程氏蠲痹汤通过调节GRK2调控GPCRs信号通路影响类风湿关节炎T淋巴细胞功能的机制研究

On the basis of the principle that Atrophic Arthritis is mainly attributed to anemofrigid-damp arthragia in Chinese medicine, Chengshijuanbi decoction, created by Xinan physicians, with assured clinical efficacy on "expelling wind and clearing away cold, resolving dampness and freeing channels" is selected and then the hypothesis, Chengshijuanbi decoction influences T lymphocyte functions by regulating GRK2 to control GPCRs signal channels, is suggested in this study. The rat models with adjuvant arthritis and T lymphocyte models of peripheral blood and synovium cultured in vitro are employed. The research is focused on T lymphocytes by investigating the regulation of GRK2 on GRCRs signal channels using multiple techniques, such as cell culture in vitro, blood serum pharmacology, ELISA assay, MTT assay, immunoblotting and immunofluorescence, etc.Subsequently, comprehensive evaluation methods in vivo and vitro with image analysis were adopted to observe the normal morphological indicators of the AA rats (swelling level of the toe arthrosis and arthritis exponent), the normal functional indices of T lymphocytes (migration ability and multiplication capacity), cytokines (IL-1 and TNF-α), the indicators of GRCRs signal channels (CCR2 and GRK2 expressions and phosphorylation of the receptors, subunit expressions of Gα and cell distribution, cAMP level and PKA activity) and then discuss the action mechanisms of Chengshijuanbi decoction on RA T lymphocytes. The proposed study could provide technical platform for filtration of new Chinese medicines for RA treatment and finding of new drug targets, and also afford academic and experimental foundations to carry on and develop Xinan medical science.

本研究选用具有"祛风散寒，化湿通络"功效且临床疗效确切的新安医家自创方程氏蠲痹汤，并据此提出"程氏蠲痹汤通过调节GRK2调控GPCRs信号通路而影响类风湿关节炎T淋巴细胞功能"的假说。本研究拟选用佐剂关节炎大鼠模型以及体外培养外周血和滑膜T淋巴细胞模型，以T淋巴细胞为切入点，从GRK2调控GPCRs信号通路的途径入手，借助多种技术，采用在体和离体相结合的综合评价方法，并结合图像分析，观察AA大鼠一般形态学指标（关节肿胀度、关节炎指数），T淋巴细胞一般功能（迁移能力、增殖能力），细胞因子（IL-1、TNF-α），GPCRs信号通路指标（CCR2、GRK2的表达及受体磷酸化，Gα各亚基表达及细胞分布，cAMP水平，PKA活性），探讨程氏蠲痹汤影响RA T淋巴细胞的作用机制。从而为筛选新的治疗RA中医药和寻找新的药物作用靶点提供技术平台；也为继承和发扬新安医学提供理论和实验依据。

本研究选用佐剂关节炎大鼠模型以及体外培养外周血T淋巴细胞模型，以T淋巴细胞为切入点，采用在体和离体两种实验模式，研究程氏蠲痹汤加减方对佐剂关节炎大鼠一般形态学指标，T淋巴细胞一般功能、细胞因子，以及GPCRs信号通路指标的影响，取得了一些创新性成果，特别是在程氏蠲痹汤加减方的药效和作用机制方面的探讨。为程氏蠲痹汤加减方作用机制的进一步探讨奠定了基础，也为新安医学有效自创方的临床运用提供了理论和实验依据。