Effect Of Lactobacillus On TLR4-MyD88-NF-?B Signal Pathway And Apoptosis In HT-29 Cells

S-layer protein is a protein component that is located on the outer layer of some bacterial cell walls.Lactobacillus S-layer protein plays an important role in its pro-life function.Many studies have confirmed that lactic acid bacteria coloniz e the host's intestine and can play an important regulatory role in the host's immune system.There are few studies on the effects of S-layer proteins on the TLR4-My D88-NF-?B signaling pathway in host cells.Therefore,this study uses lipopolysaccharide(LPS)as an inflammatory stimulator to study that the effact two kinds of S-layer proteins isolated from Lactobacillus paracasei subsp.paracasei M5 and Lactobacillus casei Q8-L influence on TLR4-My D88-NF-?B signaling pathway in HT-29 cells.The S-layer protein was extracted with 5 mol/L Li Cl,and the crude protein was purified by salting out with 1 mol/L Li Cl.SDS-PAGE showed that both strains had a single band at 45 k Da.Cells with adjusted S-layer protein concentration were finally determined to have a concentration of 150 ?g/m L by WST-8 assay.The concentration of viable and heat-killed strains was 107 cfu/m L and 108 cfu/m L,and lipopolysaccharide(LPS)was used,the optimal concentration for cell interaction was 10 ?g/m L and the time of action was 6 h for subsequent experiments.WST-8 experiments confirmed that the effect of LPS on the proliferation of HT-29 cells was inhibited by the two S-layer proteins,viable strains and heat-killed strains,and the prevention group had significant effect at the 6 h treatment time.By measuring NO content in cell supernatants,it was demonstrated that the two S-layer proteins,viable strains,and heat-killed strains can reduce LPS-induced excessive cell NO secretion,and this inhibition is related to the concentration.The levels of IL-8,IL-10,TNF-? and IL-1? secreted by the cells were determined by ELISA,and it was confirmed that the two S-layer proteins,viable strains and heat-killed strains can inhibit the secretion of LPS-stimulated cells secrete inflammatory cytokines IL-10 and IL-1?,the protein levels of key enzymes in TLR-My D88-NF-?B signaling pathway of HT-29 cells were further studied by Western Blot.The results showed that S-layer proteins,viable strains and heat-killed strains all inhibited the inflammatory cytokines IL-10 and IL-1?,and regulates the key proteins in the cell were TLR2/4,My D88,TRAF6,I?B-? and NF-?B-p65.In turn,the immune function of the host cells is regulated..Flow cytometry was used to detect the apoptosis of HT-29 cells.It was confirmed that the two S-layer proteins could regulate the cell cycle disorder caused by LPS,and inhibited the massive increase of apoptotic cells induced by LPS.S-layer proteins protect host cells by inhibiting LPS.Studies have also shown that S-layer proteins,viable strains,and heat-killed strains can inhibit the activity of Caspase-1,thereby inhibiting LPS-induced cell inflammatory activity,and by decreasing the activity of Caspase-3 and Caspase-9,reduce LPS-induced apoptosis.