补肾柔肝法调控miR-140介导软骨基质稳态失衡抑制骨关节炎筋骨失养的机制研究

The liver and kidney deficiency leading to the tendons and bones malnutrition is a pathological character of osteoarthritis. Syndrome differentiation from the liver and kidney has very distinct effects on treatment of osteoarthritis. Previously, we found that the invigorating kidney and softening liver inhibited the cartilage matrix homeostasis imbalance to improve the tendons and bones malnutrition of osteoarthritis by decreasing the inflammatory responses and increasing the cell autophagy, meanwhile the miR-140 controls the inflammation mediated the cartilage matrix homeostasis imbalance, indicating that miR-140 may be a potential target for the invigorating kidney and softening liver inhibited the tendons and bones malnutrition of osteoarthritis. Therefore, establishment of animal and cell model as research subjects by the modified Hulth’s method and the miRNA interference, investigate the effect of Tougu Xiaotong capsule with the invigorating kidney and softening liver on the morphological changes of cartilage and the expressions of miR-140, IL-1β, ERK1/2, p-ERK1/2, MMP13, ADAMTS5, Collagen II and Aggrecan in the osteoarthritis, to explore the pathomechanism of the liver and kidney deficiency leading to the tendons and bones malnutrition that is the miR-140 mediated the cartilage matrix homeostasis imbalance, using syndrome differentiation by effects of drug. It provides an experimental basis for the miR-140 as a potential target which the invigorating kidney and softening liver inhibits the tendons and bones malnutrition of osteoarthritis by regulation of the cartilage matrix homeostasis imbalance.

肝肾亏虚则筋骨失养是骨关节炎的病理基础，临床多从肝肾论治，疗效可靠。课题组前期研究发现，补肾柔肝法通过降低炎症反应，促进细胞自噬，从而抑制软骨基质稳态失衡，改善骨关节炎筋骨失养；且miR-140通过调控炎症反应介导软骨基质稳态失衡，提示miR-140可能是补肾柔肝法抑制骨关节炎筋骨失养的一个重要靶点。因此，本研究以改良Hulth法建立骨关节炎动物模型和miRNA干扰技术建立细胞模型为研究对象，采用以法测证识病的方法，观察以补肾柔肝为主的透骨消痛胶囊对骨关节炎软骨形态结构的变化与软骨细胞miR-140、IL-1β、ERK1/2、p-ERK1/2、MMP13、ADAMTS5、Collagen II、Aggrecan表达的影响，探讨miR-140介导软骨基质稳态失衡是肝肾亏虚则筋骨失养的病理机制，为初步阐明miR-140是补肾柔肝法调控软骨基质稳态失衡抑制骨关节炎筋骨失养的作用靶点提供实验基础。

补肾柔肝法是中医治疗骨关节炎的常用治法，但作用机制尚未完全阐明。软骨基质稳态失衡是软骨退变的关键病理过程，miR-140调节炎症介导软骨基质稳态失衡，为揭示骨关节炎肾虚的病因病机提供新切入点。本项目以骨关节炎动物模型与细胞模型为研究对象，采用病证结合与方证对应的方法，探讨补肾柔肝法抑制骨关节炎软骨基质稳态失衡的作用机制，结果显示：（1）透骨消痛胶囊通过降低滑膜炎症因子IL-1β、TNF-α、MMP-13含量，下调软骨Col5a1、Col1a1、Postn、ADAMTS-5、MMP-3/9/13表达，改善软骨下骨代谢与软骨基质稳态的失衡，从而延缓骨关节炎软骨退变；（2）透骨消痛胶囊通过降低炎症因子IL-1β、TNF-α、IL-6、MMP-9含量，下调ERK1/2与p38MAPK信号通路关键调控因子p-ERK1/2/ERK1/2、p-p38MAPK/p38MAPK、MEK1/2、Raf、Ras、ADAMTS-4/5、MMP-3/13、miR-34a表达，上调miR-140、miR-27b、miR-92a-3p表达，从而抑制炎症介导软骨基质稳态失衡；（3）透骨消痛胶囊通过上调内质网应激反应关键调控因子Xbp-1、miR-211表达，下调Bip、Atf4、Chop、Caspase-3/9表达，从而抑制软骨细胞凋亡介导软骨基质稳态失衡，从不同层次初步揭示软骨基质稳态失衡的复杂调控网络与补肾柔肝中药复方的多向药理特性，丰富骨关节炎肾虚的病因病机，进一步诠释从肾论治骨关节炎的科学内涵。.本项目发表学术论文12篇，其中SCI论文2篇。