| Over the past decade, there has been increasing recognition of an association between the pathophysiology of inflammatory bowel disease (IBD) and intestinal barrier function. IBD patient’s intestinal barrier function compromised. Barrier dysfunction can predispose and contribute to the pathogenesis and progression of IBD, and increase the risk of IBD patients relapse. Studies spanning the past3decades demonstrated that methionine restriction (MetR) slowed the rate of aging and extend the average maximal life span. One theory was that MetR diet in rodents decreased mitochondrial ROS (mtROS) production and oxidative damage to mitochondrial DNA and proteins thus result in a low rate of generation of endogenous damage, and a macromolecular composition highly resistant to oxidative modification. The level of interest had been increasing in using MetR as an adjunct to conventional cancer chemotherapy, MetR diet seemed to tumor-suppressing activities as demonstrate by cell culture and clinical studies. It had been very well established that MetR diet reduces the paracellular permeability, lead to improve epithelial barrier function by altering tight junction protein composition. Patients with Crohn’s disease on the MetR diet (4-week meat-and fish-free diet) showed significant change in CDAI scores before vs. end of the diet. However it did not show the mechanism and whether MetR diet also work on ulcerative colitis patients. The intestinal epithelial cell layer and cell conjunction constitute the first barrier against the external environment. Thus, we hypothesize that to increase the intestinal barrier function benefit for maintaining the immune balance and stimulating the reparation of the mucosa.. In such case, we hypothesize:Methionine diet seems to be benefit for increasing resistant to oxidative stress and the overall health and function of an organism; thus we deduce that MetR diet many also be benefit for the enhancement of barrier function of IBD patients and decreasing the sverity of inflammation.Objective:Methionine restriction diet alters tight junction (TJ) structure and composition, decreases TJ permeability and thereby enhances epithelial barrier function. We hypothesize that specifically methionine restriction will induce improved barrier function at the TJ level across gastrointestinal epithelial tissues of inflammatory bowel disease (IBD), thereby improve their gastrointestinal barrier fuction. Thus we study the effect of methionine restriction (MetR) on mucosal repair and tight junction composition in a model of inflammatory bowel disease (IBD) model. And to analysis and discuss the possibility of application of methionine restriction in IBD patients.Methods:Rats consumed3%dextran sulfate sodium (DSS) in drinking water for6days, followed by tap water. During the recovery period, the rats were provided with Metro diet containing0.17%methionine. The rats were fed a complete amino acid (AA), or Metro diet divided in to4groups:DSS+MetR, DSS+AA, MetR and AA, each group had15rats. Morphological changes in colonic mucosa were evaluated and scored by light electronmicroscopy; Myeloperoxidase (MPO) activity was measured; the effect of MetR on paracelluar permeability of colon strips was detected by Ussing chamber. RT-PCR、western blot and immunofluorescence technique analysis the tight junction protein expressions, and claudin2, occludin, claudin3, ZO-1expression was also quantified in immublots and western blot.Result:①The early clinical findings in the DSS treated rats occurred on day2, such as loose stool or diarrhea, hemoccult positive. The maximal disease activity was on day13. The DSS treated rats showed a29.01%weight loss(n=15,, P<0.01)compared to controls. And the whole colon length appeared to be significant shorter. HE observation showed prominent colitis in distal colon. On day7, DSS treated rats characterized by dilations of gland crypt, ulcer, and inflammatory cell infiltration, the inflammatory score were17.95±2.62, n=6; The most severity showed on dayl4(inflammatory score were22.01±4.69, n=6, DSS treated rats showed transmural inflammation, extension ulceration, the crypts showed more dilation. On day21, the treated rats still showed crypt dilation, focal erosion and lymph follicle experiment hyperplasia. inflammatory score were15.00±4.89, n=9②Rats on MetR diet group showed a decrease of body weight(P<0.01), But there was no significant difference of serum album and plasma electrolytes and blood creatinine level;③HE observation showed prominent colitis in distal colon, Although MPO activity and WBC account between the DSS+MetR and DSS+AA group did not significant changed; treatment with MetR diet significantly decreased the extent and severity of epithelial injury(n=9, P=0.01.④Colon strip from rats on MetR diet showed increased TEER (n=6, P=0.007), however, rats from DSS+MetR group did not show significant changes on TEER but markly decreased Isc(n=6, P=0.046.⑤DSS induced claudin2expression. RT-PCR and Western blot both showed an increase in the abundance of claudin3but decreased the amount of claudin2in DSS+MetR group. There was no significant difference of the expression of occluding and ZO-1.Conclusion:1. DSS treated rats showed an active inflammation during the early stage, and then chronic reparation. In terms of symptoms and pathologic findings, DSS induced colitis in SD rats shared most feature with classic experimental IBD model. The establishment of IBD mode is successful;2. The intestinal epithelial barrier function compromised and an alteration tight junction complex protein expression in rat intestine in the experimental IBD model. The tight junction protein may played an very important role in barrier dysfunction because its transcription and translation were markly changed;3.Over all, Low methionine diet leads to improve epithelial barrier function, The tight junction proteins play an very important role on dietary restriction because its transcription and translation and were markly changed;4.Reduction in dietary intake of methionine result in decreasing the extent and severity of intestinal inflammation, An significant alteration of TJ claudin composition and decease mucosa permeability. We hypothesize that this improvement may be related to MetR diet-induced alteration of gastrointestinal TJ claudin composition... |
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