刺玫根多糖对环磷酰胺诱导的免疫抑制小鼠的免疫调节作用

Immunomodulatory effect of polysaccharides from Rosa davuricaroots on immunosuppressed mice induced by cyclophosphamide

采用水提醇沉法提取刺玫根粗多糖,以Sevage法和TCA法脱蛋白,进一步分级醇沉法分离纯化得到较纯的60%醇沉物(RDRP)进行结构鉴定和活性评价。苯酚浓硫酸法测定出刺玫根多糖RDRP总糖含量为94.1%;高效凝胶渗透色谱法测定表明主要含有重均分子量为13.1 kDa,相对分散度为1.2的多糖;通过高效离子色谱法和红外光谱测定,鉴定刺玫根多糖为由葡萄糖、阿拉伯糖、半乳糖、甘露糖、木糖等5种单糖组成的含有α-构型的中性吡喃多糖。环磷酰胺诱导的免疫抑制小鼠模型实验结果表明,与模型组相比,刺玫根多糖各剂量组小鼠的脾脏和胸腺指数明显升高(P<0.05,P<0.01);血细胞分析仪检测发现白细胞数、红细胞数、血小板显著升高(P<0.05);ELISA法检测血清中免疫相关蛋白IgG、IgM、IgA以及细胞因子TNF-α、IFN-γ、IL-6与IL-2的分泌水平,结果显示,与模型组比较,刺玫根多糖组显著上调小鼠血清中免疫球蛋白IgA、IgG、IgM浓度水平(P<0.01)及IL-2、IFN-γ、TNF-α、IL-6的含量(P<0.01);Western blot法测定各组TLR4、MyD88、TRAF-6和NF-κB p65蛋白的表达水平,发现刺玫根多糖各剂量组脾脏中TLR4、TRAF-6蛋白极显著上升(P<0.01),高剂量组的MyD88蛋白极显著上升(P<0.01),其他组呈现上升趋势;刺玫根多糖各剂量组NF-κB p65蛋白均有上升趋势,因此其免疫调节作用机制可能与TLR/NF-κB通道有关。本研究初步阐明刺玫根多糖的基本结构特征和其免疫调节作用及其机制,为刺玫根多糖的开发应用提供一定价值的实验依据。

The crude polysaccharide was obtained from Rosa davurica root via hot water extraction method and ethanol precipitation,followed by deproteinization using the Sevage and TCA methods,respectively.After deproteinization,the crude polysaccharide was purified by graded ethanol precipitation method.Herein,the 60%ethanol precipitate was labeled RDRP,and its structure and activity of were further studied.The total sugar content of polysaccharides RDRP was 94.1%,and mainly contained a polysaccharide with an average molecular weight of 13.1 kDa with narrow polydispersity at 1.2,as well as comprised of five monosaccharides including glucose,arabinose,galactose,mannose,and xylose.IR spectrum indicate that it was a neutral glucopyranoside in theα-configuration.In cyclophosphamide-induced immunosuppressive mice test,the spleen and thymus index of mice in each dose group of RDRP were significantly increased(P<0.05,P<0.01),respectively,compared to model group;the white blood cells,red blood cells and platelets in each dose group of RDRP were significantly increased(P<0.05)compared to model group;the immunoglobulin IgG,IgM,IgA and IFN-γ,TNF-α,IL-6 and IL-2 in each dose group of RDRP were significantly increased(P<0.01)compared to model group;the TLR4 and TRAF-6 proteins in each dose group of RDRP were increased significantly(P<0.01),and MyD88 was increased significantly in high-dose group(P<0.01)and showed the increasing trend in other groups;the NF-κB p65 protein also showed the increasing trend in the RDRP groups.Those results implied that the immunomodulatory mechanism of RDRP may be related to the TLR/NF-κB.In this research,the basic structure and immunomodulatory effect and its mechanism of polysaccharide from R.davurica roots were clarified and those data will provide a valuable experimental basis for its development and application.