| ?Objective? Inflammatory bowel disease(IBD)is characterized by chronic relapsing inflammation of the gastrointestinal tract and includes two main clinical phenotypes: Crohn's disease(CD)and ulcerative colitis(UC).The etiopathogenesis of IBD is not completely understood.The intestinal epithelial barrier(IEB)dysfunction takes part in the process of human IBD and experimental colitis.Meanwhile,IEB,which could act as the first line of defense against invasion of pathogenic substances,is mainly composed of intestinal epithelium and tight junction(TJ).The TJ complex consists of transmembrane and intracellular scaffold proteins.The transmembrane proteins(occludin,claudins and junctional adhesion molecules [JAM])are connected to the actomyosin cytoskeleton filaments through scaffolding proteins(zonula occludens [ZO]).Myosin light chain kinase(MLCK)is associated with the contraction of peri-juctional actomyosin rings induced after phosphorylation of myosin light chain(MLC),which has been shown to lead to enhanced permeability in intestinal epithelia.Aryl hydrocarbon receptor(Ah R),a ligand-dependent transcription factor,plays an important role in maintaining intestinal immune barrier and mediating inflammatory responses.In this study,we investigated the effect of loss of Ah R on intestinal barrier dysfunction in 2,4,6-trinitrobenzenesulfonic acid(TNBS)-induced mouse colitis and explored its possible mechanisms.?Methods? 1.In vivo1.1 Male C57BL/6 wild type(WT)mice and Ah R knockout(Ah R-/-)mice(8-10 weeks old)were randomly divided into 6 groups,including the WT control group,Ah R-/-control group,WT ETOH group,Ah R-/-ETOH group,WT TNBS group and Ah R-/-TNBS group(n=6,each group).1.2 The TNBS-induced mouse colitis was established by administrating the TNBS in 50% ethanol.Mice in ethanol control group and blank control group were treated with 50% ethanol solution and normal saline for enema administration,respectively.1.3 Body weight,stool form and occult blood were monitored every day.Disease activity index(DAI)was determined by averaging scores of weight loss,stool form,and occult blood.Intestinal histologic scores were assessed by hematoxylin-eosin(HE)staining.1.4 Colonic mucosal barrier permeability was measured by fluorescein isothiocyanate-dextran(FITC-Dextran).The structure of TJ was observed by transmission electron microscope(TEM).The expression of the TJ protein in the intestinal epithelium was detected by Western blot and immunofluorescence assay.1.5 Western blot was used to observe the expression of MLCK,p-MLC and MLC.2.In vitro 2.1 Caco2 monolayers were divided into 5 groups as follows: control group,TNF-? group(TNF-? 50ng/m L),DMSO/TNF-? group(DMSO,TNF-? 50ng/m L),FICZ/ TNF-? group(FICZ 100 n M,TNF-? 50ng/m L),CH223191/TNF-? group(CH223191 100 n M,TNF-? 50ng/m L).2.2 Caco2 monolayers permeability was measured by FITC-Dextran.Western blot and immunofluorescence assay were used to detect the TJ proteins.Western blot was used to observe the expression of MLCK signaling pathway associatedproteins.?Results? 1.Compared with the control group,epithelial mucosa in TNBS-induced groups exhibited severe colon congestion,edema and bloody stools,and the DAI was significantly increased(P<0.05).In addition,the DAI of Ah R-/-TNBS group was notably higher than that of WT TNBS group(P<0.05).HE staining showed that a large number of inflammatory cells infiltrated,the cells were significantly edematous,degeneration,crypt structure disappeared,and the intestinal histologic scores were significantly increased(P<0.05).What's more,mucosal damage was more severe in Ah R-/-TNBS group than in WT TNBS group and so was the intestinal histologic score(P<0.05).2.Compared with the control group,the mucosal barrier permeability of FITC-Dextran was higher in TNBS-induced groups(P<0.05);TEM revealed the disordered microvillus structure,widened intercellular space and TJ formed a “vacuum-like” structure.Immunofluorescence assay showed weakened TJ fluorescence intensity and disrupted integrity.WB also showed a significant decrease in TJ protein expression in TNBS-induced groups(P<0.05).In addition,the mucosal barrier permeability in Ah R-/-TNBS group was significantly higher than that in WT TNBS group(P<0.05).The ultrastructure of TJ was more severe and the expression of TJ proteins were more reduced in Ah R-/-TNBS group than in WT TNBS group.3.The expression of MLCK and p-MLC in TNBS-induced groups significantly increased compared with the control group(P<0.05);and their expression in Ah R-/-TNBS group was marked higher than that in WT TNBS group(P<0.05).4.In vitro,Caco2 monolayers were treated with TNF-? to induce inflammation.To some extent,activation of Ah R with FICZ repaired the integrity of TJ,up-regulated the expression of TJ protein,reduced the paracellular permeability and down-regulated the expression of MLCK signaling pathway associated proteins.While treated with Ah R inhibitor CH223191 showed the opposite effect as follows: damage on the TJ structure was more severe;expression of TJ proteins was significantly decreased;paracellular permeability and expression of MLCK and p-MLC were both notably increased(P<0.05).?Conclusion? A protective role of Ah R was found against intestinal barrier dysfunction both in TNBS-induced colitis and Caco-2 monolayers induced by TNF-?.Ah R attenuated intestinal barrier dysfunction and inflammation may be relevant to the activation of MLCK-MLC phosphorylation pathway. |
PDF Full Text Request