Veronika Leláková
email: lelakova@ipmc.cnrs.fr

Sophie Béraud-Dufour

Jan Hošek
email: jan.hosek@upol.cz

Karel Šmejkal
email: karel.mejkal@post.cz

Vilailak Prachyawarakorn
email: vilailak@cri.or.th

Phanruethai Pailee

Catherine Widmann
email: widmann@ipmc.cnrs.fr

Jiří Václavík

Thierry Coppola
email: coppola@ipmc.cnrs.fr

Jean Mazella
email: mazella@ipmc.cnrs.fr

Sophie Beraud-Dufour

Nicolas Blondeau
email: blondeau@ipmc.cnrs.fr

Catherine Heurteaux
email: heurteau@ipmc.cnrs.fr

Therapeutic potential of prenylated stilbenoid macasiamenene F through its anti-inflammatory and cytoprotective effects on LPS-challenged monocytes and microglia

Keywords: natural stilbenoids, neuroinflammation, microglia, monocytes, prenyl

niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Abbreviations:

Aβ, amyloid beta; AD, Alzheimer's disease; AP-1, activator protein 1; BBB, blood-brain barrier; COXs, cyclooxygenases; DAMPs, damage-associated molecular pattern molecules; HD, Huntington's disease; IкB, inhibitor of NF-kappa B transcription factor; IL-1β, interleukine 1 beta; LDH, lactate dehydrogenase; LPS, lipopolysaccharide; MAPKs, mitogen activated protein kinases; NF-кB, nuclear factor of kappa light polypeptide gene enhancer in B-cells; NSAIDs, non-steroidal anti-inflammatory drugs; PAMPs, pathogen associated molecular patterns; PD, Parkinson's disease; RA, rheumatoid arthritis; SEAP, secreted embryonic alkaline phosphatase; TLR4, toll-like receptor 4; TNF-α, tumor necrosis factor alpha.

Introduction

Inflammation represents an adaptive reaction of body tissues to invading pathogens, injuries, or endogenous signals from stressed, damaged, or dead cells. It is a physiological process that is beneficial, well-coordinated, and self-regulated. However, when the response to inflammatory stimuli becomes uncontrolled and continuous, chronic inflammatory conditions may develop and lead to severe pathologies [START_REF] Medzhitov | Origin and physiological roles of inflammation[END_REF]. Non-resolving inflammation contributes to the pathogenesis of both peripheral (neuropathic pain) and central nervous system diseases, such as stroke, traumatic brain injury, multiple sclerosis, amyotrophic lateral sclerosis, Alzheimer's disease, Huntington's disease, Parkinson's disease, or depression [START_REF] Degan | The Role of Inflammation in Neurological Disorders[END_REF][START_REF] Miller | Inflammation and Its Discontents: The Role of Cytokines in the Pathophysiology of Major Depression[END_REF][START_REF] Skaper | An Inflammation-Centric View of Neurological Disease: Beyond the Neuron[END_REF]. Moreover, a prolonged exposure to pathogens or an ongoing systemic inflammation of the kind observed in metabolic diseases such as diabetes or obesity can lead to a chronic state of neuroinflammation and ultimately result in neuronal cell death [START_REF] Nicolas | Globular Adiponectin Limits Microglia Pro-Inflammatory Phenotype through an AdipoR1/NF-κB Signaling Pathway[END_REF][START_REF] Skaper | An Inflammation-Centric View of Neurological Disease: Beyond the Neuron[END_REF].

Unrecovered and repeated acute inflammation, as well as long-term moderate exposure to noxious stimuli at the central or systemic level may result in a chronic state of disease. Nowadays, chronic low-grade inflammation is often related to diseases of modern civilization, such as metabolic syndrome, which is often characterized as a cluster of conditions such as hypertension, hyperglycemia, insulin resistance, obesity, and high levels of serum triglycerides, that increase the risk of diabetes mellitus type 2, cerebrovascular disease, and stroke [START_REF] Lopez-Candales | Linking Chronic Inflammation with Cardiovascular Disease: From Normal Aging to the Metabolic Syndrome[END_REF][START_REF] Minihane | Low-grade inflammation, diet composition and health: current research evidence and its translation[END_REF]. Some inflammatory autoimmune disorders, such as rheumatoid arthritis, are also based on chronic inflammation [START_REF] Smolen | Rheumatoid arthritis[END_REF]. Systemic and CNS immune system crosstalk during inflammatory response is a critical consideration not to be overlooked [START_REF] Minihane | Low-grade inflammation, diet composition and health: current research evidence and its translation[END_REF]. Systemic macrophages and brain microglia together with CNS-associated macrophages play a crucial role in surveillance of the internal environment and control of homeostasis [START_REF] Ginhoux | Monocytes and macrophages: developmental pathways and tissue homeostasis[END_REF][START_REF] Yin | The Role of Microglia and Macrophages in CNS Homeostasis, Autoimmunity, and Cancer[END_REF]. Through toll-like receptors (TLRs), they transmit a signal during bacterial infection, injury, or brain damage, including that triggered by hypoxia/ischemia [START_REF] Lehnardt | Activation of innate immunity in the CNS triggers neurodegeneration through a Toll-like receptor 4-dependent pathway[END_REF].

Acute inflammation is often associated with a microbial infection that exemplifies itself in a form such as meningitis [START_REF] Hoffman | Pathophysiology and Treatment of Bacterial Meningitis[END_REF], bronchitis [START_REF] Wark | Bronchitis (acute)[END_REF], pneumonia [START_REF] Percopo | Critical Adverse Impact of IL-6 in Acute Pneumovirus Infection[END_REF], myocarditis [START_REF] Blasi | Immortalization of murine microglial cells by a v-raf / v-myc carrying retrovirus[END_REF], or cystitis [START_REF] Flores-Mireles | Urinary tract infections: epidemiology, mechanisms of infection and treatment options[END_REF]. Moreover, Gram-negative bacteria such as Klebsiella pneumoniae, Escherichia coli, or Pseudomonas sp., possess in their outer membrane the endotoxin lipopolysaccharide (LPS), responsible for their pathogenic potential and massive clinical manifestations in humans [START_REF] Sperandio | Antimicrobial Photodynamic Therapy to Kill Gram-negative Bacteria[END_REF]. High peripheral concentrations of LPS can disrupt the blood-brain barrier (BBB) and promote neuroinflammation [START_REF] Banks | Lipopolysaccharide-induced blood-brain barrier disruption: roles of cyclooxygenase, oxidative stress, neuroinflammation, and elements of the neurovascular unit[END_REF]. LPS triggers an innate immune system response through TLR4 receptors linked with several inflammatory signaling pathways leading to the production of cytokines and the onset of inflammation [START_REF] Pålsson-Mcdermott | Signal transduction by the lipopolysaccharide receptor, Toll-like receptor-4[END_REF]. We therefore used LPS from E. coli to induce TLR4-mediated inflammatory cascades in our experimental set up.

