Mecanismo molecular y farmacocinético del efecto de los flavonoides en inflamación intestinal

Laburpena

1. INTRODUCTION Inflammatory Bowel Disease (IBD) is a chronic relapsing inflammatory disorder of the gastrointestinal tract that encompasses two idiopathic and major inflammatory diseases: Crohn's disease and ulcerative colitis. Both forms of IBD significantly impair quality of life, and require prolonged medical and/or surgical interventions. What makes it particularly challenging is its still unknown cause, its unpredictable presentations and symptoms, the less than optimal treatments, and a rise in its incidence and prevalence in many areas of the world. Several nutraceutical agents have been described to present intestinal antiinflammatory activity. Flavonoids are polyphenolic compounds of natural origin that are consumed as part of the normal human diet. As much as 200-500 mg may be ingested daily in Western countries (1, 2). There are many different flavonoids in nature, which differ in their degree of oxidation, substituents, polymerization, etc. Many different biological properties have been ascribed to these compounds, including immunomodulatory/antiinflammatory activity (3). Quercetin is a major flavonoid species in vegetables included in the human diet, although it is mostly present in its glycosylated forms, such as quercitrin and rutin. Rutin is a quercetin rhamnoside whose antiinflammatory properties have been documented using in vivo experimental models of IBD, asthma and rheumatoid arthritis (3). Since flavonoids in general, and rutin in particular, are considered to have very low toxicity, they may be valuable alternatives for the management of intestinal inflammation. A number of aglycones, including apigenin, kaempferol, daidzein, genistein or crhysin have been shown to exert intestinal antiinflammatory effects in preclinical models of IBD (4-11). However, a characteristic regarding the use of quercetin is that oral administration of this flavonoid is essentially inactive on experimental models of colitis (12, 13). Instead, the glycoside derivatives of quercetin, rutin (13) and quercitrin (12, 14) exert significant effects in similar in vivo models. The commonly accepted hypothesis is that these compounds are hydrolysed by bacterial enzymes in the colon to yield quercetin (12, 15). Thus rutin (and quercitrin) would act effectively as a prodrug, preventing premature absorption of the flavonoid in the small intestine but releasing it in the colon, where quercetin antiinflamatory ability goes into action. According to this hypothesis, quercetin should be therapeutically active if administered intrarectally, and indeed this is what happens (15). Quercetin is also predicted on this basis to be effective in ileitis, while rutin ought to be inactive. In this study we set out to verify mechanistic aspects of rutin intestinal antiinflammatory action by assessing the influence of the inflammatory site (ileitis vs. colitis), and route of administration (p.o. vs. i.p.) on its anticolitic effect, plus the levels of the flavonoid in the intestinal tissue. In general, flavonoids target different cell types including macrophages, lymphocytes or intestinal epithelial cells (IEC) and regulate the expression of a plethora of inflammation related molecules. The antiinflammatory effect of many flavonoids has been largely connected to the inhibition of NF-¿B and MAPKs. Nevertheless, stimulation of these signal transduction pathways by those has been observed in basal, noninflammatory conditions (3, 16). In spite of flavonoids share that profile in most of cellular populations, the information available on IEC is more diverse. The intestinal mucosa, the innermost layer of the intestine, plays an important physiological role by mediating water and nutrient transport and acting as interphase with the complex luminal milieu, which comprises a combination of diverse bacteria and their products as well as derivative products of the diet. Specific perturbation of the intestinal epithelium can lead to intestinal inflammation (17). Thus intestinal epithelial cells (IEC) express various immune receptors and accordingly, they can produce a wide array of immunomodulatory substances such as cytokines and complement factors. Intestinal epithelial