基于多靶标网络调控研究三种南蛇藤属植物抗RA药效物质基础及作用机制

Celastrus genus has been used in the treatment of rheumatoid arthritis (RA) in China for a long time, but their effective substances is unclear. Current pharmacological studies on the Celastrus genus of anti-RA are carried out for single target. Three kinds of Celastrus genus which are conventional folk medicine to treat RA in Western Hubei Province were chosen as the research object, Preliminary research on them Showed that they can inhibited the excessive proliferation in RA FLS, and the EtOAc fraction was the active part of C. rugosus by the MTT method, zebrafish embryos method,flow cytometry in vitro and the CIA mice model in in vivo. 23 compounds were isolated from the EtOAc fraction of C. rugosus, 17 of them were tritepenoids. Among them, 5 were new compounds, 21 were first isolated from this plant. The material basis of medicinal effectiveness and action mechanism of three kinds of Celastrus genus of anti-RA will be studied comprehensively and systematically from the following aspects: the influence of active compounds on apoptosis of RA FLS, the cellular activity in maerophage, VEGF in RA synovial membrane newborn blood vessel, the regulatory Protein of these processes, the influence on the important signaling pathway such as GFs、NF-kB、VEGF、MAPKs family、Bcl-2 family、MMPS family、cyclin etc base on multi-targets.

南蛇藤属植物作为抗风湿药应用已久，但其抗RA的物质基础尚不明确，且目前有关该属植物抗RA的药理研究多是针对单一靶点进行的。申请者选择鄂西民间治疗风湿习用的三种南蛇藤为研究对象，体外采用 MTT 法，体内采用 CIA 小鼠模型研究其抗RA活性，从不同靶点筛选出藤木抗RA的活性部位为乙酸乙酯部位。本项目将细胞筛选、在体模型动物作用评价与分子靶点筛选相结合，针对 RA 复杂的发病机制中起主导作用的 RA FLS 增殖，根据 GFs、NF-kB、VEGF等主要信号通路的变化，确定三种南蛇藤与临床疗效切实相关的抗 RA 物质基础。

南蛇藤属植物作为抗风湿药应用已久，但其抗RA的物质基础尚不明确。本项目对藤木和毛枝南蛇藤进行了化学成分研究及体内外抗RA活性筛选。从两种植物中分离并鉴定了50个化合物，利用波谱和化学方法鉴定了它们的结构，分别为：三萜16个、二氢沉香呋喃倍半萜29个、神经酰胺类2个、甾体类2个、芳香化合物1个，其中有新化合物10个（2个三萜，8个倍半萜）、新天然产物1个，其余均为首次从该植物分离得到。体外采用MTT 法观察南蛇藤醇提物及不同极性部位对人正常滑膜细胞和 RA FLS 增殖的影响，实验结果表明两种南蛇藤醇提物及藤木P.E.、EtOAc部位均能抑制RA FLS的增殖，且EtOAc 部位的抑制作用最强，抑制率强于藤木醇提物。采用流式细胞仪观察藤木EtOAc部位诱导RA FLS 凋亡作用，实验结果显示藤木EtOAc部位能剂量依耐性的诱导RA FLS凋亡。以斑马鱼胚胎动物为模型，观察到藤木EtOAc部位（10、50ug/ml）显示出剂量依耐性血管生成抑制作用。体内采用胶原诱导的CIA小鼠模型考察藤木的抗RA作用，实验结果表明藤木醇提物均可显著抑制CIA模型关节炎小鼠足垫和关节的肿胀程度，降低小鼠脾指数，并通过病理切片观察，有一定的保护关节损伤的作用；在细胞因子水平上，可显著降低血清中TNF-α，IL-6，IL-8和NF-κB的水平。 以上结果表明藤木醇提物具有抗风湿性关节炎的活性，其抗RA活性部位为乙酸乙酯部位，该活性部位通过降低血清部分炎性细胞因子水平（TNF-α，IL-6，IL-8和NF-κB）发挥抗RA作用。