Stilbenoids are natural phenolic compounds found in foods (grapes, peanuts, berries, passion fruit, rhubarb), beverages (wine, white tea), and medicinal plants, including Morus spp., Artocarpus spp., and the less well known Macaranga spp. [START_REF] Dvorakova | Anti-inflammatory activity of natural stilbenoids: A review[END_REF][START_REF] Hošek | Prenylated Stilbenoids Affect Inflammation by Inhibiting the NF-κB/AP-1 Signaling Pathway and Cyclooxygenases and Lipoxygenase[END_REF]. Structurally, they share a common diarylethylene core and differ in its substitution, which may include prenyl group. Stilbenoids serve the plant as phytoalexins, protecting it against infection or harm. The strong anti-oxidant and anti-inflammatory properties of stilbenoids have multiple health benefits. Cardioprotective, neuroprotective, anti-diabetic and cancer preventive properties have been reported [START_REF] Akinwumi | Biological Activities of Stilbenoids[END_REF][START_REF] Dvorakova | Anti-inflammatory activity of natural stilbenoids: A review[END_REF][START_REF] Eräsalo | Natural Stilbenoids Have Anti-Inflammatory Properties in Vivo and Down-Regulate the Production of Inflammatory Mediators NO, IL6, and MCP1 Possibly in a PI3K/Akt-Dependent Manner[END_REF][START_REF] Hornedo-Ortega | Phenolic Compounds Characteristic of the Mediterranean Diet in Mitigating Microglia-Mediated Neuroinflammation[END_REF]. The neuroprotective potential of stilbenoids has been tested mostly on neurodegenerative disorders, such as AD. For instance, veraphenol, cis-scirpusin A and oxyresveratrol, stilbenes obtained from Smilax spp., showed significant inhibitory effects in vitro against β-secretase, the enzyme crucially responsible for the amyloid beta (Aβ) formation in AD [START_REF] Jeon | β-secretase (BACE1)-inhibiting stilbenoids from Smilax Rhizoma[END_REF]. Other than the most widely known and structurally simple stilbene trans-resveratrol, and its hydroxyderivates oxyresveratrol and piceatannol (found, e.g., in grapes), whose antioxidant properties and therapeutic potential in cardiovascular diseases arouse great interest, have been studied very little to date. Little or no information is available about the biological activities of less-known stilbenoids, including those of the genus Macaranga [START_REF] Hošek | Prenylated Stilbenoids Affect Inflammation by Inhibiting the NF-κB/AP-1 Signaling Pathway and Cyclooxygenases and Lipoxygenase[END_REF].

The genus of Macaranga comprises around 300 species and is native to Africa, Australia, Asia, and various islands of Indian and Pacific Oceans. Some species, such as M. tanarius (L.) Muell. Arg., in Thailand known as "Mek", have been used in traditional Thai medicine as anti-inflammatory treatment. Whereas the fresh leaves were used to cover wounds, a decoction from the dried root was administered against cough and fever [START_REF] Kamarozaman | Prenylated dihydrostilbenes from macaranga heynei (Euphorbiaceae)[END_REF]Phommart et al., 2005, Phupattanapong and[START_REF] Phupattanapong | Thai Medicinal Plants[END_REF].

M. sampsonii

Hance has been used for the treatment of swellings, cuts and sores [START_REF] Quynh | Chemical constituents of MeOH extract from the fruits of Macaranga sampsonii[END_REF]. The European expeditioners surveying the traditional medicinal plants of Papua New Guinea islands recorded the use of M. aleuritoides F. Muell. fruits and seeds by aborigine people to relieve abdominal pain. Various species of Macaranga were used for the treatment of diarrhea, constipation and stomach complaints in several islands, such as Tonga, Fiji, Java, Philippines, Bougainville Island and Malaysia [START_REF] Holdsworth | Traditional Medicinal Plants of New Ireland, Papua New Guinea Part. II. New Hanover Island[END_REF][START_REF] Nick | Biological screening of traditional medicinal plants from Papua New Guinea[END_REF]. M. harveyana (Muell. Arg.) Muell. Arg. known as "Loupata" was used in Tonga folk medicine for treatment of obstetric and gynecological disorders, such as secondary amenorrhea, post-partum hemorrhage and abdominal pain [START_REF] Singh | Folk medicine in tonga. A study on the use of herbal medicines for obstetric and gynaecological conditions and disorders[END_REF].

Several studies trying to describe the phytochemical profile of Macaranga species identified this genus as a rich source of polyphenols such as prenylated and geranylated stilbenes and flavonoids, terpenes, tannins, steroids and coumarins. Most of the described biological activities are attributed to stilbenes and flavonoids found in high amounts [START_REF] Magadula | Phytochemistry and pharmacology of the genus Macaranga: A review[END_REF][START_REF] Ngoumfo | Inhibitory Effect of Macabarterin, a Polyoxygenated Ellagitannin from Macaranga barteri, on Human Neutrophil Respiratory Burst Activity[END_REF].

M. siamensis S. J. Davies is a large-leaved plant, common in central and northern Thailand, firstly identified in 2001. The first phytochemical investigation provided by [START_REF] Pailee | Cytotoxic and cancer chemopreventive properties of prenylated stilbenoids from Macaranga siamensis[END_REF] demonstrated the high content of novel stilbenes, including macasiamenenes A, B, F, K, L, and P that have been isolated from dichloromethane extract of leaves and twigs. But, little information about their biological effects has been reported. Based on our previous study suggesting the anti-inflammatory properties of several prenylated stilbenoids contained in M. siamensis [START_REF] Hošek | Prenylated Stilbenoids Affect Inflammation by Inhibiting the NF-κB/AP-1 Signaling Pathway and Cyclooxygenases and Lipoxygenase[END_REF], by the present work we undertook to compare the antiinflammatory effects of seven compounds obtained from Macaranga to identify the therapeutic potential value of the most promising candidate, macasiamenene F (MF), for treatment of both systemic and central inflammation. The characterization of MF led us to describe for the first time its anti-inflammatory effects on microglial inflammation.

Materials and methods

Animals

Seven week-old male C57BL/6JRj mice (Janvier, France) were housed at 22°C with a 12-h light-dark cycle and free access to water and food. All animal care and use were performed in accordance with the policies of European Community Directive 86/609/EEC.

The ex vivo experiments were approved by the local committee. Every effort was made to minimize the number of animals used and their suffering. [START_REF] Pailee | Cytotoxic and cancer chemopreventive properties of prenylated stilbenoids from Macaranga siamensis[END_REF] isolated the macasiamenene F (MF) along with L (1), K (2), P (3), A (5), B (6) and 2,6-diprenyl-resveratrol (4) from leaves and twigs of M. siamensis S. J. Davies (Euphorbiaceae) by chromatographic methods and identified them by spectroscopic methods, including NMR analysis. The voucher specimen (No. CRI 466) was deposited in the Laboratory of Natural Products, Chulabhorn Research Institute, Thailand [START_REF] Pailee | Cytotoxic and cancer chemopreventive properties of prenylated stilbenoids from Macaranga siamensis[END_REF].

Isolation of compounds and treatments

The plant name has been checked with http://www.theplantlist.org. Compound 4, 2,6diprenyl-3,5,4´-trihydroxystilbene (4) was already known [START_REF] Verotta | Biologically-active stilbene derivatives and compositions thereof[END_REF], but compounds MF, 1-3, 5, and 6 were isolated for the first time. The purity of compounds was confirmed to be more than 98% using HPLC (Figure S1). DMSO was used as a vehicle; its concentration in cell culture never exceeded 0.1%. Apart from the dose-response studies, treatments were performed using appropriate serum-free medium at a concentration of 1 µmol/L (0.1 µmol/L for compound 1), which would not affect cell viability. At these concentrations, the viability measured by WST-1 was 100 % compared to non-treated cells (Fig. 1B). LPS from E. coli 0111:B4 (Sigma-Aldrich) freshly dissolved in PBS was used at a concentration of 1 µg/mL [START_REF] Taka | Anti-inflammatory effects of Thymoquinone in activated BV-2 microglia cells[END_REF][START_REF] Triantafilou | A CD14-independent LPS receptor cluster[END_REF].

