Research On The Immunoregulation Function Of Two Kinds Of Lactobacillus Surface Layer Protein Regulate HT-29 Cells

S-layer protein is surface protein component of some bacteria. Lactobacillus S-layer protein plays an important role in the live function of Lactobacillus. Many studies have confirmed that Lactobacillus play an important role in intestinal through the ability of adhesion and colonization, especially the function of immunoregulatory effect. S-layer protein is the outermost layer component, its tolerance to digestive enzymes and bile salts, role in inhibiting host cell adhesion and intrusion by pathogens have been reported. However, the research of immune regulation to host cell by S-layer protein is less. We use Shigella sonnei as inflammatory stimuli, separate S-layer protein from Lactobacillus paracasei subsp. paracasei M5-L and Lactobacillus casei Q8-L,study the immunoregulatory effect.S-layer protein was extracted by 5 mol/L LiCl and purified by 1 mol/L LiCl, their single band at 45 kDa manifested by SDS-PAGE. Adjust the purified S-layer protein to some certain density, eventually using MTT experiment determined the density was 100μg/mL, the multiplicity of infection between S.sonnei and HT-29 cell was 20:1, the time of exposure was 6 h. Through MTT experiment confirmed that the two kinds of protein can inhibit S.sonnei influence the proliferation of HT-29 cells. When the time of exposure was 6 h, the prevention and treatment group effect were better. When the time of exposure was 12 h, the competition group effect was better. Through observation of cell morphology under inverted microscope and validation of trypan blue dyeing experiment, once again confirmed that S-layer protein can rivalry the harmful effect of pathogenic to host cells.Based on the detection of NO content in the cell supernatant, it showed that the two kinds of S-layer protein can reduce S.sonnei induce host cells excessive secrete NO,and this inhibition was related to the concentration of protein. The experiment using ELISA to detect the cytokines concentration in cell supernate and RT-PCR to detect the mRNA express level of cytokines, the cytokines contain IL-8、IL-10、TNF-α and IL-1β,confirmed that the two kinds of S-layer protein can induce the cells express IL-10 both in gene and protein level and at the same time reduce the excessive secrete of IL-8、TNF-α and IL-1β caused by S.sonnei. Further research the MAPK signal pathways by Western Blot, found that the two S-layer protein can reduce host cells express the p-JNK protein to antagonism S.sonnei activate JNK signal pathway. The experiment confirmed the S-layer protein can control the host cell immune function through the regulation of cytokines and signaling pathways.The flow cytometry detection HT-29 cell cycle and cell apoptosis, confirmed thatthe two S-layer protein can be used to adjust the cell cycle disorder and reduce the number of apoptosis cells caused by S.sonnei, further confirmed the S-layer protein can protect the host cell through inhibition S.sonnei. Research has shown that S-layer protein can reduce the activity of Caspase-1, thereby inhibited the inflammatory of cells caused by S.sonnei, and the activity of Caspase-3 is reduced to antagonism apoptosis of host cells.