The Mechanism Of Probiotics Protecting The Intestinal Mucosal Barrier By Affecting Autophagy

Part 1Objective:To investigate the protective effect of Bifidobacterium infantis on intestinal mucosal barrier function in mice acute colitis induced by Dextran Sulfate Sodium(DSS).To explore the effect of Bifidobacterium infantis and Lactobacillus acidophilus on mucosal barrier after LPS challenging in HCT116 cells.Methods:1.Male C57BL/6 mice aged 6-8 weeks were divided into normal control group,DSS group and DSS+B.infantis group.DSS group and DSS+B.infantis group were given 2.5%DSS for 5 days and normal drinking water for 2 days.At the same time,normal control group and DSS group were given distilled water by gavage for 7 days,and DSS+B.infantis group were given B.infantis by gavage group for 7 days.The degree of intestinal inflammation was evaluated by body weight,disease activity index(DAI),colon length,colon tissue HE staining and inflammation scoring.The expression level of mucosal barrier proteins were evaluated by Western blotting.2.HCT116 cells were divided into normal control group,LPS challanging group,LPS+B.infantis co-culture group and LPS+L.acidophilus co-culture group.The expression level of the mucosal barrier proteins were detected by Western blotting.The barrier function was evaluated by measuring the permeability of monolayer cells.Scratch test was used to evaluated the wound healing ability of the monolayer cells.Results:1.Mice given B.infantis showed significantly reduced DAI score on the 7th day(p=0.0037),increased colon length(p=0.0208),and decreased inflammation scoring(p=0.0008).Western blotting showed decreased expression levels of ZO-1,Occludin,E-cadherin,and Claudin-1 in the DSS group,while the expression levels of the above proteins increased significantly after B.infantis treatment.2.LPS challenging reduced the expression of ZO-1 and Occludin,while co-culture with B.infantis or L.acidophilus could slightly increase the expression of these barrier proteins.LPS challenging leads to increased monolayer cell permeability(p=0.0135).Co-culture of HCT116 cells with B.infantis or L.acidophilus can maintain monolayer cell permeability at a low level(p=0.0524 and 0.0092 respectively).Co-culture with B.infantis or L.acidophilus can promote wound healing by inducing cell migration.Conclusion:1.B.infa ntis treatment can effectively reduce the inflammation level of mice colon in DSS acute colitis model,and promote the expression of ZO-1,Occludin,E-cadherin and Claudin-1.2.B.infantis and L.acidophilus can protect the barrier function of HCT116 monolayer cells under LPS challenging by promoting the expression of barrier proteins,reducing the permeability of monolayer cells,and promoting wound healing ability.Part 2Objective:To investigate the possible mechanism of sodium butyrate on regulating tight junction proteins by affecting autophagy.Methods:LPS challenging model was used to evaluate the protective effects of sodium butyrate on the mucosal barrier.Western blot was used to examine the effect of sodium butyrate on the level of autophagy and the concomitant changes in expression of tight junction proteins.We treated cells with autophagy inhibitors and proteasome inhibitors to investigate the degradation pathway of Claudin-2.The expression and distribution of Claudin-2 after sodium butyrate treatment was observed by immunofluorescence staining.Results:Sodium butyrate could induce autophagy,accompanied by a decrease in Claudin-2 levels.Treating cells with chloroquine could inhibit the degradation of Claudin-2.Immunofluorescence showed that sodium butyrate treatment caused reduction in expression of Claudin-2 on the cell membrane and increased migration into the cytoplasm,and increased colocalization with lysosome.Conclusion:Sodium butyrate can induce autophagy.Sodium butyrate can promote the degradation of Claudin-2 via inducing autophagy.