Cell culture and differentiation

The THP-1 human monocytic leukemia cell line was purchased from the European Collection of Authenticated Cell Cultures (Salisbury, UK). The BV-2 murine microglial cells were generated from primary microglial culture by infection with v-raf/v-myc oncogene carrying J2 retrovirus [START_REF] Blasi | Immortalization of murine microglial cells by a v-raf / v-myc carrying retrovirus[END_REF]. The THP-1 and THP-1-XBlue™-MD2-CD14 (Invivogen, San Diego, CA, USA) cells were cultured in RPMI 1640 medium containing stabilized 2 mmol/L L-glutamine (Biosera, France), whereas the BV-2 cells were cultured in DMEM (4.5 g/L glucose, 4 mmol/L L-alanyl-glutamine, and w/o sodium pyruvate (Sigma).

All of the cultures were supplemented with 10 % fetal bovine serum (FBS, Gibco) and antibiotics [100 U/mL penicillin and 100 µg/mL streptomycin (Gibco)]. Cells were grown in a water-saturated atmosphere of 5% CO 2 at a temperature 37°C. The experiments were performed on passages ranging from 3 to 16 for all types of cell lines. The THP-1 monocytes were differentiated into macrophages by adding 50 ng/mL of phorbolmyristateacetate (PMA) stimulation [START_REF] Leláková | Parallel in vitro and in silico investigations into anti-inflammatory effects of non-prenylated stilbenoids[END_REF]. All subsequent experiments with all cell lines were performed after 2 h of incubation in serum-free medium.

Sorting of brain immune cells

Immune cells were isolated from the brains of adult mice using an Adult Brain Dissociation Kit (Miltenyi Biotec, USA) according to the manufacturer's instructions. Brain homogenates were filtered using 70 µm cell strainers (BD Biosciences) and centrifuged (10 min, 2,000 rpm). The resulting cell pellets were then re-suspended and washed with PBS at pH 7.2 containing 0.5 % BSA and 2.5 mM EDTA. They were then labeled with CD11b+ MicroBeads (Miltenyi Biotec, USA) and incubated for 15 min at 4°C. CD11b+ mouse microglia and CNS associated macrophages were isolated using LS columns (Miltenyi Biotec, USA) in a magnetic field according to the manufacturer's instructions.

Ex vivo culture of microglia

Brain-sorted microglia were seeded into 96-well tissue culture plate in DMEM (Gibco) containing 10 % fetal bovine serum (FBS, Gibco) and antibiotics [100 U/mL penicillin and 100 µg/mL streptomycin (Gibco)] at a density of 7 × 10 4 cells/well. Microglial cells were cultured on poly-L-lysine-covered plates at 37°C in an atmosphere containing 5 % CO 2 for 18 h. Thirty minutes after cell plating, MF at a concentration of 1 µmol/L, which has been determined to be non-toxic for both monocytes (Fig. 1) and microglia (Fig. 2), was added together with LPS (1 µg/ml) as a co-treatment. The supernatants and cell contents were collected and used subsequently to measure the levels of cytokines after18 h.

Cell viability: Dose-response relationships

Undifferentiated floating THP-1 cells in serum-free RPMI 1640 medium were seeded into 96-well plates (5 × 10 4 cells/well). After 2 h, compounds MF, and 1-6 were added at concentrations ranging from 0.125 to 20 µmol/L. The cell viability was assessed 24 h later using Cell Proliferation Reagent WST-1 kit (Roche Diagnostics, Basel, Switzerland). Based on the resulting viability curves, the IC 50 values were calculated according to four parameters logistic (4PL) analysis. In the case of adherent BV-2 cells, the dose-response assays were carried out in exponential phase of growth established by a pilot study in 24-well plates.

Briefly, BV-2 cells were seeded in the evening in complete DMEM medium at a density 1.5 × 10 5 cells/well, the next day washed by PBS, and the medium was replaced by serum-free medium. Cells were treated after 2 h with MF at concentrations ranging from 1 to 15 µmol/L.

The mitochondrial activity and cell proliferation were evaluated after 24 h of incubation using a Cell Titer 96® Aqueous One Solution Cell Proliferation Assay (MTS; Promega, France) and manual counting of the living cells excluded from Trypan Blue (Sigma) staining, respectively. Cell mortality was assessed after 24 h using a Cytotoxicity Detection Kit (LDH; Roche) and represented as the percentage of lactate dehydrogenase (LDH) released by damaged cells as compared to cells treated with only the vehicle.

Determination of NF-κB/AP-1 activity

The activity of nuclear factor κB (NF-κB) and activator protein-1 (AP-1) was measured on THP-1-XBlue™-MD2-CD14 cells (Invivogen, CA, USA) derived from THP-1 human monocytes. This cell line stably expresses the NF-κB and AP-1 inducible secreted embryonic alkaline phosphatase (SEAP) reporter gene, the activity of which was detected using QUANTI-Blue™ reagent (Invivogen, San Diego, USA). Cells in serum-free medium RPMI 1640 medium were seeded (5 × 10 4 cells/well, 96-well plate) and incubated for 2 h at 37°C. Compounds MF, 2-6, and prednisone (P) used as a positive control, were added at the non-toxic concentration of 1 µmol/L (0.1 µmol/L was used for compound 1), together with vehicle DMSO (0.1 % (v/v)). After 1 h of incubation, the cells were treated with LPS (1 µg/mL) to activate NF-κB/AP-1 and produce SEAP. After 24 h, 20 µL of the supernatant was withdrawn, mixed with 175 µL of QUANTI-Blue™ reagent and incubated for 30 min at 37°C. Spectrophotometric measurements at 655 nm were carried out in a Fluostar Omega Microplate Reader (BMG Labtech, Ortenberg, Germany).

Molecular docking

All of the simulations of ligand-protein docking on NF-κB were performed with AutoDockVina [START_REF] Trott | AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization and multithreading[END_REF]) using a PyRx virtual screening tool. Molecular dynamics were performed with a CUDA version of NAMD [START_REF] Phillips | Scalable molecular dynamics with NAMD[END_REF] and VMD was used to prepare the molecular visualization and protein preparation. Docking simulations were performed using system with Core I7 and NVIDIA GTX 850 in the same manner we described previously [START_REF] Leláková | Parallel in vitro and in silico investigations into anti-inflammatory effects of non-prenylated stilbenoids[END_REF]. The crystallographic coordinates of NF-ĸB bound to DNA were obtained from the RCSB Protein Data Bank with ID 3GUT. Only chain A (p65) and B (50) were used for simulations. All tested compounds meet the conditions of Lipinski rule of five, suggesting that tested compounds can penetrate cell membranes, pass into cell compartments, and have several chemical groups forming hydrogen bonds. All compounds formed complexes with target protein structure with high binding energy (ΔG) around -7.0 kcal/mol.

Western blot analysis of TLR4-mediated signaling pathways

THP-1-XBlue™-MD2-CD14 cells in 6-well plates at a density of 3 × 10 6 cells/well were treated with MF or the vehicle (Veh). After 1 h, LPS (1 µg/mL) was added to the cells.

Cells not stimulated with LPS represented the control (Ctrl). After 30 min, the cells were collected, washed in PBS, and homogenized in cold lysis buffer (50 mmol/L Tris-HCl, pH andA0545). The ECL signal (Bio-Rad, USA) was detected using a Syngene PXi4 chemiluminescence imaging system (Cambridge,UK). Densitometric analysis was performed using AlphaEaseFC 4.0.0 software (Alpha Innotech, USA). The relative effects of MF and vehicle (Veh) on IĸB-α were compared after normalization to β-actin.

