The Effect And Mechanism Of Bifidobacterium Longum And VSL#3~? On TNBS-induced Colitis In Mice

Background:Probiotics have beneficial effects on inflammatory bowel disease(IBD).High mobility group protein 1(HMGB1)is a very important inflammatory mediator involved in the occurrence and development of IBD.However,comparative studies on the effects of the same dose of Bifidobacterium longum(Bif)and VSL#3~?(VSL)on IBD have not been reported.Objective:To detect the level of HMGB1 protein in feces of IBD patients and to study the effect and mechanism of the same dose of Bifidobacterium longum and VSL#3~?on TNBS-induced colitis in mice.Methods:1.The levels of HMGB1 protein in feces of IBD patients and control group were detected by ELISA.2.Establishment of colitis model in mice and intervention of probiotics:2.5%TNBS enema was used to induce experimental colitis in mice.The day of induction was day 0.Three days ago,mice began to treat 4*10~9 CFU Bifidobacterium longum or VSL#3~?to day 3 by intragastric administration every day for a total of 7 days.The general situation of mice was observed and the weight of mice was measured every day.Mice were sacrificed on day 4 and samples were collected for tests.3.Observe the macroscopic view of colon and measure the length of colon.4.Histopathological changes and scores of colon were observed by HE staining.5.Fluorescence quantitative Real-time PCR was used to detect the expression level of HMGB1 gene in mouse colon tissue and the level of bacterial flora gene in mouse colon feces.6.The levels of HMGB1 protein in serum,colon feces of mice and RAW264.7 cell culture supernatant were detected by ELISA.7.Immunohistochemical staining was used to detect the content and distribution of HMGB1 in colon tissue of mice.8.Flow cytometry was used to detect the percentage of F4/80~+cells in mouse colon lamina propria mononuclear cells(LPMC).9.Western blot was used to detect the level of tight junction protein(ZO-1,occludin,claudin-1)in colon tissues of mice and Caco-2 cells.10.The percentage of TEER changes and the permeability of FITC-dextran 4 kDa(FD4)in Caco-2 cell monolayer were detected.Results:1.The levels of HMGB1 protein in feces of CD and UC patients were significantly increased.The levels of HMGB1 protein in feces of patients with active CD and UC disease were increased,especially in patients with active UC disease.2.Combining with the general situation and weight change,naked eye view,length and histopathological changes of colon specimens,the model of experimental colitis induced by TNBS in mice was successfully established.Bifidobacterium longum and VSL#3~?can improve the severity of experimental colitis induced by TNBS in mice.3.Bifidobacterium longum and VSL#3~?can decrease the level of HMGB1 protein in serum and fecal specimens of mice with experimental colitis induced by TNBS,which might be partly related to the inhibition of HMGB1 release from intestinal epithelial cells and/or macrophages in mice.4.Bifidobacterium longum and VSL#3~?can increase the level of tight junction protein(ZO-1,occludin,claudin-1)in colon tissue of mice with experimental colitis induced by TNBS.5.Bifidobacterium longum and VSL#3~?can improve the imbalance of some bacterial flora in colon feces of mice with experimental colitis induced by TNBS.6.Bifidobacterium longum and VSL#3~?can increase the percentage of TEER,the level of ZO-1 protein and decrease the permeability of FD4 in Caco-2 cells induced by rHMGB1.Conclusion:1.The levels of HMGB1 protein in the feces of active IBD patients and colitis mice were significantly increased,which might be related to disease inflammation.2.The same dose of Bifidobacterium longum and VSL#3~?have similar effects on alleviating TNBS-induced experimental colitis in mice,reducing the level of HMGB1 and increasing the level of colon tight junction protein(ZO-1,occludin,claudin-1)in TNBS-induced colitis mice.3.Combined with the results in vitro experiments,Bifidobacterium longum and VSL#3~?improve TNBS-induced colitis in mice,possibly by inhibiting the release of HMGB1 and subsequent HMGB1-mediated intestinal barrier dysfunction.