Research On The Immunoregulation Of TLR/NLRP3 Pathway By S-layer Proteins And DNA Of Two Kinds Of Lactobacillus

The S-layer protein,an extracellular component of Lactobacilli,is a special protein outside the cell wall of Lactobacilli,which has many functions in cell protection,adhesion and aggregation.It is valued for its important role in binding to the cell surface At the same time,There are a lot of free bacterial Deoxyribonucleic acid in the intestinal tract,which has strong immune stimulating properties and can inhibit the secretion of pro-inflammatory factors.Little attention has been paid to the immunological role of Lactobacilli S-layer proteins and DNA in cells.Therefore,lipopolysaccharide was used as an inflammatory stimulant in this study.The S-layer proteins and DNA isolated from Lactobacillus Paracasei subsp.paracasei M5-L and Lactobacillus casei Q8-L were of great significance in treating the immune regulatory effect of LPS-induced HT-29 cells.The S-layer protein was extracted and purified with Li Cl of 5 mol/l and 1 mol/l,and identified by SDS-PAGE electrophoresis.A single band appeared at 45 k Da.Bacterial strain DNA was extracted with DNA kit.Agarose electrophoresis was used for qualitative analysis.The concentration of two kinds of DNA were detected by nucleic acid instrument.The inhibitory rate of S-layer protein?DNA and lipopolysaccharide on HT-29 cell proliferation were determined by CCK-8 experiment,and the action time was determined to be 6 h.The concentration of the two S-layer proteins was 150 ?g/ml,the concentration of the two DNA was 10 ?g/ml,and the concentration of LPS was determined to be 9 ?g/ml.The effect of DNA on cell proliferation was observed and validated under an inverted microscope.We detected inflammatory factors in the cell supernatant by ELISA.The anti-inflammatory effects of S-layer protein and DNA of M5-L and Q8-L strains on HT-29 cells were determined by protein level assay.Both S-layer proteins showed antagonistic effects against LPS to varying degrees,alleviating the secretion of pro-inflammatory cytokines IL-8?IL-1??TNF-??IL-18 and IL-6,while DNA of the two strains can produce superposition effect or antagonism with LPS-induced HT-29 cells,and the secretion level of inflammatory factors was significantly different.RT-PCR was used to determine m RNA expression of inflammatory factors in HT-29 cells by S-layer protein and DNA of M5-L strain.Studies have shown that the effect of S-layer protein was basically consistent with the protein level,but there were great differences in DNA.Finally,the S-layer protein of M5-L was selected to explore the immunoregulation study between TLRs and NLRP3.RT-PCR was used to analyze the TLRs gene level of HT-29 cells stimulated by S-layer protein of M5-L strain and TLR4 protein level by flow cytometry.It was confirmed that the levels of TLR2,TLR4,TLR6 and TLR9 were up-regulated in different degrees.TLR4 gene level was the most significantly up-regulated,and TLR9 regulation was more closely related to LPS and DNA.It has been found that TLR4 is mediated by LPS and regulates the secretion level of IL-1?,which indirectly leads to the activation of NLRP3,recruitment of ASC and Caspase-1 for immune regulation.Whether S-layer protein regulates TLR4/NLRP3 through NF-?B and other pathways remains to be further explored.