Isolation of RNA and qRT-PCR

Total RNA was extracted using TRI Reagent ® (Sigma), followed by treatment with TURBO DNAse (Invitrogen). First-strand cDNAs were synthetized from 0.5 µg of total RNA using 200U/µL Superscript

Immunodetection of inflammatory cytokines

Differentiated THP-1 macrophages were pretreated for 1 h with the test compounds or prednisone 1 µmol/L (in the case of compound 1 0.1 µmol/L) dispersed in 0.1 % DMSO or with a 0.1 % DMSO solution alone (the vehicle). The release of TNF-α and IL-1β was evaluated by testing the supernatant after 24 h, using Human TNFα and IL-1β ELISA kits (Diaclone, France). For BV-2 cells, the expression of TNF-α and IL-1β was determined by testing cell contents and supernatants, using AlphaLISA mIL1β (AL503C) and mTNFα (AL505C) kits (PerkinElmer, MA, USA). The Amicon® Ultra 0.5mL filters (10K) for protein concentration (Merck Millipore, France) were used to concentrate supernatants before detection of IL-1β released from BV-2 cells. The total quantities of cytokines produced were calculated and normalized to the amounts of total proteins determined by Bradford protein assay (Biorad, France).

Statistical analysis

Statistical analysis was carried out using GraphPad Prism 6.00 software. Data were expressed as the mean±SEM. Statistical analyses of differences between groups were performed using the parametric (n>30) Student's t-test (for comparisons of 2 groups) and non-parametric (n<30) Mann-Whitney (for 2 groups), and Kruskal-Wallis analysis, together with Dunn's post-hoc test (for more than 2 groups). Values of p less than 0.05 were considered statistically significant.

Results

Effects of Macaranga stilbenoids on the viability of THP-1 cells

The prenylated stilbenoid MF has been found in the plant M. siamensis S. J. Davies, isolated by chromatographic methods, and identified by NMR analysis and other spectral methods [START_REF] Pailee | Cytotoxic and cancer chemopreventive properties of prenylated stilbenoids from Macaranga siamensis[END_REF]. We are the first to evaluate its effects on the viability of THP-1 human monocytes. Figure 1B shows that MF displays one of the safest profiles. Its IC 50 was calculated at 5.7±1.1 µmol/L. The IC 50 values of compounds 1-6 were 1.8±1.1, 3.3±1.1, 4.9±1.1, 12.8±1.1, 2.7±1.1, and 3.9± 1.1 µmol/L, respectively [START_REF] Hošek | Prenylated Stilbenoids Affect Inflammation by Inhibiting the NF-κB/AP-1 Signaling Pathway and Cyclooxygenases and Lipoxygenase[END_REF].

According to previous studies on humans evaluating the impact of oral supplementation of stilbenoids on their circulating concentration in plasma, targeting the concentration of 1 µmol/L that is accepted for the most studied stilbene resveratrol as lowest dose with a significant anti-inflammatory effect on peripheral cells [START_REF] Chen | Resveratrol alleviates lysophosphatidylcholine-induced damage and inflammation in vascular endothelial cells[END_REF]) is a pharmacologically and clinically relevant (Boocock et al., 2007;Smoliga et al., 2011).

Therefore to identify the most interesting anti-inflammatory candidate at this dose, we performed a non-exhaustive screening of the anti-inflammatory activities on stimulated macrophages of the seven compounds isolated from the plant M. siamensis.

MF reduces the secretion of inflammatory cytokines by acting on the IкBα/NF-кB pathway

We first evaluated and compared to prednisone, a corticosteroid used in the management of inflammatory conditions or diseases, the effect of the seven compounds isolated from the plant M. siamensis at the concentration of 1 µmol/L on the release of the pro-inflammatory cytokine TNF-α (Fig. 1C) and IL-1β (Fig. 1D) in PMA-differentiated THP-1 macrophages. Interestingly, MF reduced the secretion of TNF-α from macrophages stimulated by LPS by 22%, which was more than any of the other prenylated Macaranga stilbenoids (range of 3-16%) tested by [START_REF] Hošek | Prenylated Stilbenoids Affect Inflammation by Inhibiting the NF-κB/AP-1 Signaling Pathway and Cyclooxygenases and Lipoxygenase[END_REF]. This anti-inflammatory effect of MF was comparable to that of prednisone (24 %), a routinely used corticoid antiinflammatory drug (Fig. 1C). Moreover, a similar anti-inflammatory effect of MF was also observed on the secretion of IL-1β from macrophages stimulated by LPS (Fig. 1D). We next focused on the regulation of TNF-α and IL-1β upstream, particularly on the transcription factors NF-кB and AP-1, which concomitantly control the expression of several pro-inflammatory cytokines, including TNF-α and IL-1β. The canonical inflammatory pathway that was triggered by TLR4 and led to overexpression of TNF-α involved NF-кB/AP-1 activation [START_REF] Lawrence | The Nuclear Factor NF-κB Pathway in Inflammation[END_REF][START_REF] Macintyre | Activator protein 1 is a key terminal mediator of inflammation-induced preterm labor in mice[END_REF]. Again in this case MF showed one of the strongest inhibitory effects of the tested prenylated M. siamensis stilbenoids (Fig. 1E), with a therapeutic potential greater than that of prednisone. Overall at the clinically transferable dose of 1 µmol/L, the anti-inflammatory activities of compounds 4 and 5 are weaker than MF justifying it to be the most promising candidate for further evaluation of antiinflammatory and protective effects. Then, to gain some mechanistic insights we investigated MF effect on the upstream activation of NF-кB/AP-1 which includes IкBα [START_REF] Lawrence | The Nuclear Factor NF-κB Pathway in Inflammation[END_REF] and MAPKs (MacIntyre et al., 2014). Interestingly, MF reduced the degradation of IкBα, a direct inhibitor of NF-κB, but did not inhibit the activation (phosphorylation) of MAPKs known for leading to the activation of AP-1 (Fig. 1F). Therefore it is tempting to believe that MF could inhibit the inflammation in LPS-stimulated macrophages primarily through IкBα/NF-кB signaling, rather than by acting via the MAPK/AP-1 pathway.

Since inflammatory response of NF-κB is driven by its nuclear translocation and its binding activity, interfering with each of these steps or with both may account for MF antiinflammatory effect. Since IкBα primary role is to bind to NF-κB for preventing its translocation to the nucleus, our results showing MF potential to inhibit IкBα degradation suggests that it could interfere with NF-κB nuclear translocation. Therefore we further investigated whether MF could also directly interfere with NF-κB to dampen its binding activity. Thus, we evaluated the interaction of MF with NF-кB DNA binding site by simulating a ligand-protein docking into DNA binding pocket of the NF-κB heterodimer p65/p50 according the method by [START_REF] Trott | AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization and multithreading[END_REF] (Figure 1A). The hydroxyl groups of MF tend to form conventional hydrogen bonds (O˗H…O) with Thr502 and Ser508 residues. Nonclassical hydrogen bond (N˗H…π) was observed with Leu507. The alkyl hydrophobic interactions were detected with residues His441, Lys444, Val447, and Leu507. Two phenyl rings of MF typical for stilbene core tend to form π-alkyl hydrophobic bonds with Lys444 and Leu507 (Figure 2B and2C). Overall MF displayed a high affinity (ΔG = -6.7 kcal/mol) for NF-κB DNA binding site similarly to other biologically active stilbenes inhibiting the NF-κB/AP-1 pathway [START_REF] Leláková | Parallel in vitro and in silico investigations into anti-inflammatory effects of non-prenylated stilbenoids[END_REF]. Altogether our results suggest that MF may not only inhibit NF-κB activity by indirectly targeting its translocation preventing IкBα degradation but also directly by interfering with the DNA binding site of NF-κB.

