrial electron transport chain

		Complex I (NADH dehydrogenase):	Mitochondria
		O2 + NADH → O2 •-+ NAD +
		Complex III (cytochrome bc1):
		O2 → O2 •-
	Xanthine oxidase	Xanthine + O2 + NADPH →	Plasma and cytoplasm of epithelial cells
		O2 •-+ H2O2 + NADP + + uric acid
	NADPH oxidase	2O2 + NADPH → 2O2 • + NADP + + H + Cell membrane
	Haber-Weiss reaction	H2O2 + O2 •-→ O2 + OH + OH •	Plasma and cell's cytoplasm
	Fenton reaction	H2O2 + Fe 2+ → Fe 3+ + OH + OH •	Plasma and cell's cytoplasm

Table 2 :

 2 Experimental design.

	Non-diabetic animals
	With CRC induction	Without CRC induction
	CRC (CRC)	Normal controls (NC)
	CRC + metformin (CRC+M)	Metformin (M)
	CRC + probiotics (CRC+P)	Probiotics (P)
	CRC + metformin and probiotics (CRC+MP)	Metformin and probiotics (MP)
	Diabetic animals
	With CRC induction	Without CRC induction
	Diabetic CRC (DCRC)	Diabetic (D)
	Diabetic CRC + metformin (DCRC+M)	Diabetic + metformin (D+M)
	Diabetic CRC + probiotics (DCRC+P)	Diabetic + probiotics (D+P)
	Diabetic CRC + metformin and probiotics (DCRC+MP) Diabetic + metformin and probiotics (D+MP)

3. Induction of CRC

It is well established that the Azoxymethane (AOM)/Dextran sulfate sodium (DSS) model is able to induce CRC in mice and this model is widely used in experimental colitis-associated colorectal cancer studies, in our laboratory and elsewhere

[124]

.

Table 3 :

 3 Histological changes.

	Structural Change	0	1	2		3
	Mucosal architecture	Normal Focal surface	Zonal surface	Diffuse destruction
		destruction	destruction
	Glandular crypt	Absent Mild atrophy	Atrophy +	Atrophy + Branching + Crypt
	architecture			Branching	abscess
	Loss of goblet cells	Absent Mild		Moderate	Extensive
	Edema	Absent Mild		Moderate	Extensive
	Crypt abscesses	Absent Focal		Zonal	Extensive
	Inflammatory cells	Absent Mild		Moderate	Extensive	(to submucosa
	infiltration	(only Mucosa)	(to muscularis	and musculosa)
				mucosa)
	Dysplasia	Absent Focal		Zonal	Diffuse

Table 4 :

 4 Deparafinization protocol adapted from Abcam IHC.

	Solution	Incubation time
	Xylene	3 min
	Xylene	3 min
	1:1 Xylene: 100% Ethanol	3 min
	100% Ethanol	3 min
	100% Ethanol	3 min
	95% Ethanol	3 min
	70% Ethanol	3 min
	50% Ethanol	3 min

Table 5 :

 5 Effects of metformin and probiotics on fecal occult blood (FOB). Note the color scale extending from strong to faint red whereby the shade of the red color indicates the value of the cell.

	Group						Experimental period (weeks)
		0	1	2	3	4	5	6	7	8	9	10	11	12	13
	NC	0% 0% 0% 0%	0%	0%	0%	0%	0%	0%	0%	0%	0%	0%
	D	0% 0% 0% 0%	0%	0%	0%	0%	0%	0%	0%	0%	0%	0%
	CRC	0% 0% 0% 17% 33% 100% 100% 50% 100% 100% 100% 100% 100% 100%
	DCRC	0% 0% 0% 17% 17% 100% 100% 80% 100% 100% 100% 100% 100% 100%
	CRC+M	0% 0% 0% 0%	0%	100% 83%	33% 100% 83%	67%	100% 100% 100%
	DCRC+M	0% 0% 0% 0%	0%	100% 33%	17% 100% 50%	33%	100% 100% 100%
	CRC+P	0% 0% 0% 0%	0%	83%	33%	33% 100% 100% 67%	100% 100% 100%
	DCRC+P	0% 0% 0% 0%	0%	100% 50%	33% 100% 33%	33%	100% 100% 60%
	CRC+MP	0% 0% 0% 0%	0%	67%	17%	0%	67%	50%	33%	67%	67%	50%
	DCRC+MP	0% 0% 0% 0%	0%	67%	17%	17% 100% 17%	17%	100% 100% 33%
	M	0% 0% 0% 0%	0%	0%	0%	0%	0%	0%	0%	0%	0%	0%
	D+M	0% 0% 0% 0%	0%	0%	0%	0%	0%	0%	0%	0%	0%	0%
	P	0% 0% 0% 0%	0%	0%	0%	0%	0%	0%	0%	0%	0%	0%
	D+P	0% 0% 0% 0%	0%	0%	0%	0%	0%	0%	0%	0%	0%	0%
	MP	0% 0% 0% 0%	0%	0%	0%	0%	0%	0%	0%	0%	0%	0%
	D+MP	0% 0% 0% 0%	0%	0%	0%	0%	0%	0%	0%	0%	0%	0%

  100% of the animals, with tubular adenoma, dysplasia, and colitis, preferably in male mice of susceptible strains at week 12 from the start of the treatment. Moreover, it is important to shed light one more time on the fact that tumor development in this model closely mirrors the pattern seen in human CRC[143].

