Effect Of Lactobacillus S-layer Protein On H₂O₂-induced Mitochondrial Apoptosis Pathway In HT-29 Cells

The S-layer protein is the main component of the surface structure of lactobacillus,accounting for 10%-15%of the total protein of the cell and 70%of the surface of the grid.It has been reported that the S-layer protein has a positive contribution to the probiotics of lactobacillus.Currently,there are few studies on the effect of S-layer protein on the mitochondrial apoptosis pathway of host cells.Therefore,in this study,H₂O₂ was used as an oxidative damage stimulant to study the effects of S-layer proteins isolated from Lactobacillus paracasei subsp.paracasei M5-L,Lactobacillus casei Q8-L and Lactobacillus rhamnosus GG?LGG?on the mitochondrial apoptosis pathway of HT-29 cells.In this study,5 mol/l LiCl was used to extract S-layer protein,and 1 mol/l LiCl was used to purify crude extracted protein by salting out.SDS-PAGE test showed that both strains had a single band at 43 kDa.After adjusting the concentration of S-layer protein,the concentration of active and heat-inactivated bacteria was finally determined to be 150?g/ml by MTT experiment,the concentration of live and heat-inactivated bacteria was 10⁷ cfu/ml and 10⁸ cfu/ml,and the optimal concentration of H₂O₂ to interact with cells was 0.8 mM,and the action time was 6 h for follow-up experiments.MTT experiment confirmed that three S-layer proteins,live bacteria and heat-inactivated bacteria could inhibit the effect of H₂O₂ on HT-29cell proliferation at the action time of 6 h.The five indexes of T-AOC,SOD,CAT,MDA and LDH of the antioxidant enzyme system in HT-29 cells were detected.The experiments showed that the S-layer proteins of the three strains could increase the content of T-AOC,SOD and CAT and reduce the content of MDA and LDH,thereby antagonizing the effect of H₂O₂ on HT-29 cells.Each treatment group of the three S-layer proteins had a significant positive regulatory effect on the abnormal activity of caspase-3 and caspase-9 caused by H₂O₂.The results showed that the S-layer proteins of M5-L,Q8-L and LGG could slow down H₂O₂-induced HT-29 apoptosis by reducing their activation.By flow cytometry of three strains of S-layer of proteins and H₂O₂ in the cells of HT-29 in the change of mitochondrial membrane potential and cellular Ca²⁺two apoptosis index of the study,the influence of layer,in turn,determined the M5-L S-layer protein group?prevention?antagonism H₂O₂ for HT-29 cell function,the optimal,so eventually chose the M5-L S-layer protein group?prevention?for studying the mechanism of apoptosis part.To improve the mechanism of three S-layer proteins antagonizing H₂O₂,real-time fluorescence quantitative PCR was used to detect the expression of mRNA related to mitochondrial apoptosis pathway in HT-29 cells.The results showed that the S-layer protein of lactobacillus can increase the expression of XIAP and Bcl-2genes by reducing the expression levels of APAF-1,Caspase-3,Caspase-9,CytC,Smac and Bid genes,and then inhibited the apoptosis induced by H₂O₂ through the mitochondrial cell apoptosis pathway.Due to the low expression content of AIFM1and ENDOG genes compared with other genes,the change trend of AIFM1 and ENDOG in each group was not very regular according to the results of this experiment.Whether the S-layer protein of M5-L inhibited cell apoptosis through AIFM1 and ENDOG pathways that were not caspase-dependent remained to be further studied.