Anti-inflammatory dose of MF does not alter the proliferation and viability of BV-2 microglial cells

We then tried to determine if this anti-inflammatory effect observed on monocytes could be extended to microglia, the immune cells of the brain. To evaluate whether the antiinflammatory effect of MF could be obtained on the safe side within a concentration range similar to the active one used for monocytes, we first performed a dose-response study to assess whether MF had an effect on the proliferation of BV-2 microglia (Fig. 2A) and their corresponding mitochondrial activity (Fig. 2B), that are accepted indicators of BV-2 vital cellular functioning. Not only changes in mitochondrial activity reflect an alteration of the energy homeostasis of the cell physiology often associated with a switch form physiological to pathological states, but mitochondria alteration is known for triggering inflammation and to play a central role in pro-inflammatory signaling and inflammatory response [START_REF] Meyer | Mitochondria: An Organelle of Bacterial Origin Controlling Inflammation[END_REF]. Therefore it was crucial to know whether and at which dose MF may alter the physiological functioning of BV-2. The high dose of 15 µmol/L altered the proliferation and activity of BV-2, but doses below 6 µmol/L had no negative effect (Fig. 2A-B). We therefore chose the concentration 1 µmol/L, previously identified and used to reduce the LPS-induced inflammatory response in monocytes, and used it for all subsequent experiments to evaluate the protective and anti-inflammatory potential of MF on microglia challenged by LPS [START_REF] Kacimi | Endotoxin-activated microglia injure brain derived endothelial cells via NF-κB, JAK-STAT and JNK stress kinase pathways[END_REF] At this concentration, pretreatment with MF decreased the LPS-stimulated loss of microglia by 20% relative to the vehicle after 24 h (Fig. 2C andD). This cellular protection was accompanied by a 20% reduction in the release of LDH by cells pretreated with MF as compared to cells treated only with the vehicle (Fig. 2D).

Pretreatment with MF dampens the LPS-induced pro-inflammatory response of BV-2 microglial cells

The important anti-inflammatory properties of MF at 1 µmol/L, established on LPSstimulated monocytes, along with its protective action for BV-2 brain immune cells, led us to investigate its anti-inflammatory action in reducing the production of IL-1β and TNF-α. MF reduced the TNF-α and IL-1β protein expression in both intracellular content and release of proteins at the basal level, with no harmful stimuli (Fig. 3A).

Moreover, BV-2 cells pretreated with MF showed an anti-inflammatory effect in the form of dampened BV-2 response to LPS-induced inflammation (Fig. 3D). Indeed, the intracellular content and release of both cytokines were also substantially reduced in cells pretreated with MF as compared to those treated only with the vehicle (Fig. 3D). This effect was correlated with a reduced level of gene expression for both pro-inflammatory cytokines:

1 h of pretreatment of BV-2 cells with MF significantly reduced the expression of LPSstimulated TNF-α and IL-1β mRNA by 26 % and 20 %, respectively (Fig. 3C).

Post-treatment with MF for resolving LPS-induced inflammation

Establishing the proof of the anti-inflammatory MF efficiency of MF as post-treatment is a mandatory steep toward clinical translation. Given as post-treatment, anti-inflammatory drugs are expected to resolve an ongoing inflammatory state by lowering the levels of cytokines such as TNF-α and IL-1β. After LPS challenge, the expression of such proinflammatory molecules is been initiated within a 3-6h time window, that also correlates with their in vivo expression, observed in severe acute CNS pathologies with massive inflammatory reaction, like stroke (Le [START_REF] Thuc | Central CCL2 signaling onto MCH neurons mediates metabolic and behavioral adaptation to inflammation[END_REF][START_REF] Nilupul Perera | Inflammation following stroke[END_REF]. As inhibition of inflammation is of interest to change the course of the pathology and improve stroke outcome, especially if starting the treatment after the recommended therapeutic window for thrombolysis with tPA (after 3-4.5 hours after the onset of stroke), we therefore investigated the effects of administering MF as a post-treatment, 3 or 6 h after the onset of LPS-induced inflammation (Figs. 5A and 5B, respectively) on TNF-α and IL-1β inflammatory response, observed after 6 h or 24 h, respectively.

The amount of TNF-α were found to be reduced by approximately 20 % in both the cellular content and external medium, whether MF was applied 3 or 6 h after LPS (Fig. 5A).

Treating the inflammation with MF 3 h after its induction reduced the IL-1β response to LPS found after 6 h, but treatment delayed by 6 h did not influence IL-1β levels 24 h after the onset of inflammation (Fig. 5B). This result may be explained by the fact that as IL-1β is known to be expressed rapidly during the first hours after LPS stimulation [START_REF] Kwon | The time-dependent effect of lipopolysaccharide on kainic acid-induced neuronal death in hippocampal CA3 region: possible involvement of cytokines via glucocorticoid[END_REF] the association of late post-treatment and a low dose of MF would be too stringent conditions to show any anti-inflammatory effect. Therefore, increasing the dose and/or shorten the timing of administration may be anticipated option to further investigate for revealing and improving the MF anti-inflammatory effect on this particular cytokine. Moreover while several group have shown that BV-2 cells produce detectable quantity of interleukin, in our settings, IL-1β was hardly induced and secreted as compared to TNF-α at the selected measurement times (6 and 24 h). In these stringent settings, it is most likely that the difference in MF effects at the dose of 1 µmol/L on TNF-α and IL-1β could be explained by the timing and level of the protein release in response to LPS challenge.

Discussion

Inflammation accompanies many acute and chronic pathologies at both the peripheral and CNS levels. Acute inflammatory diseases are mostly infectious, but inflammation may also be a part of non-infectious pathologies, such as stroke, traumatic brain injury [START_REF] Skaper | An Inflammation-Centric View of Neurological Disease: Beyond the Neuron[END_REF], and hemorrhage [START_REF] Goerge | Inflammation induces hemorrhage in thrombocytopenia[END_REF]. Chronic inflammation is usually characterized as a slowly progressing mild inflammatory reaction that is linked with several long-term disorders, including atherosclerosis [START_REF] Libby | Inflammation and Atherosclerosis[END_REF], inflammatory bowel diseases [START_REF] Vochyánová | Diplacone and mimulone ameliorate dextran sulfate sodiuminduced colitis in rats[END_REF], chronic obstructive pulmonary disease [START_REF] Barnes | Chronic obstructive pulmonary disease[END_REF], osteoarthritis [START_REF] Robinson | Low-grade inflammation as a key mediator of the pathogenesis of osteoarthritis[END_REF], or autoimmune disorders, such as allergic asthma [START_REF] Barnes | Chronic obstructive pulmonary disease[END_REF] or rheumatoid arthritis [START_REF] Smolen | Rheumatoid arthritis[END_REF].

Monocytes and microglia are major phagocytes of the innate immune system, providing the first line of defense. The presence of Toll-like receptors (TLRs) enables them to respond to different noxious stimuli, exogenous pathogen-associated molecular patterns (PAMPs) and some endogenous damage-associated molecular pattern molecules (DAMPs).

