骨碎补总黄酮通过软骨细胞生物钟PI3K/AKT/Bmal1通路调控骨关节炎软骨细胞凋亡的机制研究

With a high morbidity, osteoarthritis (OA) is the most common type of chronic arthritis that causes deformity and loss of function in advanced stage, which seriously influence patient's quality of life. OA is characterized by cartilage degeneration and damage. Cartilage cell hypertrophy and apoptosis, regulated by the abnormal genes expression of circadian clock in chondrocytes, is the main pathological basis of OA. Kidney deficiency is the main pathogenesis of OA. According to previous studies, we found that a traditional Chinese medicine (TCM) prescription with drynaria total flavonoids as core drug can regulates circadian clock in chondrocytes of OA. On the basis of the theoretical consistency among “circadian clock in chondrocytes”, “kidney governs bone” and “kidney governs growth, development and reproduction”, we hypothesize that: Kidney deficiency may be accompanied by the changes of circadian clock in chondrocytes, drynaria total flavonoids can regulate apoptosis of OA cartilage cell through the PI3K/AKT/Bmal1 signaling pathway. In this study, mice with OA and kidney deficiency and SW1353 cell line will be introduced as objects, animal and cell experiments will be conducted to explore changes of PI3K/AKT/Bmal1 in mice with OA and kidney deficiency, and effects of drynaria total flavonoids. A cell blocking experiment will also be conducted to further explore the special targets in PI3K/AKT/Bmal1 pathway. In a word, through the animal - cellular - molecular system, we will explore the relationship between kidney deficiency and circadian clock in chondrocytes of OA, as well as the effects of drynaria total flavonoids, in order to provide an experimental basis for the clinical application of tonifying kidney TCM in treating OA.

骨关节炎(OA)发病率高，危害大，严重影响患者生活质量，晚期可致残。OA以软骨变性、破坏为特征，软骨细胞的肥大和凋亡是其主要病理基础，受软骨细胞生物钟基因异常表达调控。肾虚是OA的主要病机，课题组前期发现以骨碎补为君药的中药复方具有调节OA软骨细胞生物钟的作用。根据“软骨细胞生物钟”与中医“肾主骨”、“肾主生长发育”理论的一致性，我们提出：肾虚伴随着软骨细胞生物钟变化，骨碎补总黄酮通过调节软骨细胞生物钟PI3K/AKT/Bmal1通路调控OA软骨细胞凋亡。本项目拟以肾虚OA大鼠和SW1353细胞株为对象，观察肾虚OA大鼠PI3K/AKT/Bmal1通路的变化及骨碎补总黄酮对通路的影响，以细胞水平阻断实验验证该通路的特异性，从动物-细胞-分子体系逐层解析肾虚与OA软骨细胞生物钟的关系以及骨碎补总黄酮调控OA软骨细胞凋亡的作用机制，以期为补肾中药临床治疗OA提供实验基础。

骨关节炎(osteoarthritis，OA)是一种以软骨变性、破坏为特征慢性关节疾病，软骨细胞的凋亡与软骨基质的异常降解是其主要病理基础。既往研究表明，软骨细胞的肥大、凋亡与软骨细胞生物钟的变化密切相关。 “软骨细胞生物钟”与中医“肾主生长发育”及“人体生物节律”的理论存在某种密切关系，研究两者的关系可望更好地揭示肾虚在 OA 中的生物学基础。本研究通过建立肾阳虚OA 复合模型，以 PI3K/AKT/Bmal1 通路为切入点，探明肾阳虚 OA 大鼠软骨细胞生物钟的变化，并探究骨碎补总黄酮对肾阳虚OA的调节作用。进一步在体外实验中阻断 PI3K/AKT/Bmal1通路，观察骨碎补总黄酮对生物钟通路、软骨基质分解酶的影响，阐明骨碎补总黄酮通过调节软骨细胞生物钟调控OA的可能作用机制。结果表明，OA大鼠模型中p-AKT（ser473）水平明显升高、生物钟因子Bmal1表达降低、软骨基质分解酶MMP13表达升高、软骨基质保护蛋白TIPM4表达降低，提示OA大鼠出现生物钟的异常及软骨基质代谢的紊乱。经过肾阳虚造模后软骨退变进一步加重且上述指标的异常更加的明显，提示肾阳虚造模进一步加重了OA大鼠的生物钟异常及软骨基质代谢的紊乱。使用骨碎补总黄酮降低了p-AKT（ser473）的表达的同时在一定程度上逆转了生物钟的异常及软骨基质代谢的紊乱，提示在肾阳虚OA大鼠模型中骨碎补总黄酮可能是通过抑制PI3K/AKT通路发挥了对Bmal1及MMP13的调节作用，进而改善了肾阳虚相关症状及关节软骨的退变。在体外实验中，IL-1β刺激软骨细胞进行干预后Cry1、Per1、Bmal1表达降低，MMP13、MMP14、Adamts4及p-AKT（ser473）表达升高，使用骨碎补总黄酮干预后能在一定程度上逆转Cry1、Per1、Bmal1、MMP13、Adamts4及p-AKT（ser473）的异常改变。使用雷帕霉素作为PI3K/AKT下游通路mTOR的抑制剂对IL-1β刺激软骨细胞进行干预后，Bmal1、MMP13升高，TIPM4降低，提示mTOR参相关调节作用。雷帕霉素与骨碎补总黄酮共同干预后，较之于单独使用雷帕霉素，Bmal1、MMP13进一步升高，TIPM4进一步降低，提示骨碎补总黄酮除PI3K/AKT/mTOR途径外仍具有其他的途径参与Bmal1、MMP13、TIPM4的调节作用。