Potential Role Of MAPK In The Production Of Nitric Oxide And Tumor Necrosis Factor-alpha By β-glucan In Macrophages

β-Glucans constitute one of the structural components of fungus cell walls , make the initiation of innate immunity, such as phagocytosis and release cytokine . As the most important polysaccharides of cell wall, it has attracted more and more attention in the field of biology.Macrophages play a critical role in defenses against many microbes by directly ingesting and killing them and indirectly by production of cytokines and by presenting antigens that activate CD4 T lymphocytes to initiate the adaptive immune response. Innate immunity is the first line of host defense against microbial pathogens. Pattern recognition receptors that recognize conserved microbial products are an important componentof innate immunity. so it's important to know the immune activity of macrophage stimulated byβ-Glucan.Previous investigations focus on the intact animal studies about protein released stimulated byβ-Glucan, but it's upper stream hasn't been touched .however, through the study in vitro with some molecular biology skills, we observe thatβ-Glucan induce macrophages to purduce nitric oxide (NO) and tumor necrosis factor (TNF), and whether mitogen-activated protein kinase (MAPK) signal is involved in the course of the activation. Macrophage-like line RAW264.7 cells were cultured. The cells were then stimulated with various concentrations and incubation times ofβ-Glucan to study dose-dependent and time-dependent manners. The effect ofβ-Glucan on the production of NO released by RAW264.7 cells was evaluated by Griess method, Western blot was performed to detect the protein content of MAPK in the cytoplasm. The activity of inducible nitric oxide synthase (iNOS) was determined using iNOS assay kit.The data we obtained show was that the effect ofβ-Glucan in the proliferation and the phagocytosis of RAW264.7 cell line is diphasic (fig.2). β-Glucan (less than 100μg/mL) enhance the proliferation and the phagocytosis of RAW264.7 cell line in a dose-dependent and time-dependent manners.However,up to 400μg/mL,β-Glucan completely suppressed them. The iNOS activity , NO production and TNF-αsecretion can be significantly induced in response toβ-Glucan stimulation in a dose-dependent manner.β-Glucan(100μg/mL) highly induced the release of NO and TNF-α24h after incubation, but the increasement was lower.Western blot showed ERK1/2 and ERK5 in MAPK cell signal has phosphorylation in RAW264.7 stimulated byβ-Glucan. The inhibiter of ERK1/2 (PD98059) obviously depressed the production of NO and the secretion of TNF-α.In this paper, we observed thatβ-Glucan(less than 100μg/mL)caused marked the proliferation and phagocytosis in a dose-dependent manner. But when it's up to 400μg/mL, the rate of growth step dowm. So dose the production of NO and the secretion of TNF-α. ERK5 and ERK1/2 has been phosphorylated protein after stimulated byβ-Glucan. It's also one of most important cell signal in the activation of macrophage.