基于微阵列双靶细胞膜二维色谱和DARTS技术的防风抗类风湿性关节炎“多成分-多靶点”调控机制研究

针对防风抗类风湿性关节炎药效物质基础不清，作用机制不明的问题，在前期证实防风醇提物可通过调节成纤维样滑膜细胞和巨噬细胞的活化发挥抗类风湿关节炎作用的基础上，本项目以成纤维样滑膜细胞膜和巨噬细胞膜为活性成分和作用靶点识别载体，从现有活性成分筛选策略局限于单靶细胞的不足出发，整合阵列-多维色谱技术，构建阵列式-多靶细胞膜亲和色谱全二维在线筛选系统，鉴定防风在两种靶细胞膜上的结合组分，并验证其活性。采用药物亲和反应靶标稳定（DARTS）技术，鉴定并解析防风活性成分在两种靶细胞膜上的结合靶蛋白，通过分子对接表征膜结合组分与结合靶蛋白的潜在对应关系。遴选2～3种活性成分，采用siRNA干扰等手段进行靶点反向试验验证，明确防风抗类风湿关节炎活性组分与靶标的对应关系，为抗类风湿关节炎的新药开发提供活性先导化合物和潜在靶点。本项目将为基于多靶细胞膜识别的中药活性成分高通量同步表征提供新思路和方法。

In order to solve the problem of unclear pharmacodyamic material basis and unclear mechanism of anti-rheumatoid arthritis effect of Saposhnikovia divaricate, based on the previous confirmation that ethanol extract of Saposhnikovia divaricata can exert anti-rheumatoid arthritis effect by regulating the activation of fibroblast like synoviocytes and synoviocytes macrophages, the two kinds of cell membranes were chosen as active components and target recognition carrier in this project. In order to identify the binding components of Saposhnikovia divaricata on the two kinds of target cell membrane, a two-dimensional array multi-target cell membrane affinity chromatography online screening system is constructed by integrating array and multi-dimensional chromatography technology. After verifying the activity of the compounds, the drug affinity responsive target stability (DARTS) technology will be used to identify and analyze the binding target proteins of the active components of Saposhnikovia divaricata on the two target cell membranes, and the potential correspondence between the membrane binding components and the binding target proteins will be characterized by molecular docking. Then, two or three active components are selected to perform target reverse test verification such as siRNA interference and surface plasmon resonance. The corresponding relationship between anti-rheumatoid arthritis active components of Saposhnikovia divaricata and targets is clarified, so as to provide active lead compounds and potential targets for the development of new anti-rheumatoid arthritis drugs. This project will provide new ideas and methods for high-throughput simultaneous characterization of active components of traditional Chinese medicine based on multiple target cell membrane recognition.