蜂针治疗RA穴效关系及其基于调控OPG/RANK/RANKL系统的骨保护机制研究

Rheumatoid arthritis(RA) is an autoimmune disease with unknown etiology and pathogenesis. There is no radical drug being developed and applied. As one of the most unique therapies for the treatment of RA in traditional Chinese medicine, bee needling has significant efficacy of analgesia, anti-inflammation, prevention of joint deformation. Our previous studies have shown that bee needling plays an important role in the treatment of RA through immune regulation of cytokines and inhibition of abnormal apoptosis of synovial cells, and has a dose-effect and time-dependent relationship. In our study, we found that the efficacy of bee needling in the treatment for RA was related to acupoints selection, and long-term bee needling treatment could significantly inhibit bone damage. Therefore, we intend to investigate the relationship with efficacy and mechanism of bee needling and acupoints selection for RA, on the basis of dose-effect and time-effectiveness of bee needling, ainming to provide powerful experimental evidence of acupoints prescriptions of bee needling treatment for RA, as well as the establishment of standardized RA treatment prescription. And in the further experiment, we want to validate our hypothesis that bee needling plays roles in bone protection through improving the level of OPG and OPG/RANKL, promoting OPG expression, downregulating the expression of osteoclasts RANK receptor and the downstream signaling pathways, inhibiting OC maturation and differentiation, and provide a theoretical basis for bone protection of bee needling against RA.

类风湿关节炎（RA）是一种病因病机未明的自身免疫性疾病，迄今未有根治性药物成熟和面世应用。蜂针作为中医药治疗RA的众多疗法中较为独特的一种，其止痛、消炎、防止关节继续变形的作用非常明显。申请者前期研究表明：蜂针治疗RA是通过对细胞因子的免疫调控和抑制滑膜细胞的异常凋亡来发挥作用的，并且具有量效、时效关系。我们在研究中发现蜂针治疗RA的疗效跟蜂刺的腧穴选择有一定关系，且长期接受蜂针治疗能明显抑制骨损害。基于此，我们申请了本项目，拟在量效、时效关系的研究基础上，探讨蜂针治疗RA的穴效关系及机制，为蜂针治疗RA最佳选穴处方以及标准化方案的制定提供有力的实验依据；并进一步实验验证我们提出的“蜂针是通过提高OPG和OPG/RANKL水平，促进OPG表达，下调破骨细胞RANK受体及下游相关信号通路的表达，抑制OC成熟分化，从而起到骨保护作用的”假说，为蜂针对RA的骨保护作用提供理论依据。

项目背景：蜂针对RA的止痛、消炎、防止关节继续变形作用显著。我们的前期研究证实了蜂针治疗RA的量效、时效关系，且其疗效与经穴的选择密切相关，此外，长期接受蜂针治疗可减轻骨损害。基于此，我们探究了蜂针治疗RA的穴效关系及其骨保护的机制。.研究内容、结果：建立佐剂性关节炎大鼠模型探讨蜂针治疗RA的穴效关系：在中医针灸学原理指导下，确立近部取穴、远部取穴及远近结合取穴法进行活蜂螫刺干预；采用H&E染色观察关节滑膜病理改变；免疫组化法检测关节滑膜TNF-α表达；WB检测关节滑膜蛋白含量；ELISA法检测血清IL-6和IL-17含量。结果表明三种取穴方法均降低了关节肿胀程度，抑制关节炎症，减轻关节损害，以近部取穴组疗效最为显著，机制可能与下调IL-6、IL-17水平，抑制滑膜中TNF-α表达相关。体外培养RAW264.7诱导其分化为破骨细胞，制备含蜂毒液血清，CCK-8法检测含蜂毒液血清对RAW264.7细胞增殖的影响，WB法检测下游关键信号通路蛋白。CCK-8法显示含蜂毒液血清对RAW264.7细胞的增殖呈梯度依赖性抑制，浓度在2.5%时，RAW264.7细胞增值率最高。采用RANKL刺激诱导RAW264.7细胞为破骨细胞，取2.5%浓度含蜂毒液血清进行干预，检测下游OPG、NFATc1蛋白表达，结果表明含蜂毒液血清可增加OPG表达，降低NFATc1蛋白水平，抑制破骨细胞分化。建立胶原诱导性关节炎小鼠模型，在穴效关系的基础上，予蜂毒肽穴位注射验证蜂针对RA的骨保护作用：ELISA法检测血清ALT、AST、Crea进行安全性评价；关节H&E染色、micro-CT检查评估骨破坏情况；TRAP染色评估破骨细胞分化情况。结果表明蜂毒肽穴位注射可以减轻滑膜组织增生，减少炎性细胞浸润和软骨、骨破坏，抑制RA破骨细胞分化相关基因表达，且具有明确的安全性。.科学意义：我们在已取得的蜂针治疗RA的时效、量效关系的基础上，设计了本实验方案，深入探讨蜂针治疗RA的穴效关系及骨保护机制，为蜂针治疗RA最佳选穴处方以及标准化方案的制定提供有力的实验依据。