cells express cyclooxygenase (COX2) when stimulated by pro-inflammatory factors, including lipopolysaccharide (LPS), tumour necrosis factor-¿ (TNF-¿), oxidative stress, etc. (18). Epithelial prostaglandins seem to be involved in the resolution of inflammation and the healing process (19) as well as in intestinal homeostasis (20). Hence it is possible that agents that promote COX2 induction could be useful in the therapy of inflammatory bowel disease by hastening the resolution of the inflammatory process. Because of the need of clarify the effects of flavonoids on IEC and the mechanism of action of these flavonoids we study the effects and structure-activity relationship of nine different flavonoids (Table 1) on COX-2 expression in IEC18 cells, a non-tumour model IEC line. The different categories of flavonoids assayed differ mainly in the presence or absence of a double bond between C2 and C3, the 3-hydroxyl, and the position of the phenol group (also known as B ring). The substitutions in these basic structures give rise to the different flavonoid compounds. The role of microbiota in inflammatory bowel disease has been long established. Sensing microbiota antigens involves the stimulation of a series of non specific receptors that mediate the innate immunity. These receptors are termed pathogen-recognition receptors (PPRs) and bind, not specific molecules, but types of molecules that substantially differ from those of eukaryotic cells. PPRs comprise Toll-like receptors (TLRs), nucleotide-binding and oligomerization domains (NODs)-like receptors (NLRs) and the helicase family (retinoic inducible gene I (RIG-I) and differentiation associated gene or MDA5). Although activation of NOD2 and TLRs leads to an inflammatory response, mainly mediated by NF-¿B and MAPK, the role and regulation of these receptors is much more complex since their absence leads in many cases to intestinal inflammation, as described for NOD2 mutations in Crohn¿s disease patients or in knockout mice for different TLRs. IL-1ß and TNF-¿ are proinflammatory cytokines of the innate immune system whose effects are mediated though their respective receptors. As in the case of NODs, the MAPK and NF-¿B are activated after the stimulation of these receptors. Inhibition of the activation of these receptors has been widely used as a therapeutic tool. In fact, antibodies against TNF are successfully used to treat inflammatory conditions like inflammatory bowel disease or rheumatoid arthritis. In order to characterize the effect of flavonoids in modulating the activity of different receptors that finally signal through NF-¿B and MAPK we chose to study two proinflammatory cytokines monocyte chemotactic protein-1 (MCP-1) and chemokine C-X-C motif ligand 1 (GRO-¿ or CXCL-1) and the regulation of TLR2, 4, 5, 9, TNF receptor (TNFR), IL1 receptor (IL1R) and NOD2. Furthermore we aimed to ascertain the structural requirements for these effects. 2. AIMS Based on all the above, we proposed 3 main objetives in this Doctoral Thesis: 1. To study the rutin and quercetin pharmacokinetic and their anti-inflammatory effect relationship on small and large intestine. 2. To validate the anti-inflammatory effect of rutin, by using a chronic model of intestinal inflammation T lymphocyte-dependent. 3. To characterize the molecular and cellular mechanism of action, and structure-activity relationship of flavonoids. 3. MATERIAL AND METHODS To carry out these objectives, we use a number of techniques, including tissue culture techniques, RT-qPCR, magnetic cell separation, histology techniques, flow cytometry, shRNA gene silencers, gel electrophoresis and ELISAs as well as experimental models of colitis (TNBS, DSS, lymphocyte transfer model). 