基于PI3K/AKT/mTOR通路探讨骨痹通方调控骨关节炎软骨细胞自噬的机制

The chondrocyte homeostasis maintains the normal form and function of articular cartilage, while a normal level of autophagy is a basic element sustaining the chondrocyte homeostasis. The decrease of chondrocyte autophagy may contribute to the initiation and development of osteoarthritis (OA), while PI3K/AKT/mTOR pathway is the key pathway which is associated with chondrocyte autophagy. “Kidney governs bone” and kidney deficiency is the main pathogenesis of OA. Results from our previous study showed that Gu-Bi-Tong recipe could improve the pain, stiffness and function of joint for patients with OA through tonifying kidney. However, the mechanism could not be explained fully only by inhibiting chondrocyte apoptosis from the regulation of Wnt/β-catenin pathway. In this case, we hypothesize that “the decrease of chondrocyte autophagy may be a biological foundation of kidney deficiency, tonifying kidney can improve the level of chondrocyte autophagy, so as to cure OA”. In this study, experiment in vivo and vitro will be conducted to explore the relationship of kidney deficiency and chondrocyte autophagy, and the effect of Gu-Bi-Tong recipe on chondrocyte autophagy layer by layer in animal-cellular-molecular system by PI3K/AKT/mTOR pathway, partly reveal the mechanism of Gu-Bi-Tong recipe regulating chondrocyte autophagy, so as to provide a further experiment evidence for clinical application of kidney-tonifying traditional Chinese medicine in treating OA.

软骨细胞内稳态维持着关节软骨的正常形态和功能，而正常的自噬水平是维持软骨细胞内稳态的基本要素。骨关节炎(OA)的发生、进展与软骨细胞自噬水平下降有关，PI3K/AKT/mTOR通路是调控软骨细胞自噬的关键通路。“肾主骨”，肾虚是OA的关键病机。前期研究提示，以补肾立法的骨痹通方能改善OA患者的疼痛、僵硬和关节功能，但仅从调控Wnt/β-catenin通路抑制软骨细胞凋亡尚不能完全解释骨痹通方治疗OA的作用机制。基于此，我们提出“软骨细胞自噬水平下降也可能是肾虚的生物学基础，补肾可使低下的软骨细胞自噬水平升高，达到治疗OA的作用”的假说。本研究拟采用体内与体外实验相结合的方法，以软骨细胞PI3K/AKT/mTOR通路为切入点，从动物-细胞-分子体系逐层解析肾虚与软骨细胞自噬的关系及补肾中药骨痹通方对其的影响，部分揭示骨痹通方调节软骨细胞自噬的作用机制，为补肾中药治疗OA提供进一步的实验依据。

骨关节炎(osteoarthritis, OA)是最常见的慢性关节病，以软骨细胞的凋亡与软骨基质的降解为主要病理特点。既往研究表明，过度自噬能导致软骨细胞凋亡。中医认为，肾虚是OA的关键病机。因此，我们推测“软骨细胞自噬”可能与中医“肾虚”存在关联，以补肾立法的骨痹通方可能通过调节软骨细胞自噬改善OA的软骨退变。本研究建立肾阳虚OA复合模型，以PI3K/AKT/mTOR通路为切入点，观察肾阳虚OA大鼠软骨细胞自噬水平的变化，探索骨痹通方对自噬相关蛋白及软骨基质分解酶的影响，明确骨痹通方能否改善OA软骨退变，部分阐明该方抑制软骨细胞过度自噬以治疗OA的作用机制。研究结果表明：（1）相较于正常大鼠，OA大鼠存在软骨退变，软骨细胞中自噬相关基因Atg5、Atg7、Atg12表达升高，p62蛋白表达减少，LC3B-I向LC3B-II转化，自噬小体大量出现，提示OA大鼠存在软骨细胞过度自噬。同时，肾阳虚OA大鼠软骨退变与上述指标异常更加显著，提示肾阳虚能加重OA大鼠的软骨退变和软骨细胞过度自噬。（2）骨痹通方能改善OA大鼠膝关节软骨病理Mankin's评分，在降低p-PI3K和p-AKT（ser473）表达的同时，一定程度上逆转软骨细胞的过度自噬、凋亡及软骨基质代谢的紊乱，提示在肾阳虚OA大鼠模型中，骨痹通方可能通过抑制PI3K/AKT/mTOR通路发挥抑制OA软骨细胞过度自噬和凋亡的作用，进而可以改善OA软骨退变。（3）在体外实验中，LPS刺激软骨细胞后，MMP-13、Beclin1、p-PI3K、p-AKT的表达和Bax/Bcl-2比率上升，细胞自噬和线粒体自噬的荧光信号明显增强，表明LPS刺激可引起软骨细胞过度自噬和凋亡率增加，骨痹通方干预能在一定程度上逆转上述改变，提示骨痹通方能够抑制LPS诱导的软骨细胞过度自噬和凋亡，且该作用可能与其抑制PI3K/AKT/mTOR通路有关。综上所述，本研究结论：OA大鼠存在软骨退变和软骨细胞过度自噬，而肾阳虚能加重上述改变；骨痹通方能通过抑制软骨细胞过度自噬和凋亡改善OA软骨退变，且该作用可能与其抑制PI3K/AKT/mTOR通路异常激活有关。