基于Wnt信号通路研究“虚、瘀、毒”病机下兔膝骨关节炎软骨细胞凋亡及中药的干预机制

Knee osteoarthritis (KOA) is a common disease in the elderly, which is a major factor in developing countries to lead the elderly disabled as complex etiology, mechanisms are not very clear, about the pathological point of view, which was considered to be a kind of the articular cartilage degeneration and around the joint hyperosteogeny characterized by chronic progressive bone and joint disease. At present, the curative effect is not stable by the modern medicine or TCM, so it is great significance to study how to prevention and treatment the disease. The subject on the basis of Wnt signal pathway, to study the pathogenesis “deficiency, blood stasis, toxin” disease condition of rabbit knee osteoarthritis based, and explore the invigorating the kidney and promoting blood circulation, removing painkillers act intervention effect. Uses the Japanese big ear rabbit osteoarthritis model, from two aspects of both in vivo and in vitro experiments to investigate whether Wnt signal pathway of apoptosis and osteoarthritis of the knee joint cartilage cells associated with osteoarthritis of the knee, is “deficiency, blood stasis, toxin” pathogenesis have correlation and consistency, and the adoption of invigorating the kidney and promoting blood circulation, numbness analgesic compound Chinese medicine (Modified Duhuo Jisheng mixture) intervention anyway, verification of “virtual, stasis, toxin” disease condition of rabbit knee osteoarthritis with classical pathway (Wnt pathway) signal factor in the activation and inhibition, resulting in the molecular mechanism of osteoarthritis of the knee, for invigorating the kidney and promoting blood circulation, removing painkillers act traditional Chinese medicine to provide a scientific basis for the treatment of osteoarthritis of the knee osteoarthritis, rich scientific connotation of “deficiency, blood stasis, toxin” pathogenesis.

膝骨关节炎(Knee Osteoarthritis，KOA)是中老年人常见疾病，是发展中国家导致老年人病残的主要因素，病因复杂，机制尚不完全清楚，从病理角度来看，是一种以关节软骨退变和关节周围骨质增生为特征的慢性进行性骨关节病。目前中、西医缺乏特异性治疗，疗效欠稳定，因而阐明KOA的发病机制及研究有效防治具有重要的意义。本课题在前期工作的基础上，采用日本大耳家兔骨性关节炎模型，从体内和体外实验两方面探讨Wnt信号通路是否与膝骨关节炎关节软骨细胞的凋亡有关，是否与膝骨关节炎“虚、瘀、毒”病机具有相关性和一致性，并采用补肾活血、祛痹止痛中药复方干预进行反正，验证“虚、瘀、毒”病机状态下兔膝骨关节炎Wnt通路中的信号因子激活与抑制，从而导致膝骨关节炎的发生的分子机制，为补肾活血、祛痹止痛法类中药治疗膝骨关节炎提供科学依据，丰富骨性关节炎“虚、瘀、毒”病机的科学内涵。

本课题在前期工作的基础上，采用新西兰大白兔骨性关节炎模型，从体内和体外实验两方面探讨Wnt信号通路与膝骨关节炎关节软骨细胞的凋亡有关，膝骨关节炎“虚、瘀、毒”病机具有相关性和一致性，并采用补肾活血、祛痹止痛的加味独活寄生合剂干预进行反证。结果表明（1）采用石膏伸直位固定兔膝关节6周，是建立稳定的KOA模型的有效方法；采用石膏固定，结合氢化可的松灌胃与环境因素干预可建立具有“虚、瘀、毒”病机特点的兔KOA病证结合模型。（2）加味独活寄生合剂可从整体上改善KOA病证结合模型兔的中医证候体征，并且可通过抑制Wnt/β-catenin通路，调控Wnt3a、β-catenin、MMP-13、Bax和Bcl-2的mRNA和蛋白表达，进而有效抑制软骨细胞过度凋亡，起到保护关节软骨的作用。（3）加味独活寄生合剂含药血清能促进兔正常软骨细胞及骨髓间充质干细胞成软骨诱导细胞的增殖，其作用机制可能通过影响Wnt信号通路中的Wnt5a、β-catenin、SOX9等基因的表达，调控Wnt信号通路，进而调节细胞周期的G1期，促进正常软骨细胞的增殖。（4）加味独活寄生合剂含药血清能促进兔退变的软骨细胞凋亡，其作用机制可能通过影响Wnt信号通路中的Wnt5a、β-catenin、Casp3/8等基因的表达，调控Wnt信号通路，从而促进退变的软骨细胞凋亡，结合加味独活寄生合剂临床治疗KOA患者，其作用机制与中医“瘀去新生”理论相符合。（5）效液相色谱-四级杆-时间飞行质谱仪分析加味独活寄生合剂和含药血清的主要成分，其体内直接作用物质很可能出自血清中的33个药源性成分, 结合血清药理学对其作进一步研究有助于阐明该制剂药效物质基础。（6）加味独活寄生合剂的单体成分牛膝多糖能促进人KOA软骨细胞的增殖，能够调控软骨细胞自噬的水平和抑制KOA软骨细胞凋亡，其机制可能是通过抑制Caspase-3/9途径发挥作用。.本项目初步证实了Wnt信号通路与膝骨关节炎关节软骨细胞的凋亡有关，加味独活寄生合剂通过调控Wnt信号通路发挥药效作用，为中医药治疗骨关节疾病提供了一定的科学依据，同时丰富了骨关节炎“虚、瘀、毒”病机的科学内涵。