Effect Of Polysaccharide From Inonotus Obliquus On Mouse Immunocyte

Objective:To extract and measure the polysaccharide from inonotus obliquus, to detect the effects of this polysaccharides on the mRNA expression of immunocytes’ cytokines in mice, and secretion of cytokines, offered experimental basis to clarify the immunoregulatory effects and mechanism about the polysaccharide of inonotus obliquus.Methods:Inonotus obliquus was weighed and smashed, it was steeped in distilled water for12h, then heated and extracted twice, its filtrate was filtered, merged and concentrated, ethanol precipitation twice, filtered, then sediments were merged, after the sediments were dissolved in distilled water, decolored by actived carbon, filtered, trichloroacetic acid was used to take off the protein which from filtrate, then the filtrate was dialysed, concentrated, freeze-dried to powder, the powder was weighed and stored at-20℃, it was dissolved in distilled water for experiment. Raw264.7cell lines, abdominal cavity macrophage and spleen cells of mice were cultivated to measure and select suitable dosage and administration time by RT-PCR, under these conditions, to measure the effects of polysaccharide from inonotus obliquus on the mRNA expression of cytokine IL-6, IL-1β, TNF-a by RT-PCR, their grayscale of electrophoresis figures were analysed and compared with ImageJ2x, statistical analysis with SPSS19, then the supernate of Raw264.7cell lines was collected at48h, detected with ELISA, to calculate the concentration of cytokine TNF-a and IL-1β with standard curve, statistical analyse with SPSS19.Results:1965.98g inonotus obliquus were extracted10.28g polysaccharides, external standard method was used to measure that the content of polysaccharides from inonotus obliquus was41.83mg/mL (836.6mg/g). To detection inhibition of polysaccharides from inonotus obliquus to mRNA expression of proinflammatory cytokine IL-6which from Raw264.7cell lines that stimulated by LPS, found that40μg/mL polysaccharides acts on cells for6h was the best concentrations and administration time of drug. The polysaccharides (40μg/mL) had the most obvious inhibition to mRNA expression of proinflammatory cytokine TNF-a and IL-1β which from Raw264.7cell lines, macrophages and spleen cells of mice that stimulated6h by LPS or PMA (P<0.05). The polysaccharides had the most obvious inhibition to secretion of proinflammatory cytokine TNF-α and IL-1β which from Raw264.7cell lines that stimulated48h by LPS (P<0.05).Conclusion:1. High polysaccharides content which from inonotus obliquus is obtained with digestion, alcohol precipitation, dialysis, freeze drying technique.2.40μg/mL polysaccharides acts on cells for6h, has the most obvious inhibition to mRNA expression of proinflammatory cytokine IL-6which from Raw264.7cell lines that stimulated by LPS.3.40μg/mL polysaccharides acts on cells for6h, has the most obvious inhibition to mRNA expression of proinflammatory cytokine TNF-α and IL-1β which from stimulated Raw264.7cell lines, macrophages and spleen cells of mice by LPS or PMA.4.40μg/mL polysaccharides acts on cells for48h, has the most obvious inhibition to secretion of proinflammatory cytokine TNF-α and IL-1β which from stimulated Raw264.7cell lines by LPS.