紫锥菊多糖及其硫酸化衍生物对鸡树突状细胞免疫增强作用机制的比较研究

Echinacea purpurea as a medicinal plant introduced to China for nearly two decades, now Echinacea purpurea formulation has been approved as a new class of veterinary drugs. Echinacea purpurea polysaccharide as the main active ingredient of Echinacea purpurea, will play an important role in regulating immunity, antiviral, antitumor and other biological effects. The sulfation modification could further enhance the biological activity of polysaccharide and produce new pharmaceutical value in our previous study. In this study, purified Echinacea purpurea polysaccharide are obtained by modern separation and purification technology and sulfated Echinacea purpurea polysaccharides are prepared by sulfation modification. The structure of polysaccharide is analyzed by instruments and chemical means. The immune-enhancing activity and mechanism of sulfated Echinacea purpurea polysaccharide is studied by the morphology of Chicken bone marrow dendritic cells(chBM-DCs), the rate of phagocytosis, the induction activity of T lymphocytes and the detection of signaling pathways associated proteins with molecular biology methods. The structure-activity relationship of active polysaccharide is studied in combined with the structure and the immune-enhancing effects of Echinacea purpurea polysaccharide. In this project, the preparation and structure of sulfated Echinacea purpurea polysaccharide is systematically studied. Structural characteristics of sulfated and unsulfated polysaccharides and their immune-enhancing activity for chBM-DCs is discussed in depth and compared, aiming at illustrating the relationship between the structure and the biological activity of polysaccharide. It will provide theoretical supports for the design, research and development of polysaccharide medicines as well as new ideas for development of new veterinary medicines.

紫锥菊为我国新引种的药用植物，紫锥菊制剂已被批准为一类新兽药；紫锥菊多糖为紫锥菊的主要活性成分，具有增强免疫、抗病毒、抗肿瘤及消炎等作用。前期研究证明硫酸化修饰能进一步提高中药多糖的生物活性、增强树突细胞免疫功能，本研究在此基础上，拟分离纯化紫锥菊多糖，并进行硫酸化修饰，测定其含量及结构，以鸡骨髓源树突细胞（chBM-DCs）为靶细胞，比较修饰前、后的紫锥菊多糖对chBM-DCs形态、吞噬功能、诱导T细胞活化的影响，验证紫锥菊多糖的免疫增强作用，探讨硫酸化修饰提高紫锥菊多糖的免疫增强活性的可能性，筛选出活性最强的紫锥菊多糖，进一步测定活性紫锥菊多糖对chBM-DCs相关免疫调控基因和蛋白的影响，并开展活性紫锥菊多糖的构效关系研究，阐明紫锥菊多糖增强免疫的作用机理。本研究不仅为探讨紫锥菊的活性成分和进一步提高紫锥菊多糖的免疫增强活性提供试验数据，而且为研制开发紫锥菊类免疫增强剂提供理论依据。

本试验通过采用现代分离技术制备紫锥菊多糖（EPP），通过硫酸化修饰制备硫酸化紫锥菊多糖（SEPP）,测定其含量及通过红外结果分析，表明硫酸化修饰成功。体外试验以鸡骨髓源树突状细胞（chBM-DCs）比较EPP与SEPP的免疫活性，结果表明，三种剂量的SEPP作用chBM-DCs后，CD11c和CD80的表面标志物表达增加，尤其是在SEPPM组;三种剂量的SEPP、EPPL和LPS可显著增强chBM-DCs对同种异体混合淋巴细胞转化的影响;体外应用EPP或SEPP作用chBM-DCs后，SEPPH组和EPPM组的IL-2水平显著高于LPS组（P<0.05）；各SEPP和EPP组均能提高IFN-γ水平，下调IL-4和IL-10水平。结果表明，sEPP和EPP对chBM-DCs均有免疫调节作用，与EPP相比，SEPP具有更好的免疫调节作用。体内试验比较了EPP与SEPP对环磷酰胺（CY）致雏鸡免疫抑制状态的调节作用，在给药后第7、14、21和28d测定鸡群的体重，肝脏、脾脏、肾脏的脏器指数，外周血淋巴细胞增殖活性、血液生化指标、细胞因子和抗氧化因子指标。结果表明，SEPP和EPP均能显著提高环磷酰胺诱导免疫抑制鸡体重、肝指数、脾指数和肾指数，以及提高外周血淋巴细胞转化能力，降低GLU、UREA浓度和ALP、LDH活性，升高AST、TP、ALB浓度，且能显著提高IL-2、IL-6浓度。说明雏鸡口服一定剂量的EPP和SEPP对CY引起的细胞免疫和体液免疫抑制具有一定的颉颃作用，其中在各采样时间点SEPP对鸡外周血淋巴细胞转化效果优于EPP，给药21d、28d SEPP对鸡外周血细胞因子IL-2的含量水平高于EPP。并以RT-qPCR技术检测用药后chBM-DCs NF-κB mRNA和TLR4 mRNA的表达量。结果表明，各EPP和SEPP组能上调NF-κB mRNA的表达水平，而下调TLR4 mRNA的表达水平。本研究不仅为硫酸化修饰提高紫锥菊多糖的免疫活性作用提供试验数据，而且为研制开发紫锥菊类免疫增强剂提供理论依据。