基于JAK2/STAT3信号通路探讨补肾通络法改善类风湿关节炎骨吸收的作用机制

Rheumatoid arthritis (RA) is one of Bi syndrome in Traditional Chinese Medicine, and kidney deficiency is the main pathogenesis of it. As the research focus of bone resorption, JAK2/STAT3 pathway not only can be evoked by IL-6 to promote osteoclast (OC)-mediated bone resorption, but also can be blocked by the JAK2 inhibitor AG490 to suppress OC-mediated bone resorption via multiple approaches. In our previous grant, the data showed that typical decoction “yi shen juan bi pill” of Bushen Tongluo Method could ameliorate OC differentiation and OC-mediated bone resorption by regulating IL-6 and Th17/Treg in CIA model rats as well as by regulating Treg cytokine secretion in Treg/OC co-culture in vitro. Based on the above conclusions, we put forward to the new hypothesis that typical decoction “yi shen juan bi pill” of Bushen Tongluo Method can directly and/or indirectly ameliorate OC-mediated bone resorption by regulating the JAK2/STAT3 pathway to show therapeutic effects on RA. In this grant, the Treg/OC co-culture model will be esblished, JAK2 inhibitor and drug-containing serum will be intervened. Meanwhile, immunology, and molecular biology analysis will be used to intensive study the mechanism that how typical decoction “yi shen juan bi pill” of Bushen Tongluo Method ameliorate bone resorption via JAK2/STAT3 signaling pathway. This research will provide experimental basis and new idea to the treatment of RA.

类风湿关节炎（RA）属中医“骨痹”范畴，肾虚是其主要病机。JAK2/STAT3通路作为目前骨吸收研究热点，其不仅可被IL-6激活进而促进OC介导的骨吸收，而且可被JAK2抑制剂AG490多重阻断OC介导的骨吸收。申请人前个结题基金研究发现，补肾通络法代表方益肾蠲痹丸可通过调节CIA模型大鼠IL-6、Th17/Treg和Treg/OC共培养中Treg分泌功能，改善OC分化和骨吸收。据此我们提出以下假说：补肾通络法代表方益肾蠲痹丸通过调节JAK2/STAT3信号通路，直接和/或间接改善OC介导的骨吸收，发挥对RA的治疗作用。本项目拟通过Treg/OC共培养体系，用JAK2抑制剂及含药血清进行干预，借助免疫学、分子生物学等手段，深入探讨益肾蠲痹丸调节JAK2/STAT3信号通路改善骨吸收的机制，为补肾通络法治疗RA提供新的实验依据。

破骨细胞（csteoclast, OC）介导的骨吸收是类风湿关节炎（rheumatoid arthritis, RA）患者的常见特征。骨吸收功能亢进，导致骨破坏和骨丢失是RA致残的重要原因。通过调节OC的分化和功能，抑制骨吸收是治疗RA的有效手段。益肾蠲痹丸（Yi Shen Juan Bi Pill, YSJB）是补肾通络法治疗RA的经典复方，可改善RA引起的骨质破坏。本课题组前期研究发现YSJB可显著减轻CIA大鼠关节骨侵蚀表现，抑制CIA大鼠的局部关节炎症和OC分化。在体外，YSJB不仅能直接抑制破骨细胞分化和其介导的骨吸收功能，还能够通过提高调节性T细胞（regulatory T cell, Treg）的功能，间接抑制破骨细胞分化和其介导的骨吸收功能，但其作用机制目前尚不完全清楚。JAK/STAT信号通路几乎在所有细胞中表达，参与调节机体内细胞生长发育、免疫应答等重要生物学过程。作为JAK2特异性抑制剂，AG490可通过抑制JAK2/STAT3信号通路降低NFATc1表达，阻断RANKL诱导的破骨细胞分化。此外，AG490还可调节T细胞稳态。本研究基于JAK2/STAT3信号通路，通过建立OC-Treg共培养、OC单培养、Treg单培养三个培养体系，采用YSJB含药血清和JAK2/STAT3信号通路阻断剂AG490以及靶向JAK2的siRNA干预进行体外实验。实验结果表明YSJB可直接调节OC中JAK2/STAT3信号通路，从而下调OC中RANK，NFATc1，c-fos基因表达，抑制破骨细胞分化及其介导的骨吸收功能。此外，YSJB还可通过调节Treg中JAK2/STAT3信号通路，上调IL-10表达，增强Treg免疫抑制功能，间接发挥抑制破骨细胞分化及其介导的骨吸收功能的作用。该发现阐明了JAK2/STAT3信号通路与OC介导的骨吸收在YSJB改善RA骨破坏过程中的机用，进一步明确补肾通络代表方剂益肾蠲痹丸改善RA骨吸收的机制，为该疾病的药物治疗提供新的靶标。