| Background:Inflammatory bowel disease (IBD), including ulcerative colitis (UC) and Crohn's disease(CD), are chronic relapsing disorders affecting the gastrointestinal tract. The pathogenesis of IBD in human remains inadequately understood. However, considerable evidence indicates that IBD may result from an interaction of genetic, environmental and immune factors.Intestinal immunodeficiency was regarded as the main etiology of IBD. The intestinal mucosal barrier (IMB) plays an important role in intestinal epithelial to resistance exogenous antigen. The intestinal mucosal barrier consists of intestinal epithelial cells(IEC) and tight junctions(TJ). Studies have shown that there is a closely relationship between intestinal inflammation and TJ. Intestinal epithelial cell permeability was significantly increased in IBD patients, meanwhile, epithelial tight junction was damage. In addition, the intestinal barrier dysfunction result in the toxic molecules invade the intestine, induce the inflammatory response and intestinal diseases, even lead to the systemic diseases such as systemic inflammatory response syndrome(SIRS) and multiple organ failure(MOF). Therefore, the effective inhibition of inflammation and maintenance of intestinal mucosal barrier function are considered to be the main strategy for the treatment of IBD.Aryl hydrocarbon receptor (AhR), a transcription factor, is extensively expressed in vertebrate cells. In the cytosol, AhR is presented in an inactive form that binds to several co-chaperones. Following ligand binding, AhR dissociates from its chaperones and dimerizes with AhR nuclear translocator(ARNT). The AhR/ARNT complex then activates the transcription of target genes such as cytochrome P450 (CYP)1A1. 6-formylindolo (3,2-b)carbazole(FICZ) is an endogenous ligand of AhR. Studies have shown that FICZ involved in the many cell biological processes, including regulation of cell proliferation and apoptosis and inhibition of immune response by activating AhR.Recently, numerous studies have shown that AhR activation could inhibits intestinal inflammation, however, the detailed underlying mechanism remains unclear. Our previous study suggests that AhR activation could ameliorates intestinal mucosal barrier dysfunction through regulating the expression and distribution of tight junction proteins in an intestinal obstruction model.In this study, we investigated the mechanism of AhR in ameliorating intestinal inflammation and intestinal barrier dysfunction in in vivo and in vitro models by Western blotting(WB), Real-time quantitative polymerase chain reaction (Real-time Q-PCR),immunohistochemical (IHC), immunofluorescence (IF) and Ussing Chamber.Methods:1. Colitis was induced by the administration of 3% dextran sulphate sodium(DSS) for 7 days. FICZ was given by intraperitoneal injection daily beginning 2 days after the start of DSS administration. The mouse body weight changes, colon lengths, morality, histological examination and the expression of the inflammatory factors were used to assess the intestinal inflammation. Western blotting, Real-time Q-PCR, IHC were used to detected the expression of CYP1A1, TTP, ZO-1, claudin-1 and occluding. Ussing Chamber was used to detected the transepithelial electrical resistance(TER) of the colon.2. LoVo human intestinal epithelial cells were incubated with lipopolysaccharide(LPS)or FICZ for 8 h. SiRNA transfections was used to transient knockdown of AhR gene expression. Western blotting, Real-time Q-PCR, IF were used to detected the expression of CYP1A1, TTP, MAPKAPK2(MK2), p-MAPKAPK2(p-MK2).3. Caco-2 human intestinal epithelial cells were treated with TNF-a/ IFN-y for 48 hours,with or without FICZ, and altered expression and subcellular localization of TJ proteins were studied by Western blotting analysis and immunofluorescence staining. The expression of MLCK and phosphorylation level of myosin light chain (p-MLC) were measured by Western blotting analysis. Meanwhile, the TER of the cells were detected.Results:1. In the DSS-induced colitis model, The DSS-treated mice exhibited significant weight loss after 3-4 days of DSS treatment. At day 7, there was a 30% rate of mortality in these mice.At the same time, the length of colon shortened in these animals. In contrast, the DSS+FICZ-treated mice exhibited less weight loss, mortality (10%) and the shortened length of colon than the DSS-treated mice. In addition, histological examination of colonic tissues in the different groups revealed that mice treated with FICZ developed lesser severity of colitis.The expression of TNF-a and IFN-y was significantly increased in the colons of mice with DSS-induced colitis in comparison with vehicle-treated mice. However, mice treated with FICZ had reduced levels of TNF-a and IFN-y. The colonic TER was significantly reduced in the DSS group as compared to the control group, however, FICZ prevented the decline of TER induced by DSS.2. In the DSS-induced colitis model, FICZ could activated the AhR and significantly up-regulated the expression of CYP1A1. Meanwhile, the expression of RNA-binding protein TTP was also increased.3. FICZ can significantly up-regulated TTP expression in LPS-treated cells. The expression of TTP was decreased in cells in which AhR expression was silenced. Meanwhile,FICZ could not up-regulated the expression of TTP after knockdown the AhR gene expression.Meanwhile, FICZ could inhibits the phosphorylation of MK2. However, FICZ could not inhibits MK2 phosphorylation after knockdown the AhR gene expression.4. In the DSS-induced colitis model, the expression of tight junction proteins such as ZO-1, claudin-1 and occludin in colonic epithelium of colitis were decreased induced by DSS.However, FICZ could upregulate the expression of ZO-1, claudin-1 and occludin.5. Compared with TNF-?/IFN-?-treated group, FICZ could increased the TER of the cells. Moreover, the disrupted distribution of ZO-1 were decreased. Futhermore, FICZ could increased the phosphorylation of MLC.Conclusion:1. AhR could be activated by FICZ both in vivo and in vitro models.2. AhR activation could reduce DSS-induced inflammation of the colon and amelirote intestinal mucosal barrier dysfunction.3. MK2/p-MK2/TTP pathway involved in the AhR regulation of intestinal inflammation process.4. AhR activation could significantly up-regulate the expression of the tight junction proteins and maintain the tight junction proteins location.5. MLCK/p-MLC pathway may be involved in the AhR regulation of tight junction proteins, alleviate the process of abnormal intestinal mucosal barrier function. |
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