The Research On Signal Mechanism Of Surface Layer Protein Fromlactobacillus Acidophilusactivating Regulation Of Immunity In RAW264.7 Macrophages

Surface layer protein (S-layer protein, SLP) is one kind of bioactive molecule iin Lactobacillus.Main functions of SLP include inhibiting pathogenic bacteria from adhering and immunomodulatory effects. As a sort of immune cells, macrophages can mediate nonspecific immune response, and its activation plays an important role in immune regulation. Researches show that in macrophages lipopolysaccharides can improve the phagocytosis of macrophages.produce toxins that can kill cancer cells, and promote the secretion of pro-inflammatory cytokines like TNF-? and IL-6 to modulate immune response. For now most researches about SLP focused on adhesion and colonization, while few researches concerned about the mechanism of immunoregulation.1. The expression of TNF-? mRNA and IL-10 mRNA were measured by Real-time PCR. The results showed a markedly higher expression of TNF-? mRNA and IL-10 mRNA than that in control group. After the macrophages were pretreatment with Chelerythrine, the content of target genes was significant lower than SLP-stimulated group. These indicated that SLP can promote the expression of inflammatory cytokines by activating PKC.2. In order to study the signal pathways during SLP activating macrophages. IFA was used to detect the nuclear translocation of transcription factors NF-?B. The protein expression of I?B? and phosphorylation of ERK1/2 in cells was detected by using western blot. NF-?B and AP-1 DNA binding activity of macrophages was evaluated by EMSA. The results showed that the protein expression of I?B? was markedly lower than control group.NF-KB was significantly activated and translocated from cytoplasm to nucle, so was AP-1.The expression level of phosphorylation of ERK1/2 increased when stimulated by SLP. These indicated that NF-?B pathway and MAPK/ERK pathway participate in activating macrophages. Furthermore, we found that SLP has bidirectional regulation on activating cells.3. iTRAQ was used to analyze Proteome of SLP-stimulated RAW264.7 macrophages, and we found 149 differential proteins of which above 90% were up-regulated. Through KEGG online database, 10 signal pathways about immune system and 10 signal pathways about signal transduction were selected. Results moreover confirmed that MAPK signal pathway and NF-?B signal pathway were involved in macrophages activation by stimulate of SLP. And we also found that PI3K-Akt pathway may participate too.This project mainly studied the impact of L. acidophilus S-layer protein and signal pathways in activating macrophages. The target of this study was to clatify the mechanism of immunoregulation taken by S-layer protein in macrophages, and provide theory basis of the application of functional food.