| As the outermost layer component,S layer protein plays an important role in the live function of Lactobacillus,such as adhesion and colonization in intestinal epithelial cells and inhibiting host cell adhesion and intrusion by pathogens.It has been confirmed that S layer protein have immunomodulatory effects on intestinal inflammation caused by pathogens.Considering the properties,we speculated the effect of S layer protein due to coaggregation ability with pathogens and competing with the pathogen for binding sites.In order to study the immunoregulation effect of S layer protein on host cells,we used Shigella sonnei as inflammatory stimuli,separated S layer protein from L.casei Q8-L,studied the immunoregulatory effect of Lactobacillus casei Q8-L S layer protein on HT-29 cells.Two extraction methods for S layer protein were compared by SDS-PAGE and BCA,and two-step extraction method was determined as the optimal extraction method: first extracting with 1 mol/L LiCl for 30 min and then re-extracting with 5 mol/L LiCl for 1 h,and the S layer protein extraction of the L.casei Q8-L fermentation broth was 600 μg/ml.The purified protein after 1 mol/L LiCl treating showed a single band at 45 kDa by SDS-PAGE.MTT assays showed that L.casei Q8-L S layer protein could reduce the inhibitory effect of S.sonnei on the proliferation of HT-29 cells,and the following experimental conditions were determined: the total time was 6 h,the concentration of S layer protein was 200 μg/mL,and the MOI was 1: 1.Under these conditions,the growth status and morphology of cells and pathogens were observed by optical microscopy.The contents of PGE2 and NO were measured by PGE2 assay kit and NO assay kit,and the results showed that the S layer protein of L.casei Q8-L could significantly inhibit S.sonnei-induced cells to produce PGE2 and NO.In addition,the expression levels of NF-κB pathway-related genes were measured by RT-qPCR and the expression of TLR2 and TLR4 were measured by flow cytometry.All these results showed that S layer protein could regulate the levels of TLR2/4 and NOD-1/2,,furthermore regulate the NF-κB pathway.The expression of COX-2 and PPAR-γ mRNA was also analyzed and it was found that S layer protein could inhibit the activation of NF-κB pathway induced by pathogens to a certain extent.Meanwhile,NF-κB activation was observed by immunofluorescence and ImageJ software was used to analyze the expression of NF-κB p65 protein to verify this conclusion.These results demonstrated that the S layer protein had a certain ability to regulate NF-κB pathway in inflammatory cells induced by S.sonnei.In order to study the role of TLR2/4 in the regulation of NF-κB pathway in HT-29 cells stimulated by S layer protein,the TLR2/4 of HT-29 cells were blocked by antibodys,the levels of TNF-α and IL-8 stimulated by S layer protein were measured by ELISA,the expression levels of NF-κB pathway-related genes were measured by RT-qPCR,and NF-κB activation was observed by immunofluorescence,Through comparing the experimental results after with before blocking,the effects of blockade on the regulation of S layer protein on HT-29 cell and the inflammatory response induced by pathogens was studied.The results showed that the regulation ability of S layer protein on TNF-α、IL-8 and NF-κB pathway-related genes was reduced after TLR2/4 blocked,as well as the ability of inhibiting the activation of NF-κB pathway induced by pathogens to a certain extent.These results indicate that TLR2 and TLR4 play an important role in the regulation of S layer protein on NF-κB pathway. |