TLR4 is a major receptor implicated in the inflammatory response recognizing bacterial LPS [START_REF] Zhu | Toll-like receptor signaling pathways--therapeutic opportunities[END_REF]. Activation of TLR4 signaling leads to the expression of several proinflammatory cytokines, including TNF-α and IL-1β, mediated by the activation of NF-κB signaling pathway [START_REF] Pålsson-Mcdermott | Signal transduction by the lipopolysaccharide receptor, Toll-like receptor-4[END_REF]. Both TNF-α and IL-1β are potent regulators of the innate immune system important for host defense. Especially, there is an increasing evidence pointing out a prominent role of IL-1β in acute neuronal injuries and many chronic brain diseases [START_REF] Allan | Inflammation in central nervous system injury[END_REF]. Targeting the TLR4/NF-κB pathway therefore represents a potential therapeutic strategy for both acute and chronic inflammationbased diseases that are linked with abnormal secretion of cytokines [START_REF] Kuzmich | TLR4 Signaling Pathway Modulators as Potential Therapeutics in Inflammation and Sepsis[END_REF].

agreement with the ability of non-prenylated stilbenoids to reduce the phosphorylation of MAPKs [START_REF] Leláková | Parallel in vitro and in silico investigations into anti-inflammatory effects of non-prenylated stilbenoids[END_REF], whereas prenylated stilbenoids have been shown to affect the degradation of IкBα [START_REF] Hošek | Prenylated Stilbenoids Affect Inflammation by Inhibiting the NF-κB/AP-1 Signaling Pathway and Cyclooxygenases and Lipoxygenase[END_REF].

Like monocytes at the peripheral level, microglia and CNS infiltrated monocytes are responsive to neuronal damage. Their activation is an alarming sign of the brain pathology that can occur in conditions such as microbial infection, traumatic brain injury, or stroke, and also degenerative disorders of the CNS [START_REF] Dheen | Microglial activation and its implications in the brain diseases[END_REF]. In this work, we demonstrate the ability of MF to improve the in vitro native state of microglia by reducing the basal release of cytokines. This beneficial effect is associated with protection against LPS-induced loss of microglia.

Moreover, under inflammatory conditions, MF dramatically inhibits the LPSstimulated gene and protein expressions of both TNF-α and IL-1β, whatever the stage of treatment (pre-, co-, and post-treatment). The greatest reduction of cytokines (up to 48 %) is achieved when the microglia are pretreated with MF before inflammation is induced.

Although the up-stream signalization, such as level of NF-κB and IκB was not investigated in BV-2 cells, due to the phenotypic similarity of monocytes and microglia [START_REF] Orihuela | Microglial M1/M2 polarization and metabolic states[END_REF], it is tempting to anticipate that the response in mouse microglia may be similar. These results rivet attention on the potential for developing MF as a nutraceutical or dietary supplement, aimed at preventing any type of systemic and/or CNS inflammatory disorder, and thereby maintaining or improving organ function, preventing chronic disorders, promoting good health, or even delaying the process of aging [START_REF] Nasri | New concepts in nutraceuticals as alternative for pharmaceuticals[END_REF][START_REF] Tauskela | Failure and rescue of preconditioning-induced neuroprotection in severe stroke-like insults[END_REF].

In the acute and chronic states of inflammatory disease, the ability of MF to reduce the secretion of the cytokines TNF-α and IL-1β may be of particular interest, because these cytokines are known to be involved in the activation of resident microglia and astrocytes during infection, inflammation, and the development of brain injury [START_REF] Norden | Sequential activation of microglia and astrocyte cytokine expression precedes increased Iba-1 or GFAP immunoreactivity following systemic immune challenge[END_REF].

Their dysregulation and sustained activation can create a toxic circle, elevating the release of inflammatory factors that endanger the functioning and survival of neurons [START_REF] Ramesh | Cytokines and chemokines at the crossroads of neuroinflammation, neurodegeneration, and neuropathic pain[END_REF].

Our results show, that MF could be interesting as a supplementary compound used against chronic inflammatory diseases that would correspond to the so called "early phase of inflammation" in the present work. Low-grade systemic inflammation is like CNS inflammation (neuroinflammation), a pathological process that accompanies many persistent diseases and also the normal aging process nicknamed "inflammaging". Inflammaging is possibly related to chronic activation of the innate immune system and changes in the functions of monocytes [START_REF] Benedetto | Contribution of neuroinflammation and immunity to brain aging and the mitigating effects of physical and cognitive interventions[END_REF]. Altered activation of microglia, characterized by elevated levels of TNF-α and IL-1β, is associated with neurodegenerative disorders, such as AD, Parkinson's disease (PD), and Huntington's disease (HD), and is a reaction to the presence of cellular aggregates and the misfolded proteins β-amyloid, α-synuclein, and huntingtin, respectively. Reducing neuroinflammation could be a way to slow the progress of these diseases [START_REF] Sastre | Neuroinflammation in Alzheimer's, Parkinson's and Huntington's Diseases[END_REF]. Inflammation underlying neurodegeneration is also present in multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), systemic lupus erythematosus (SLE), neuromyelitis optica (NMO), diabetes mellitus (DM), chronic bacterial meningitis, Lyme neuroborreliosis, and HIV-1 encephalitis [START_REF] Amor | Inflammation in neurodegenerative diseases[END_REF][START_REF] Ramesh | Cytokines and chemokines at the crossroads of neuroinflammation, neurodegeneration, and neuropathic pain[END_REF].

Some stilbenoids, such as trans-resveratrol, isorhapontigenin, gnetol, and pinosylvin, have shown significant potential to inhibit butyrylcholine esterase (BChE) enzyme, the increased activity of which is associated with the formation of Aβ plaque in patients with AD [START_REF] Sermboonpaisarn | Potent and selective butyrylcholinesterase inhibitors from Ficus foveolata[END_REF]. Oxyresveratrol exerts neuroprotective effects against Parkinsonian mimetic 6-hydroxydopamine (6-OHDA)-induced neurotoxicity in neuroblastoma cells [START_REF] Chao | Dietary oxyresveratrol prevents parkinsonian mimetic 6-hydroxydopamine neurotoxicity[END_REF]. Studies of the "Mediterranean diet", which is based on vegetables, fruits, whole grains, nuts, fish, and seafood, healthy fats, and olive oil, with moderate consumption of red wine, and is rich in polyphenols, including stilbenoids, have reported beneficial effects, mitigating age-related and other neurodegenerative pathologies [START_REF] Hornedo-Ortega | Phenolic Compounds Characteristic of the Mediterranean Diet in Mitigating Microglia-Mediated Neuroinflammation[END_REF].

The anti-inflammatory effects of MF may also be of interest for acute conditions such as cerebral ischemia, in which reduced blood flow and oxygen supply (hypoxia) lead to cerebral infarction. This condition triggers an intravascular inflammatory cascade that extends into the surrounding tissues of the injured brain, and is characterized by elevated levels of inflammatory cytokines, chemokines, adhesion molecules, reactive oxygen species, nitric oxide, extracellular proteases, and other substances flowing into and accumulating in immune cells, such as microglia [START_REF] Thuc | The complex contribution of chemokines to neuroinflammation: switching from beneficial to detrimental effects[END_REF]. A therapeutic strategy that intervened in the inflammatory process of cerebral ischemia might slow down and limit the brain damage caused by ischemia. The potential of stilbenoids to act against cerebral ischemia has been,

however not thoroughly, investigated. It is noteworthy that a study using a transient rat middle cerebral artery occlusion model has shown that oxyresveratrol reduces the size of volume of an infarction and thus prevents neurological deficits induced by the ischemia/reperfusion (I/R) injury, inhibits the release of cytochrome c, and prevents the activation of caspase-3 [START_REF] Andrabi | Oxyresveratrol (trans-2,3',4,5'-tetrahydroxystilbene) is neuroprotective and inhibits the apoptotic cell death in transient cerebral ischemia[END_REF]. This indicates that as strong anti-oxidant and anti-inflammatory agents, stilbenoids could be effective in the treatment of I/R-injury, a devastating pathology without therapeutic opportunity.

Despite their relatively wide range, most of the anti-inflammatory drugs available on the market are linked with many side effects when used in the long term. The therapeutic and adverse effects of the most commonly prescribed non-steroidal anti-inflammatory drugs (NSAIDs) are related to the inhibition of cyclooxygenases (COXs). Their gastrointestinal, renal, and cardiovascular toxicity, together with numerous drug interactions, represent a risk especially for elderly patients who take multiple medications [START_REF] Wongrakpanich | A Comprehensive Review of Non-Steroidal Anti-Inflammatory Drug Use in The Elderly[END_REF].

