不同紫锥菊多糖构效关系与生物活性的确证及机制研究

Chinese veterinary medicine plays a vital role in poultry epidemic prevention and immune enhancement. The different molecule weight and structure of polysaccharides cause diverse functions. The Echinacea purpurea polysaccharide, as the main component of Echinacea purpurea, was proved to have various bioactivities. However, reports on the precious biological activities (immune-promotion and anti-inflammatory) and structure-activity relationship of the pure polysaccharides including bioactive mechanisms in vitro and in vivo have been rarely seen so far. In our project, we first obtain the pure polysaccharides and confirm the immune-enhanced polysaccharide and the anti-inflammatory polysaccharide at the cellular level. Then we deeply study and infer the structure of different polysaccharides and confirm the mechanism of different activities via testing the binding between polysaccharides and receptors in macrophage. Further, the immunosuppressive mouse model and inflammatory mouse model are established for the evaluation and mechanism recognition of polysaccharides in vivo. Finally, we obtain purified Echinacea purpurea polysaccharides with clear mechanism of action, reliable therapeutic effect, and definite structure-activity relationship, which will supply a crucial theoretical fundamental and significant guidance for developing new veterinary drugs with clear mechanism and effective therapeutic efficacy.

中兽药是临床畜禽疫病预防及免疫增强的重要选择，多糖不同的分子量及结构有不同的疗效，免疫药草紫锥菊的主成分紫锥菊多糖被证实具有多种生物活性，但对纯化后不同分子量、不同结构的紫锥菊多糖具有的生物活性构效关系及内在作用机制研究尚未见报道。本项目从纯化获得不同紫锥菊多糖开始，在细胞水平分别确认免疫增强活性多糖与抗炎活性多糖，深入研究推测出不同活性多糖结构；通过测试多糖与巨噬细胞表面受体的结合，体外确证不同多糖产生不同生物活性的内在机制；建立免疫抑制与炎症小鼠模型，对多糖活性进行评价，并对多糖作用机制进一步研究确认，最终，获得作用机理明确、疗效确实、构效关系明晰的纯化紫锥菊多糖，为开发作用机理明确、疗效确实的新中兽药提供重要的理论基础与指导意义。

在当前抗生素在动物临床全面禁用的情况下，通过应用作用机制明确、疗效确实的中兽药提前预防保健提高畜禽免疫力而达到少得病或者通过与疫苗联合使用提高免疫有效率成为防治畜禽疫病发生的重要选择。本项目从中药紫锥菊中提取分离得到三个紫锥菊纯化多糖（EPPS-1、EPPS-2、EPPS-3），在免疫增强、抗炎、抗氧化活性方面筛选出具有较好活性的EPPS-3;结构表征研究表明，EPPS-3相对分子量13.0KDa，分子中阿拉伯糖:半乳糖:半乳糖醛酸:葡萄糖醛酸:葡萄糖以7.80:4.55:2.64:1:9.02的摩尔比例组成；EPPS-3中具有7种类型的单糖连接方式，其中以1,4-吡喃葡萄糖基和1,4-半乳糖基为主，推测其可能的结构是以→4)-α-D-Galp-(1→4)-α-D-Glcp-(1→4)-β-D-GalpA-(1→5)-α-L-Araf-(1→为骨架，以末端T-α-L-Ara-(1→连接在→4)-β-D-GalpA-(1→的C-3上为支链。体内外抗炎活性研究表明，EPPS-3可显著降低促炎因子（IL-6、TNF-α）、增加抑炎因子（IL-10）的释放，其抗炎机制表现为显著抑制P-ERK，T-ERK和p-P65的表达和活性。体内外抗氧化活性研究表明，EPPS-3可显著促进GSH-PX、SOD酶的活性、抑制MDA的分泌，促进肝组织损伤的恢复，并通过线粒体凋亡信号通路、Keap1-Nrf2-ARE信号通路缓解肝损伤。体内外免疫增强活性研究表明，EPPS-3可以显著促进细胞增殖、免疫球蛋白（IgG、IgM）和IFN-γ细胞因子的分泌，提高CD4/CD8比。本研究获得了结构明确，免疫增强、抗炎、抗氧化活性强，作用机制明确的紫锥菊纯化多糖，为能切实满足临床需求的疗效确实的新中兽药的研发与应用提供理论基础和数据支持。