4. RESULTS Our results can be divided into two main parts: IN VIVO. Rutin is active in both ileitis and colitis, while quercetin showed only marginal effects. Furthermore, rutin retained much of its colonic antiinflammatory activity when administered by the intraperitoneal rather than the oral route. Parallel experiments carried out in noncolitic rats showed that oral rutin gavage results in significant levels of both rutin and quercetin in the ileal mucosa, while only quercetin was detected in the colonic mucosa. In contrast, i.p. rutin administration increases the levels of both rutin and quercetin in the ileum and colon alike. Orally administered quercetin was found to reach both the ileal and colonic mucosa. To validate the anti-inflammatory effect of rutin, we carry out an in vivo experiment in a T cell transfer colitis model. Treatment of mice with 57 mg/Kg rutin improved colitis, as evidenced by a lower body weight loss (-0.2 ± 1.1% vs. ¿1.8 ± 0.8%, Rut vs. Control, p<0.05), colonic myeloperoxidase (42.9 ± 6.6 vs. 64.3 ± 6 mU·mg protein-1, p<0.05) and alkaline phosphatase activities (76.5 ± 12.4 vs. 117.1 ± 10.2 mU·mg protein-1, p<0.05). In addition, a decreased secretion of proinflammatory cytokines (IFN-¿, IL-17, IL-6 and TNF-¿) by mesenteric lymph node cells was observed ex vivo. The colonic expression of proinflammatory genes including IFN-¿, TNF-¿, CXCL1, S100A8 and IL-1ß was significantly reduced with rutin as assessed by RT-PCR. Rutin treatment additionally ameliorated ion transport and reduced activation of STAT4 and IFN-¿ in the spleen. The 28.5 mg dose resulted only in lower body weight loss and reduced TNF-¿ release by mesenteric lymph node cells. IN VITRO. ¿ Effects of flavonoids on enterocytes The effect of flavonoids on COX2 expression by IEC18, depended on the experimental conditions tested [non-stimulated and lipopolysaccharide (LPS)-stimulated]. Flavonoids caused an increase in COX2 expression and NF-¿B-dependent gene transcription under basal conditions. Conversely, under LPS stimulation flavonoids increased, decreased or did not affect COX2 levels depending on the specific type. Variable effects were observed on extracellular signal regulated kinase/p38/c-Jun N-terminal kinase phosphorylation and p50/65 nuclear translocation. In order to asses the effects of flavonoids on enterocyte cytokine secretion, we studied in IEC18 cells, the structure/activity relationship of 9 flavonoids on the regulation of Toll Like Receptors (TLR) 2, 4, 5, and 9, the Nucleotide-binding and Oligomerization Domain 2 (NOD2), the TNF receptor (TNFR) and the IL-1ß receptor (IL-1R). Chrysin and diosmetin alone stimulated GRO-¿ and MCP-1 production. Quercetin, daidzein and genistein inhibited it. Silencing TLR4 and MyD88 with sh-mRNA in IEC18 indicated that the effect of chrysin was independent of TLR4 but depended of MyD88. While flavones only inhibited TLR stimulated GRO-¿ and MCP-1 production, isoflavones inhibited the activation of all the receptors. Finally, flavonols had a lower inhibitory action than their correspondent flavones. ¿ Effects of flavonoids on T lymphocytes Previous studies have been established a proapoptotic/antiproliferative effect of several flavonoids on splenocytes at 50 ¿M under concanavalin A induction. Because lymphocytes are the main population on splenocytes and because concanavalin A is a T cell receptor unspecific activator, we tested the possible immunomodulatory effects of quercetin and its glycoside, rutin, on immunomagnetic isolated T cells at non toxic concentrations. Under these conditions, quercetin only showed lymphocyte immunomodulation in an epithelium-dependent manner; however, rutin showed antiproliferative effects on immunomagnetic isolated T cells. None of the mediators tested seems to be the responsible of those epithelium-dependent effects of quercetin. 5. CONCLUSIONS 1. Rutin shows intestinal antiinflamatory activity in both the colon and ileum, which is favored by oral administration of the flavonoid. 2. The advantage of rutin vs. quercetin in this regard is not related so much to prevention of absortion in the small intestine as to lower metabolization coupled to luminal secretion by the intestinal epithelium, thus extending quercetin presence in the lumen. 3. Rutin is active in a T cell colitis model, giving an additional validation of its therapeutic efficiency. 4. Quercetin exerts antiproliferative effects on rat T lymphocytes at concentrations higher than 30 µM and immunomodulatory effects at lower levels, potentiated by a paracrine action on IEC18 cells and, arguably, on the intestinal ephitelium in general. Conversely, rutin has no major direct effects on IEC18 cells or rat T lymphocytes. In vivo data support the biological relevance of this mechanism in the antiinflammatory effect of rutin. 5. 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