	Mutations in K-Ras and β-catenin as well as microsatellite instability are commonly present in AOM-induced tumors [142]. Colorectal carcinogenesis is a multistep process in which normal crypts are initiated to form aberrant crypt foci (ACF) that progress into adenocarcinomas. Such multistep tumor development was efficiently reproduced in the AOM/DSS murine model with high reproducibility in several susceptible murine strains. Commonly, 3-10 macroscopic tumors maintained blood glucose levels above 250 mg/dl. In addition, animals receiving the AOM/DSS combination developed CRC as diagnosed by several clinical and histological develop in 80%-Animals receiving STZ injection have successfully developed diabetes diagnosed by a parameters.

Acknowledgments

First and Foremost, I would like to thank God, for all the blessings and Miracles in my life.

I would like to express my sincere gratitude to my Director at the University of Limoges,

Abbreviations

.

A .

Chapter 2: Aims of the study

The concept of this research emerged after a thorough analysis of the literature, focusing on the links between colorectal cancer and diabetes and the crucial role of microbiota and dysbiosis in these links. This study was established to further investigate the interplay between colorectal cancer and diabetes in a colitis-associated colorectal cancer mouse model in the presence of probiotics and metformin in-vivo, using the Azoxymethane/Dextran sodium sulfate (AOM/DSS) colitis-associated CRC model that recapitulates key aspects of human colorectal cancer progression.

Moreover, an emphasis on the role of diabetes in this process was explored by inducing diabetes in the same mouse model using Streptozocin (STZ). On this basis, the specific aims include: microscopic evaluation and photography. DHE intensity was quantified using Zen 2.3 software [9].

Determination of nitrite levels

The classic colorimetric Griess reaction was used to measure serum nitrite levels (Figure 19).

Greiss reagent is composed by two light sensitive reagents: A and B.

Reagent A: 1% sulfanilamide (0.3g) in 30% acetic acid (9 ml acetic acid +21 ml H2O)

Reagent B : 0.1% N-naphthylethylenediamine dihydrochloride (0.1g) in 60% acetic acid (30 ml acetic acid + 20 ml H2O).

The solution used for the standard curve is sodium nitrite (NaNO2) with C0 = 10mM (the curve should include at least 7 points = 7 concentrations by serial dilution ).

Preparation of 10 mM NaNO2 solution: 690 mg of NaNO2 (powder ) dissolved in 1 L dH2O and for the serial dilutions, the used formula is: Ci µM × Vi= Cf µM × Vf µL .

Effect of metformin and probiotics on polyp formation

Polyp formation was evaluated by quantifying the number of formed bumps within the colon from the ileocecal junction until the distal end of rectum.

Animals in CRC and DCRC groups had an average number of 9.25±0.85 and 10.33±1.76 polyps, respectively. These numbers were significantly higher than the very low and negligible number obtained in the normal controls (0.67±0.33) # p<0.05 and *p<0.05, respectively.

Treatment with either drug alone in non-diabetic and diabetic CRC animals reduced the number of polyps; however, this reduction was not statistically significant. Conversely, animals receiving the MP combination therapy showed significantly less polyps with 3.33±0.56 polyps in CRC+MP group and 3.67±0.88 in DCRC+MP group ( # p<0.05, *p<0.05), respectively (Figure 28).

In D group, diabetic mice had an average of 3.5±0.56 polyp, this number was higher than their non-diabetic counterparts (0.67±0.33 polyps in NC group) with no statistical significance (Figure 28).

.

Assessment of colonic tissue proliferation

Immunohistological-staining by Ki-67 was performed on paraffin embedded colon tissue to evaluate the proliferation rate in the colon. CRC and DCRC groups showed elevated proliferation indices with the ki-67 positive cells dispersed throughout most of the crypt area, and migrating to the lumen (Figure 38).

Non-diabetic and diabetic CRC animals receiving M alone, had significantly lower proliferation rates as seen in CRC+M and DCRC+M groups ( # p<0.05, *p<0.05 respectively).

Conversely, in CRC+P and DCRC+P groups, when probiotics were administered as single drug, the decrease in proliferation index was not statistically significant (Figure 38C).

The most effective treatment regimen was the MP combination as animals in groups CRC+MP and DCRC+MP had low ki-67 levels, close to that of the normal controls ( # p<0.05, *p<0.05 respectively). Importantly, we can say that this combination was most likely capable of inhibiting colon cancer cell proliferation (Figure 38).

Modulation of nitric oxide levels with probiotics and metformin

Griess method was used to measure the concentration of nitrite, a stable metabolite of Nitric oxide (Figure 40). Non-diabetic and diabetic CRC mice showed the highest nitrite levels.

The MP combination was effective in both diabetic and non-diabetic CRC groups, whereby a significant suppression of nitrite production in CRC+MP and DCRC+MP groups were noted

Chapter 6: Conclusions and future perspectives

Clinical, histological and molecular data emanating from this study, focused on metformin and probiotics as a new therapeutic option for inflammation and its associated carcinogenesis.

Inhibition or decrease of cytokines and oxidative stress were the main mechanisms this study focused on, in the context of colitis associated colorectal cancer.

Metformin combined with probiotics were able to significantly prevent AOM/DSS-induced damage through correcting the inflamed microenvironment as well as reducing ROS and cytokines production and cellular proliferation, thus leading to the inhibition of CRC. This protective effect of metformin might relate to its interaction with the balanced microbiota corrected by probiotics (Figure 42). However, very little is currently known about the bacterial targets of metformin. It is possible that the microbiota could affect the host physiology, however, the exact mechanisms are not fully elucidated yet (Figure 42).

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