Effect Of Bupleurum Polysaccharides On The Function Of Peritoneal Macrophages In Mice

Radix Bupleuri (dried roots of Bupleurum chinense or Bupleurum scorzonerifolium), known as'Chai-Hu',is one of the most frequently prescribed crude herbs in the prescriptions of traditional Chinese medicine for the treatment of inflammatory diseases, nephrosis syndrome and auto-immune diseases. Bupleurum smithii var. parvifolium is abundantly distributed in the northwest region of China and its roots are also used as Radix Bupleuri.Previous experiments in our lab confirmed that the crude polysaccharide (BPs) isolated from this plant was a major component that contributed to the anti-complementary activity and it had a beneficial effect on acute lung injury (ALI) and systemic lupus erythematosus-like syndroma (SLE),but the effect on mononuclear phagocyte system is still in doubt. We explored the effect of BPs on the phagocytosis and chemotaxis of macrophages; as well as on the free calcium concentration in macrophages using the fluorescent technique.Base on these, we hope for gaining more fundamental information for better clinical medication.PartⅠEffect of BPs on the phagocytosis of peritoneal macrophages in mice [Aim] To investigate the effect of BPs on the phagocytosis of mouse peritoneal macrophages. [Methods] Peritoneal macrophages were isolated from adult male mice. The main groups were as follows:the blank control group and complement activated product group. Each group contained some sub-groups which treated with solvent and BPs (400,200,100,10,1μg/mL).Phagocytosis of FITC labeled E. coli by macrophages was observed by fluorescence microscopy. Percentage (PP) and the index (PI) of phagocytosis of bacteria by the macrophage were observed and evaluated, and contents of MDA, SOD in phagocytic supernatant were measured. [Results] Peritoneal macrophages treated with complement activated product showed a greater PP and PI than blank control group(P<0.001);BPs (400,200,100,10,1μg/mL) given alone significantly enhanced the phagocytosis of peritoneal macrophages(P<0.001); but BPs (400,200,100,10,1μg/mL) inhibited the increase of phagocytosis induced by complement activated product(P<0.001).Complement activated product increased significantly MDA and decreased SOD compared with the blank control group (P<0.01);however, BPs exhibited no significant effect on the content of MDA and SOD in phagocytic supernatant(P>0.05).[Conclusion] BPs could enhance the phagocytosis of macrophages, but inhibit the increase of phagocytosis induced by complement activated product with no significant effect on contents of MDA and SOD.PartⅡEffect of BPs on the chemotaxis of peritoneal macrophages in mice [Aim] To determine the effect of BPs on the chemotaxis of mouse peritoneal macrophages. [Methods] Peritoneal macrophages were isolated from adult male mice. 1)assay of BPs:a) Co-culture. The main groups were as follows:the blank control groups, MCP-1 group and complement activated product group. Each group contained some sub-groups which treated with solvent and BPs (400,200,100,10,1μg/mL). The chemotaxis of peritoneal macrophage was assayed by using 24 well transwell permeable supports.b) Pre-treated. The groups were similar with the co-culture part, and the chemotaxis of macrophage was assayed after the stimulation with chemoattractant.2)assay of BLA:The main groups were as follows:the blank control groups, MCP-1 group and complement activated product group.Each group contained some sub-groups which treated with solvent and BLA (80,40,20,10,5 ng/mL). The chemotaxis of peritoneal macrophage was assayed by using 24 well transwell permeable supports.[Results] Peritoneal macrophages treated with MCP-1 or complement activated product showed a greater chemotaxis index than blank control group(P<0.001);1)The trends of results of co-culture were similar to those of results of pre-treated:BPs (400,200,100,10,1μg/mL) given alone enhanced the chemotaxis of macrophages significantly(P<0.001),but inhibited the increase of chemotaxis index induced by MCP-1 or complement activated product(P<0.001).2) BLA (80,40,20, 10,5 ng/mL) given alone exhibited no significant effect on the chemotaxis(P>0.05); however, BLA (80,40,20,10,5 ng/mL) inhibited the increase of chemotaxis index induced by MCP-1 or complement activated product(P<0.001).[Conclusion] 1) BPs could enhance the chemotaxis of macrophages but inhibit the increase of chemotaxis induced by MCP-1 or complement activated product; 2) BLA could inhibit the chemotaxis of macrophages induced by MCP-1 or complement activated product; however, it had no significant effect on chemotaetic function of peritoneal macrophages in the absence of chemoattractant.PartⅢEffect of BPs on the [Ca2+]i of peritoneal macrophages in mice [Aim] To investigate the effect of BPs on the [Ca2+]i of mouse peritoneal macrophages. [Methods] Peritoneal macrophages were Isolated from adult male mice. 1)The effects of BPs (400,200,100μg/mL) on the [Ca2+]i of the macrophages in the absence of stimulation were observed; 2) predisposed with LPS, the effects of BPs (400,200,100μg/mL)on the [Ca2+]i of the macrophages in the stimulus condition of LPS were observed.[Results] 1)BPs could induce the calcium oscillation in the absence of stimulation; 2) [Ca2+]i in the macrophages increased significantly after the addition of LPS, while BPs could decrease the [Ca2+]i significantly after the LPS. [Conclusion] BPs could induce the calcium oscillation in the macrophages, but inhibit the increase of [Ca2+]i induced by LPS.