Steroidal drugs can have pleiotropic and severe adverse effects [START_REF] Aljebab | Systematic Review of the Toxicity of Long-Course Oral Corticosteroids in Children[END_REF].

Biological treatment in the form of anti-TNF-α and anti-IL-1β antibodies has been shown to be highly effective for chronic disorders such as RA, especially when treatment is started in the early phase of the disease. However, this treatment represents an enormous financial burden for the health care system and is not accessible to all patients [START_REF] Dinarello | Treating inflammation by blocking interleukin-1 in a broad spectrum of diseases[END_REF][START_REF] Monaco | Anti-TNF therapy: past, present and future[END_REF]. Thus, nutritional intervention and the development of functional food based on natural molecules that are not known to have profound side effects alter promise of preventing inflammation caused by several chronic and acute diseases. Many good candidates may be hidden in natural sources. MF would be among the most promising thanks to its ability to counteract both peripheral and CNS inflammation. MF is structurally a small molecule, effective at a pharmacologically relevant concentration. Synthesizing it in the laboratory could lower its production cost as compared to existing commercial treatments.

MF and its derivatives could fulfill the goal of finding a novel anti-inflammatory substance that is potent in prevention and/or supplementary treatment of inflammation-based pathologies of both systemic and central character and safe for long-term use.

Conclusion

Our current study shows that stilbenoid macasiamenene F isolated from a plant M.

siamensis has significant anti-inflammatory effects. It is therefore an interesting candidate for further investigation using in vivo models where inflammation in the brain plays a major role, e.g., cerebrovascular diseases. The increasing prevalence of chronic inflammatory disorders and more frequent occurrence of acute inflammatory diseases make the need for safe and effective anti-inflammatory therapeutic strategies. Other promising candidates may yet be found in natural sources, but with respect to the current state-of-the-art in nutraceuticals, MF

  7.5; 1 mmol/L EDTA; 1mmol/L EGTA; 1 mmol/L sodium orthovanadate; 5 mmol/L sodium pyrophosphate; 50 mmol/L sodium fluoride; 270 mmol/L sucrose) with protease inhibitors (Roche Diagnostics, Basel, Switzerland). Equal amounts of proteins (9 µg) were separated in 12 % polyacrylamide gel and transferred to a PVDF (polyvinylidene fluoride) membrane. This membrane was then incubated overnight at 4°C with the following monoclonal primary antibodies: anti-IĸB-α (1:500; Cell Signaling, USA, product No. 4814), anti-β-actin (1:5000; Abcam, Cambridge, UK, No. 8226), anti-SAPK/JNK (1:1000; Sigma-Aldrich, USA, SAB4200176), antiphospho-p44/42 MAPK (p-ERK1/2, 1:2000, No. 4370), anti-p44/42 MAPK (ERK1/2, No. 4695), anti-phospho-p38 MAPK, and anti-phospho-SAPK/JNK (1:1000; Cell Signaling, MA, USA, No. 4511 and 4668) and polyclonal antibody: anti-p38 MAPK (1:1000; Cell Signaling, MA, USA, 9212). Each membrane was then probed with the appropriate anti-mouse or anti-rabbit IgG secondary peroxidase-conjugated antibodies (1:2000, from Sigma-Aldrich, USA, No. A0168

Figure 1 :

 1 Figure 1: Effects of prenylated M. siamensis stilbenoids on NF-κB signaling of LPS-stimulated THP-1 macrophages and THP-1 XBlue™-MD2-CD14 monocytes. A, Test stilbenoids MF, 1-6; B, Dose-response curves showing viability of THP-1 after a 24 h incubation with compounds MF, 1-6; C, Effect on release of TNF-α protein; D, Effect on release of IL-1β protein; E, Activation of NF-кB/AP-1 after 24 h; F, MF upstream regulation of IкBα. Data are presented as the mean±SEM, n=3, *indicates a significant difference of *P<0.05, **P<0.01, ****P<0.0001 versus only with the vehicle treated cells (Veh) analyzed by the non-parametric tests Kruskal-Wallis in the case of C and D, and the Mann and Whitney for E.

Figure 2 :

 2 Figure 2: Molecular docking pose of MF interactions with NF-κB (PDB 3GUT). A, The bond of MF to p50 subunit of NF-κB DNA binding pocket. Red -MF, green ribbon -p50 subunit, blue ribbon -p65 subunit, orange double helix with colored sticks -DNA fragment; B, Interactions of MF with p50 NF-κB depicting hydrogen bonds (green-classical, grey-non-classical) and hydrophobic interactions (dark and light purple); C, MF forms interactions with p50 residues Thr502, Ser508 (classical hydrogen bonds), Leu507 (non-classical hydrogen bond and hydrophobic interactions), His441, Lys444, and Val447 (hydrophobic interactions).

Figure 2 :

 2 Figure 2: Determination of the dose-limiting toxicity of MF and its protective effect against inflammatory challenge in BV-2 cells. A, Effect of a 24 h-application of MF at concentrations of 1, 2, 6, and 15 µmol/L on the proliferation of cells assessed by cell counting, * indicates P<0.05, significant difference in comparison with BV-2 cells treated only with the vehicle (Veh.); B, Effect of a 24 h-application of MF on the mitochondrial activity, *** indicates P<0.001 compared to Veh.; C, Representative snapshots (Scale bar = 150 µm) showing the MF-induced improved survival of BV-2 cultures injured by a 24 h-LPS challenge; D, Quantitative assessment of the beneficial effect of MF on BV-2 cultures injured by a 24 h-LPS challenge, as determined by cell counting and the extent of LDH release, *** indicates P<0.001 compared to the respective LPS non-stimulated control, #P<0.05 and ###P<0.001 indicate significant differences between MF+LPS vs. Veh.+LPS. Data are presented as the mean±SEM; n=6 measured in sextuplicates, and analyzed by Student's t-test.

Figure 3 :

 3 Figure 3: Anti-inflammatory effects of MF pre-treatment on the expression of TNF-α and IL-1β. A, MF reduced the basal expression and release of TNF-α and IL-1β by BV-2 cells; B, Schema illustrating the protocol of pre-treatment of BV-2 cells with MF (1 µmol/L) and the timing of evaluation of its effect on the mRNA and protein expression: C, MF reduced the LPS-stimulated expression of TNF-α and IL-1β mRNA; D, MF reduced the LPS-induced expression and release of TNF-α and IL-1β by BV-2 cells. *P<0.05, **P<0.01, ***P<0.001 vs. Veh. group. Data are expressed as the mean±SEM, n=3, measured in sextuplicates, analyzed by the non-parametric Mann and Whitney test for two independent groups.

Figure 4 :

 4 Figure 4: Anti-inflammatory effects of MF cotreatment with LPS in the BV-2 cell line and the ex vivo culture of microglia cells. A, Schema illustrating the cotreatment of BV-2 cells and primary mouse microglia with MF (1 µmol/L) co-treatment with LPS (1µg/mL) together with the times at which the cytokines were measured: 6 h and 24 h for the BV-2 cell line, and 18 h for the ex vivo culture of microglia cells; B, TNF-α expressed in the cell content and protein release from the BV-2 cells at 6 and 24 h; C, IL-1β expression in cell content and protein release from BV-2 cells at 6 and 24 h; D, TNF-α and IL-1β protein contents in brain-sorted mouse microglia after 18 h of cotreatment of MF with LPS or only the vehicle. Data are expressed as the mean±SEM, n=3, measured in sextuplicates, analyzed by the Mann-Whitney test. *P<0.05, **P<0.01 vs. Veh. group.

