The Study Of Immunomodulatory Effect And Its Mechanism Of Bacillus Natto Glycopeptide In Murine Macrophages

Bacillus natto glycopeptides(BNGP) on normal and LPS-stimulated RAW264.7 macrophages was investigated in this paper.After treated with different concentration of BNGP or LPS with different concentration of BNGP, different indexes were detected. The results were shown as follows:1. Mouse peritoneal macrophage was isolated and cultured. The cell viability of macrophage was detected by MTT assay, the phagocytic activity was detected by neutral red phagocytosis experiments, the release of nitrous oxide(NO) was detected by Griess reaction, the production of cytokines(IL-1β, TNF-α) and pro-inflammatory mediator(PGE2) in culture supernatants were measured by ELISA. The results showed that,cell viability was significantly enhanced at the dose of 31.25-500μg/m L BNGP, the phagocytosis capability,the release of NO, the product of PGE2, IL-1β and TNF-α in culture supernatants were increased at the dose of 62.5-500μg/m L BNGP with a dose-dependent manner.In LPS-stimulated macrophages, the phagocytosis capability was enhanced by the synergy between BNGP and LPS, the secretion of IL-1β, TNF-α and PGE2 were inhibited by BNGP at the dose of 62.5-500μg/m L, but the effect on NO production was not discovered.2. RAW264.7 macrophage was cultured.The proliferation of macrophage was detected by MTT assay, the phagocytic activity was detected by neutral red phagocytosis experiments, the release of nitrous oxide(NO) was detected by Griess reaction, the production of cytokines(IL-1β, TNF-α) and pro-inflammatory mediator(PGE2) in culture supernatants were measured by ELISA, the expression of i NOS and COX-2 were detected by Western Blot.The results showed that BNGP promoted the proliferation of macrophage, increased the secretion of NO, IL-1β, TNF-α and PGE2, improved the expression of i NOS and COX-2 at the dose of 62.5-500μg/m L,enhanced the phagocytosis at the high dose(>125 μg/m L); In LPS- stimulated macrophages, BNGP inhibited the secretion of IL-1β, TNF-αand PGE2 at the dose of 62.5-500μg/m L, the production of NO and the expression of i NOS at the dose of 500μg/m L, also down-regulated the expression of COX-2 at the high dose(>125 μg/m L).3. Flow cytometry was applied to detect the interference effect of BNGP in FITC-LPS binding to the receptors of RAW264.7 macrophage,the translocation of NF-κB p65 into nucleus was detected by immunofluorescence, the expression of NF-κB p65 in the cytoplasm and nuclear and the expression of IκB-α, phosphoIκBα were detected by Western Blot. The results showed that,(1) BNGP significantly reduced the the amount of FITC-LPS that binding to macrophages at the dose of 500μg/m L.(2) BNGP increased the percentage of activated NF-κB cells vs. control group significantly at the dose of 62.5-500μg/m L. In LPS-stimulated macrophages, BNGP reduced the percentage of NF-κB p65 translocation cells that induced by LPS significantly at the high dose(500 μg/m L) and no significant effect at the low dose(≤250μg/m L).(3)BNGP activated the translocation of NF-κB p65 into nucleus from cytoplasm with a dose-dependent manner. In LPS-stimulated macrophages, BNGP inhibited the nuclear translocation of NF-κB in RAW264.7 cells induced severely by LPS.(4)BNGP promoted the phosphorylation of IκB-α that binding to NF-κB in cytoplasm, BNGP+LPS inhibited the phosphorylation of IκB-α that induced by LPS.