The Probiotic Mixture Based On Bifidobacteria Alleviates Dextran Sodium Sulfate-induced Colitis By Increasing The Expressions Of Tight Junction Proteins And Regulatory T Cells Response

AimInflammatory bowel diseases(IBD)comprising two main forms:ulcerative colitis and Crohn's disease,which are characterized by chronic and non-specific inflammatory diseases affecting the gastrointestinal tract.Currently,the precise etiology and pathogenesis remain incompletely clear.Recently studies showed that probiotics can be used for the prevention and treatment of human inflammatory diseases including IBD.However,the exact mechanism of this beneficial effect remains incompletely clear.To investigate whether supplementation with a probiotic mixture(bifidobacterium,lactobacillus and enterococcus)can ameliorate dextran sulfate sodium(DSS)-induced murine colitis by improving the function of mucosal barrier and regulating the immunological response of T cells involved.Methods1.Specific pathogen free female BABL/c mice(8-10 wk old)were randomly divided into five groups:NC group(received saline),BB group(mice were administered with the probiotic mixture by intragastrlc gavage for 14 days),DSS group(14-day saline intragastric administration+7-day 4%DSS exposure from day 8),the BD group(7-day probiotic mixture by intragastric administration+7-day 4%DSS exposure from day 8)and the BDB group(14-day probiotic mixture by intragastric administration +7-day 4%DSS exposure from day 8).Colonic tissues,blood specimens and spleens were rapidly obtained from mice after sacrificed at the end of experiment.2.Disease activity index(DAI)was daily recorded and evaluated.Macroscopic scores and histopathologic scores were assessed to compare intestinal inflammation.3.The levels of tumor necrosis factor(TNF)-? in colonic tissues were detected by enzyme-linked immune sorbent assay(ELISA),4.The structures of tight junctions were observed by transmission electron microscope(TEM);The expression and distribution of JAM-1?occludin?claudin-4 were examined by western blot and immunohistochemistry.5.Single-cell suspensions of blood and spleen were prepared with Red Blood Cell Lysis Buffer and CD4+CD25+Foxp3+ Treg cells were measured by FACS analysis.6.The expression of Foxp3 and CD4 protein were determined by western blot and immunofluorescence.The expression of interleukin(IL)-17 was decteted by western blot and qRT?PCR.Results1.Compared to normal control(NC)group,DSS-induced mice showed greatly increased disease activity index(DAI)score,macroscopic score(MS)and histopathological score.DSS-induced mice had the shortened lengths and decreased weights of colon.All these alterations indicated successful DSS-induced colitis models were established.2.Comapred with the DSS group,DAI,MS and histopathological scores were significant improved in mice treated with probiotic mixture(BD and BDB group).The expressions of TNF-? decreased,and improvement in intestinal inflammation was observed in BD and BDB groups than the colitis models.These results demonstrated that probiotic mixture could alleviate the activity of acute colitis,which seemed to be more pronounced in the BD group compared with the BDB group.This probiotic mixture may be opportunistic bacteria at acute damage stage by re-triggering intestinal immune system and increased inflammatory reaction in the BDB group.3.Compared with the DSS group,the damage of gut tight junction ultra-structures were improved by TEM examination.The protein expressions and distributions of JAM-1,occludin and claudin-4 were increased in the BD and BDB group.4.Mice in the BD and BDB group decreased the proportions of total CD4+ T cells both in the peripheral blood and spleen while increased the amounts of CD4+CD25+Foxp3+Treg cells in the peripheral blood,these suggested that the levels of CD4+T and Tregs cells could be regulated by probiotics and involved in inflammatory reaction.5.Copmared with the DSS group,the expression of CD4 protein in the colonic tissues was decreased while Foxp3 was increased in the BD and BDB group.The expression of IL-17 of both protein and mRNA was increased in colitis mice treated with probiotic mixtures.6.Correlation analysis showed that DAI score was negatively correlated with the amount of Treg cells in the blood and the expression of Foxp3 protein in the colon.ConclusionBifidobacteria triple probiotic mixture has beneficial effects on DSS-induced murine colitis by improving mucosal barrier function and imnune-modulating the numbers of CD4+T cells,especially increasing the numbers of Foxp3+cells and the expressions of IL-17 in colonic tissue as well as promoting the proportions of Treg cells in peripheral blood.Conversely,this kind of probiotic mixture can decrease the the expressions of CD4 protein in colonic tissue and the proportions of CD4+T cells in peripheral blood.