淫羊藿苷通过PI3K/Akt/mTOR 及ULK1 通路在骨关节炎中的作用研究

Osteoarthritis (OA) is a degenerative arthritis characterized by articular cartilage destruction, synovial inflammation, and osteophytosis. There is no sufficient treatment so far. Since autophagy is defective in OA, activation of autophagy through mTOR signaling may become a new OA treatment target. Recent animal experiments have shown that rapamycin can be used to treat OA by inhibiting mTOR, which is difficult to be popularized because of the many adverse reactions of rapamycin. Epimedium has the effect of strengthening tendons and bone, its main component is icariin, which has the effect of regulating autophagy. In order to further investigate whether the protective effect of icariin on OA is regulated by autophagy through PI3K/Akt/mTOR-ULK1 signaling or not, ULK1 is over expressed and knocked down. Then phenotype of OA models as well as mTOR-related molecules in autophagy is measured after icariin treatment. ULK1 knock out mice will also be treated with Epimedium to investigate the role of ULK1 in the Torin1-mTOR signaling pathway. The results of this study will help to understand the mechanism and therapeutic effect of icariin on OA from a new perspective.

骨关节炎(OA )是以关节软骨变性、破坏及骨质增生为特征的退行性病变。目前尚无理想的OA治疗药物。自噬参与了OA的发病机制。通过调节mTOR信号通路，上调自噬成为目前治疗OA的一个新策略。近来动物实验表明，雷帕霉素通过抑制mTOR而上调自噬用于治疗OA，由于雷帕霉素不良反应多，难以临床推广。淫羊藿具有强筋健骨之功效，其主要成分为淫羊藿苷，具有调控自噬的作用。本研究通过淫羊藿苷作用于白介素-1β诱导的细胞OA模型及关节内注射胶原酶诱导的OA小鼠模型，观察淫羊藿对OA软骨细胞的保护作用是否与自噬有关；运用ULK1过表达和基因沉默手段，以及ULK1基因敲除小鼠，研究淫羊藿对OA软骨细胞发挥保护作用是否通过调控自噬PI3K/Akt/mTOR及ULK1信号通路实现，旨在从一个新角度探索淫羊藿苷防治OA的可能机制。

目的：旨在探讨淫羊藿苷（ICA）对骨关节炎 (OA) 自噬的影响以及其潜在的作用机制。.方法：（1）细胞实验：ICA预处理SW1353软骨细胞2小时，然后用IL-1β刺激以模拟OA细胞炎症模型，使用实时PCR和Western blot检测基质金属蛋白酶（MMP-3）和胶原蛋白II（COL2）的表达水平；运用ULK1-siRNA基因沉默ULK1，转染至SW1353软骨细胞，Western blot检测转染前后自噬相关蛋白ULK1、Beclin-1、LC3和p62的表达，PI3K, Akt, mTOR, 和ULK1的磷酸化水平；（2）动物实验：通过改良Hulth法建立OA动物模型，使用HE染色、番红固绿法、免疫组化法检测软骨组织病理状态；免疫组化检测Beclin-1的表达。.结果：（1）IL-1β增加了MMP-3的表达，诱导了COL2的降解，并降低了自噬相关蛋白的表达水平，包括ULK1、Beclin-1和LC3，而ICA预处理减弱了IL-1β诱导的MMP-3的过度产生，增加COL2表达，并诱导自噬相关蛋白的表达；同时，ICA减少了PI3K、Akt和mTOR的磷酸化，增加了ULK1的产生，并诱导自噬；shRNA敲低ULK1后，激活PI3K/Akt/mTOR，从而逆转了ICA的保护作用。（2）在改良 Hulth 法构建的 OA 动物模型中，ICA 显著改善了 OA 软骨组织的病理表现，同时激活软骨组织自噬相关蛋白Beclin-1表达。.结论：ICA通过调节PI3K/AKT/mTOR/ULK1信号通路诱导自噬，改善OA炎症，ICA可能是治疗OA的有效方法。