Effects On The Immunomodulatory Activity Of RAW264.7 Cells By B-3 Polysaccharide

The research on the biological activity of polysaccharides has attracted wide attention in recent decades.Among those,immunomodulatory and anti-tumor activity of polysaccharides from different sources has been the hot topic in this field.Whether the extracellular polysaccharide of Antarctic bacteria has immunomodulatory and anti-tumor or not has triggered a heated debate.In this study,whether B-3 polysaccharide,an extracellular polysaccharide which is extracted and separated from the Antarctic psychrophilic bacteria B-3,has immunoregulatory effects on RAW264.7 cells has been investigated.The effect of B-3 polysaccharide on the proliferation of RAW264.7 cells has been determined by the MTT method.The results showed that B-3 polysaccharide promoted the proliferation of RAW264.7 cells in certain scope,and at low concentrations,this effect is more apparent.In addition,the effect of B-3 polysaccharide on the ability of RAW264.7cells' uptaking FITC-dextran has been determined by flow cytometry.It turned out that at the concentrations of 0.2,2,20 and 100 ?g/mL,B-3 polysaccharide enhanced the phagocytosis of RAW264.7 cells and the effect depended on the concentration.Griess reagent method and ELISA kit were used separately to determine the effect of B-3 polysaccharide on the NO,TNF? and IL10 production of RAW264.7 cells or mice abdominal cavity macrophages.The results showed that at the concentrations of 2,20,and100 ?g/mL,B-3 polysaccharide could significantly increase NO production by RAW264.7cells as well as TNF? and IL10 secretion by RAW264.7 cells and mice abdominal cavity macrophages.And TAK-242,the inhibitor of TLR4,significantly reduced NO production of RAW264.7 cells stimulated by B-3 polysaccharide,which illustrated that the activation of macrophages by B-3 polysaccharide had a close relationship with TLR4 on the cell surface.The change of NF-?B signalling pathway in RAW264.7 cells stimulated by B-3polysaccharide was analyzed by Western blot.According to the results,B-3 induced the degradation of I?B? and the phosphorylation of p65 remarkably.Therefore,B-3polysaccharide activated NF-?B signalling pathway in RAW264.7 cells.Transwell inserts and Snapwell inserts were used to co-culture RAW264.7 cells stimulated by B-3 polysaccharide with mice melanoma cells(B16)in a way of un-contactor partial contact.The results showed that an appropriate concentration of B-3polysaccharide enhanced kill capability of RAW264.7 cells on B16 cells.The expression of Fas and FasL of mRNA level in RAW264.7 cells stimulated by B-3 polysaccharide was measured by real-time fluorescent quantitative PCR.The experimental results showed that B-3 polysaccharide reduced Fas expression and increased FasL expression in RAW264.7cells.The change in the expression of Fas and FasL may have connection with the increased apoptosis of B16 cells in partial contact co-culture with RAW264.7 cells.Effects on gene expression of RAW264.7 cells by B-3 polysaccharide were detected by Digital Gene Expression Profiling(DGE).The results showed that the stimulation by2 ?g/mL B-3 polysaccharide caused 178 genes up-regulated and 242 gene down-regulated in RAW264.7 cells.Nos2 and metallocarboxypeptidase D,the two up-regulated genes can promote NO synthesis.