基于JAK-STAT信号转导通路利用蛋白组学技术从方病证相关角度研究风湿宁胶囊治疗类风湿关节炎的作用机制

Based on the previous study of our team, we believe that Fengshining capsule has significantly effects in treating immunological imbalance, pannus formation of synovium, and inhibition of apoptosis of the CIA rats model. The traditional Chinese medicine emphasizes the relation between prescription and syndrome when treating disease. Fengshining capsule which has the function of dispelling coldness, removing dampness, dredging meridians and relieving pains, focus on the coldness and dampness, while we also find the articulation of cold-dampness syndrome animal model has abnormal change. Hence, we can theoretically deduce that there is a certain correlation between the articular changes induced by CIA animal model and the syndrome of dampness and dampness. From the aspects of immunological imbalance, pannus formation of synovium, and inhibition of apoptosis, following the signal pathway of JAK-STAT, this research will compare the similarities and differences of pathogenic mechanism among 3 types of rats model, including the model of cold-dampness syndrome, CIA model, and a compositive model of cold-dampness syndrome and CIA, and detects the similarities and differences when the models above intervened with FSN. Meanwhile, through proteomic approach, we will evaluate and testify the differentially expressed proteins of rats' synovium which are respectively achieved from the model of cold-dampness syndrome, CIA model, compositive model of cold-dampness syndrome and CIA, and another 3 FSN intervened models, aiming at finding and comparing the relative proteins between cold-dampness syndrome and CIA model and the differentially expressed proteins after they are intervened by FSN, so that we can do a deeper research about the mechanism of FSN in treating rheumatoid arthritis in the view of combining disease and syndrome together.

基于前期研究发现风湿宁胶囊（FSN）对CIA模型大鼠免疫失衡、滑膜血管增生、凋亡受阻等方面有治疗作用。中医疗疾强调方证相关。风湿宁具散寒除湿、通络止痛功用，治疗寒湿痹证，且本课题组已发现寒湿模型关节有异常变化。故可从理论上导出CIA模型所致关节病变与寒湿证之间有一定相关性。本研究以JAK-STAT信号通路为主线，将其负反馈调节为内容，从滑膜细胞免疫失衡、血管增生及凋亡受阻方面探查FSN是否通过阻断JAK-STAT通路而起作用。并比较寒湿证、CI A模型与寒湿CIA复合模型大鼠发病机理异同，探查FSN作用于三模型异同。并通过蛋白组学方法鉴定三模型以及FSN干预后大鼠滑膜组织的差异蛋白并进行验证，以期找到寒湿证与CIA模型相关蛋白质和FSN作用后的差异蛋白，以期从病证结合角度研究FSN治疗RA的作用机制。此研究可为方病证相关研究、为类风湿关节炎病理机制探究和FSN防治RA作用机制提供实验依据。

研究目的：在前期研究风湿宁胶囊（FSN）对CIA大鼠免疫失衡、滑膜血管增生、凋亡受阻等有效的基础上，从FSN祛风散寒、活血通络功能为出发点，建立病症结合模型，研究FSN作用机制与JAK-STAT信号通路的关系，及主要差异蛋白和基因，为FSN临床用药及药物研发提供科学依据。.研究内容及结果：①通过外界因素（风、寒、湿）结合II型胶原乳化剂共同刺激方式，模拟建立CIA、寒湿CIA、风湿寒（热）CIA大鼠模型，发现风寒湿CIA大鼠模型精神萎靡、毛色不泽，趾温、体重等较其余各组降低，足爪肿胀度较余组增大，关节病理变化较CIA和寒湿模型严重，且其余RA临床吻合性更高，确定风寒湿CIA模型可靠；通过功效相反方剂（风湿宁、桂枝芍药知母汤、乌头汤）干预，发现FSN对风寒湿CIA上述指标变化有较好缓解作用，并在红外成像技术辅助下，证明其可抑制CIA模型关节炎症，因此，FSN是治疗RA的理想方剂；.②研究FSN对寒湿CIA模型作用机制，发现其可抑制IL-1和TNF-α，进一步调节机体T/B细胞介导的免疫应答，控制下游JAK-STAT信号通路JAK2、STAT2、STAT3以及NF-κB信号通路TLR4、MyD88、IκB-α与NF-κB表达，促进负反馈抑制因子SOCS2、SCOS3分泌，以抑制滑膜炎症、滑膜处血管新生，减轻软骨损伤；.③借助蛋白组学和代谢组学，对FSN干预后（寒湿）CIA模型蛋白表达差异和基因表达的不同进行筛查和功能富集分析，发现与RA发生相关功能主要涉及炎症、免疫、细胞结构蛋白、信号转导等，与RA相关通路主要有补体系统、凝血系统的激活，FSN治疗RA相关基因具体包括FDFT1、SQLE、AKAP13、KCNMB1、UBB、RPS27A等，其结果也佐证前期实验中，课题组研究认为炎症和免疫反应，以及细胞结构内线粒体为主介导的细胞凋亡程序在CIA模型中起着重要的作用的结果。.研究意义：通过在体和离体实验及蛋白组学方法证实，FSN可调节机体炎症和免疫功能，影响下游JAK-STAT和NF-κB信号通路相关因子表达，发挥抑制滑膜炎和软骨损伤的作用，为今后的药物研究提供方向；同时，课题组成功建立风寒湿CIA大鼠模型，可作为今后RA风寒湿证型研究的可靠模型。