Effects Of Weissella Confusa BSP201703 On Occludin Expression And Its Mechanism In Intestinal Epithelial Cells

With the continuous discovery and reporting of probiotics'beneficial effects in our life,coupled with its advantages in application,more and more probiotics have been used to explore their effects of improvement in preventing enteric disease.Previously work in this laboratory have isolated a lactic acid bacteria with good biological characteristics from giant panda feces,named Weissella confusa BSP201703(W.confusa BSP201703).Animal experiments preliminarily showed that W.confusa BSP201703 had a protective effect on gut barrier function,but the mechanism had not been expounded.Therefore,this study will carry out trials in vitro,using a typical cellular model to verify the protective effects of W.confusa BSP201703 on epithelial barrier and further investigate its mechanism by which W.confusa BSP201703regulates occludin(Ocld)expression in intestinal epithelial cells.In the study,Caco-2 cells were selected as experimental subject,and lipopolysaccharide(LPS)stimulation was used to establish a model of impaired intestinal epithelial barrier,the trans-epithelial electrical resistance(TEER)of monolayer and the apparent permeability coefficient(P_app)of fluorescein isothiocyanate dextran 4(FD-4)of the impaired intestinal epithelial barrier with or without W.confusa BSP201703 pretreatment were measured and calculated,the results were compared with those of total control group so that to analyze the effects of W.confusa BSP201703 on intestinal epithelial barrier function in vitro.Subsequently,according to the results of CCK-8 assay,real-time fluorescent quantitative polymerase chain reaction(RT-qPCR)and western blot(WB)were used for analyzing the effects of different factors on Ocld expression at the level of transcription and translation,in order to optimize parameters such as LPS concentration,W.confusa BSP201703 concentration and treatment time for the follow-up mechanism exploration experiment of W.confusa BSP201703.At last,Caco-2 monolayer was treated with inhibitors of specific kinases or antagonist of receptor,and the expression of target protein was analyzed by WB,by comparing with W.confusa BSP201703 treatment group and LPS treatment group,it can be shown whether specific kinases or receptor are involved in the process of W.confusa BSP201703 regulating Ocld protein expression.The results showed:in the Caco-2 cell model,W.confusa BSP201703 pretreatment could effectively alleviate LPS-induced the decrease in TEER and the increase in P_app of FD-4,and protect the intestinal epithelial barrier function.Take the expression of Ocld as an indicator,also refer to the cell proliferation-toxicity test data,the experimental parameters optimization results suggested that stimulation of Caco-2 monolayer with 100?g/m L LPS for 2 h could establish a suitable model of impaired intestinal epithelial barrier,and pretreatment of the impaired intestinal epithelial barrier with W.confusa BSP201703 at the concentration of 1×10⁶CFU/m L for 6 h had the best effect for improving intercellular Ocld expression.The mechanism exploration experimental results showed W.confusa BSP201703 pretreatment inhibited LPS-induced increase in ROCK1(P<0.01)and phosphorylated p38 MAPK(P<0.01),simultaneously enhanced cellular MLCK expression(P<0.01),and then exerting an influence on the expression of Ocld protein.However,Caco-2 monolayer pretreated with TLR2 antagonist Cu-CPT22eliminated the positive effect of W.confusa BSP201703 on the expression of Ocld protein,suggesting that TLR2 is the receptor of W.confusa BSP201703 on intestinal epithelial cells.In summary,in vitro,this study confirmed that W.confusa BSP201703 had positive effects on the expression of Ocld protein and intestinal epithelial barrier function maintenance,and we found that ROCK/p38 MAPK/MLCK signaling pathway is one of the mechanisms by which W.confusa BSP201703 regulates the expression of tight junction protein Ocld in intestinal epithelial cells,and TLR2 is the receptor of W.confusa BSP201703 on Caco-2 cells.Our work laid a research foundation for the farther application of W.confusa BSP201703.However,the effective structural elements of W.confusa BSP201703 in action have not yet been discovered,it deserves further study.