Figure 5 :

 5 Figure 5: Anti-inflammatory effects of posttreatment with MF. Schema illustrating the protocol of inducing inflammation with LPS (1 µg/mL) in BV-2 cells and treating them 3 h (A) or 6 h later (B) by applying MF post-treatment (1 µmol/L). A, Effect of MF applied 3 h after the induction of inflammation by LPS. MF post-treatment reduced both the protein content and release of the TNF-α and the IL-1β as evaluated 6 h after the application of LPS. B, When the MF posttreatment was effectuated at 6 h after the induction of inflammation by LPS, the anti-inflammatory effect of reduced TNF-α protein content was still found 24 h after the application of LPS, but that of the IL-1β protein was not. That transitory expression of IL-1β apparently occurred entirely before the post-treatment was applied. Data are expressed as the mean±SEM, n=3, measured in sextuplicates, analyzed by the Mann-Whitney test. *P<0.05, **P<0.01, ***P<0.001 vs. Veh. group.

Acknowledgment

We acknowledge Dr. Agnès Petit-Paitel and Julie Cazareth from IPMC CNRS for constructive discussions concerning the ex vivo experimental set-up. We thank to Dr. Frank Thomas Campbell for the reading and editing the manuscript.

Sources of funding

This project was supported by the Czech Grant Agency (GACR), grant no. 16-07193S, and the Ministry of Education, Youth and Sports of the Czech Republic, NPU I (Grant no. LO1305). This work was further supported by the Centre National de la Recherche Scientifique and the LabEx ICST #ANR-11 LabEx 0015 (France).

MF dampened the early stage of inflammation in a co-treatment with LPS

The anti-inflammatory effect of pretreatment with MF is of great interest for nutritional intervention and the development of functional foods. But the fact that pretreatment with MF would need to be applied pre-emptively limits its clinical usefulness, particularly for the situations arising without warning. Therefore, we investigated the possible therapeutic use of MF as an anti-inflammatory drug during (a cotreatment protocol) or after (a posttreatment protocol) the establishment of inflammation.

To study the effect of MF in the early phase of inflammation, we assessed the cellular content and protein release of the inflammatory cytokines TNF-α and IL-1β in a culture medium of BV-2 cells after 6 h and 24 h of cotreatment with MF and LPS. Co-application of MF and LPS reduced both the intracellular level and the release of TNF-α which are normally induced by the application of LPS (Fig. 4). Marked inhibition (33 %) of the LPS-induced TNF-α up-regulation was already observed after 6 hours of cotreatment. This antiinflammatory effect clearly influenced the level of TNF-α release, which was decreased by 26 % after 24 h (Fig. 4B). Similarly, severe inhibition of the IL-1β response to LPS was reflected in both the cell content and the culture medium after 6 and 24 h of cotreatment (Fig. 4C). We then determined the extent to which the beneficial effects observed in the BV-2 cell line could be extended to brain-sorted primary microglia (Fig. 4D). Whereas under the applied experimental conditions cotreatment with MF seemed not to be efficiently reduce the level of TNF-α in a statistically significant manner. Cotreatment with MF lowered the level of IL-1β in primary microglia by factor of five, as compared to the vehicle (Fig. 4D).

Plant-derived stilbenoids have demonstrated a promising ability to attenuate peripheral [START_REF] Dvorakova | Anti-inflammatory activity of natural stilbenoids: A review[END_REF][START_REF] Eräsalo | Natural Stilbenoids Have Anti-Inflammatory Properties in Vivo and Down-Regulate the Production of Inflammatory Mediators NO, IL6, and MCP1 Possibly in a PI3K/Akt-Dependent Manner[END_REF][START_REF] Leláková | Parallel in vitro and in silico investigations into anti-inflammatory effects of non-prenylated stilbenoids[END_REF] and CNS inflammation [START_REF] Akinwumi | Biological Activities of Stilbenoids[END_REF][START_REF] Hornedo-Ortega | Phenolic Compounds Characteristic of the Mediterranean Diet in Mitigating Microglia-Mediated Neuroinflammation[END_REF] in vitro and in vivo.

However, clinical trials to investigate their potential in humans are still needed. Pterostilbene, a natural derivative of trans-resveratrol, that has frequently been investigated, has shown the ability to inhibit platelet aggregation ex vivo [START_REF] Messina | Effect of substituted stilbenes on platelet function[END_REF], improve high fat-induced atherosclerosis inflammation via NF-κB signaling in vivo [START_REF] Zhang | Pterostilbene, a novel natural plant conduct, inhibits high fatinduced atherosclerosis inflammation via NF-κB signaling pathway in Toll-like receptor 5 (TLR5) deficient mice[END_REF], protect against myocardial ischemia-reperfusion (I/R) injury by reducing oxidative/nitrosative stress and the inflammatory response to it [START_REF] Yu | Pterostilbene protects against myocardial ischemia/reperfusion injury via suppressing oxidative/nitrative stress and inflammatory response[END_REF], and reduce the size of a myocardial infarction [START_REF] Wu | Protective Effects of Pterostilbene Against Myocardial Ischemia/Reperfusion Injury in Rats[END_REF]. At the CNS level, pterostilbene protects against hyperglycemia- [START_REF] Yang | Pterostilbene attenuates high glucose-induced oxidative injury in hippocampal neuronal cells by activating nuclear factor erythroid 2-related factor 2[END_REF] and glutamate-induced neuronal oxidative injury [START_REF] Wang | Neuroprotective effects of pterostilbene against oxidative stress injury: Involvement of nuclear factor erythroid 2-related factor 2 pathway[END_REF]. In contrast, the stilbenoids of Macaranga spp. have not yet been thoroughly investigated.

Among these compounds, MF displays a safe profile for therapeutic use, with low cytotoxic effects on THP-1 cells and on the BV-2, MOLT-3, HepG2, HuCCA-1, and A549 cell lines [START_REF] Pailee | Cytotoxic and cancer chemopreventive properties of prenylated stilbenoids from Macaranga siamensis[END_REF]. We focused on the activity of the transcriptional factors NF-кB and AP-1, both of which have been shown to concomitantly control the expression of various inflammatory cytokines during inflammatory response [START_REF] Macintyre | Activator protein 1 is a key terminal mediator of inflammation-induced preterm labor in mice[END_REF]. Whereas NF-кB is mostly responsible for the gene and protein expressions of TNF-α and IL-1β and is upregulated by its direct cytoplasmic inhibitor IкBα [START_REF] Lawrence | The Nuclear Factor NF-κB Pathway in Inflammation[END_REF], AP-1 is controlled predominantly by ERK1/2, p38, and JNK, members of the family of mitogen-activated protein kinases (MAPKs) [START_REF] Zhou | Effect of TLR4/MyD88 signaling pathway on sepsisassociated acute respiratory distress syndrome in rats, via regulation of macrophage activation and inflammatory response[END_REF]. In the present work, we reveal the promising potential of MF against peripheral inflammation by demonstrating its ability to: 1) intervene in the IкBα/NF-кB pathway and inhibit the degradation of IкBα, which leads to decreased activation of the transcriptional factor NF-кB, and 2) reduce secretion of inflammatory cytokine TNF-α, which is under the transcription control of NF-кB. This result is in is the most likely to prove being effective for the prevention and treatment of inflammationrelated pathologies, including secondary brain injury following a stroke, for which a therapeutic opportunity is severely lacking.

Author Contributions

Conflict of interest

The authors declare to have no conflict of interest related to this publication and there has been no financial support for this work that could have influenced its outcome.