Study On Immunomodul Atory Effect Of CGA?RPI?Polysaccharides From Pine Needles And The Compound

CGA, polysaccharides from pine needles and RPI are three widely existed, easily acquiring natural products, with a variety of physiological activities. In order to study their roles in immune response,we use mouse peritoneal macrophages as model to explore the immunoregulation effects of CGA,RPI,polysaccharides from pine needles and their compound on normal and LPS-stimulated mouse peritoneal macrophages. We used MTT assay to test the proliferation of macrophage.The phagocytic activity of macrophage was tested by neutral red phagocytosis experiments.Moreover, the production of nitrous oxide(NO) was measured by Griess reaction and the production of interleukin-1?(IL-1?), tumor necrosis factor-?(TNF-?) and interleukin-10(IL-10) in culture supernatants were measured by Enzyme-Linked Immunosorbent Method(ELISA).We also used reverse transcriptionPCR to explore gene expression of IL-1?,TNF-?,IL-10 and IFN-4?, IFN-?.West-blot determined the expression of MAPK signal pathway protein:p38MAPK?JNK?ERK and their phosphorylation protein.The results were shown as follows:(1)Macrophage stimulated by CGA with different concentrations could significantly increased the secretion of NO.At the concentration of 25~100 ?g/mL,CGA improved the proliferation of macrophage.High dose of CGA could improve the phagocytic rate of macrophage,increased the secretion of IL-1?,TNF-? and inhibited the secretion of IL-10 in a dose independent.When macrophage was stimulated by LPS,CGA could cooperate with LPS,improving the phagocytic rate and the production of IL-1?.It also increased the release of TNF-? and reduced the secretion of IL-10.(2)Macrophage stimulated by RPI with different concentrations could significantly increased the secretion of IL-10.When the concentration of RPI was greater than 12.5 ?g/mL,it inhibited the production of NO and TNF-?.High dose of RPI(50~100 ?g/mL) could significantly inhibited the phagocytosis and increased the secretion of IL-10.When macrophage was stimulated by LPS,RPI could inhibited the release of NO and IL-1? at the high dose.When the concentration was less than 25 ?g/mL,RPI had no effect on the production of IL-10 and TNF-?.(3)Macrophage stimulated by polysaccharides from pine needles with different concentrations could significantly increased the secretion of NO.When the concentration was more than 25 ?g/mL,it could promoted the release of TNF-? and IL-1?.Polysaccharides from pine needles could also inhibited the production of IL-10.Different concentration of polysaccharides from pine needles could significantly release a large number of TNF-? in cooperation of LPS.In LPS-stimulated macrophage When the concentration was greater than 50 ?g/mL,the phagocytosis capability of LPS-stimulated macrophage was enhanced by the synergy of polysaccharides from pine needles and LPS,the secretion TNF-? and IL-1? were promoted,and the production of IL-10 was inhibited in a dose dependent.(4)By using orthogonal design method we fond that when the concentration of CGA was 100?g/mL,the concentration of RPI was 50 ?g/mL,the concentration of polysaccharides from pine needles was 200 ?g/mL,their mixture could significantly enhance the capacity of macrophage immune regulation and the result was the best. The RT-PCR results showed that compoud could promote the relase gene expression of IL-1??TNF-? ? Type I interferon,inhibit the gene expression of IL-10 on normal and LPS-stimulated macrophage,and the effect was remarkable.(5)We used West-blot to explore the expression of p38 MAPK,ERK,JNK and their phosphorylation protein of macrophage stimulated by compound or compound co-stimulated with LPS for a certainly time.The result showed that the expression of ERK and p-ERK were down-regulated;JNK,p-JNK,p38 MAPK,p-p38 MAPK were up-regulated after being stimulated for 4 hours.