金匮肾气丸通过NOX4-ROS通路调控糖尿病肾病足细胞自噬的机制研究

Podocyte injury plays an important role in the development of diabetic nephropathy and proteinuria. And there is closely relationship between oxidative stress and autophagy or apoptosis in podocyte. In previous research we found that Jinkuishenqi pill can reduce glomerular fibrosis and urine albumin of db/db mice, and activate autophagy in podocyte. So we speculate that Jinkuishenqi pill could attenuate podocyte injury and improve diabetic nephropathy by the activation of autophagy and the inhibition of apoptosis through NOX4-ROS pathway. Based on this hypothesis, we will use db/db mice as type II diabetic animal models and podocyte models induced by high glucose, to observe the effect of Jinkuishenqi pill on podocyte autophagy and apoptosis by detecting autophagy flow, autophagosome formation or TUNEL technology, to abserve the effect of Jinkuishenqi pill on intracellular ROS and oxidative stress response in podocyte by immunofluorescence labeling, coimmunoprecipitation and siRNA, and then to discuss the effect of Jinkuishenqi pill on the activation of NOX4 subunit and the formation or dissociation of Bcl-2/Beclin1 complex. We want to clarify the molecular mechanism of Jinkuishenqi pill on the activation of autophagy by AMPK/mTOR pathway, the inhibition of apoptosis by JNK1/Bcl-2 pathway and the protection of the podocyte keeping from injury, and then this project may provide the experimental basis for clinical treatment.

足细胞损伤是导致糖尿病肾病蛋白尿发生和病程进展的重要原因，而氧化应激与足细胞自噬、凋亡关系十分密切。前期工作发现，金匮肾气丸能减轻db/db小鼠肾小球硬化、减少尿蛋白，降低高糖诱导的足细胞ROS产生，并激活自噬，推测金匮肾气丸可能通过足细胞NOX4-ROS通路，激活自噬及抑制凋亡，减轻足细胞损伤，延缓糖尿病肾病病理进程。因此，我们拟采用db/db小鼠建立糖尿病肾病模型及高糖诱导永生化足细胞损伤模型，应用自噬流检测、自噬体形成及TUNEL染色观察金匮肾气丸对于足细胞自噬与凋亡的影响；采用免疫荧光标记、免疫共沉淀及RNA干扰技术，观察金匮肾气丸对足细胞ROS含量及氧化应激的作用，探讨其对NOX4亚基活化及Bcl-2/Beclin1复合体形成或解离的影响，阐明金匮肾气丸激活AMPK/mTOR通路依赖的自噬，抑制JNK1/Bcl-2途径依赖的凋亡，减轻足细胞损伤的分子机制，为临床治疗提供实验依据。

足细胞损伤是导致糖尿病肾病蛋白尿发生和病程进展的重要原因，而氧化应激与足细胞自噬、凋亡关系十分密切。本项目主要研究金匮肾气汤对db/db糖尿病小鼠足细胞损伤、自噬、凋亡及氧化应激的影响，通过体内和体外实验，探讨金匮肾气汤延缓糖尿病肾病进展的机制。 通过透射电子显微镜观察糖尿病小鼠肾脏足细胞内自噬体情况，通过Western blot检测糖尿病小鼠肾组织和足细胞自噬相关指标LC3I/II、p62、p-mTOR/mTOR、p-AMPK/AMPK，凋亡相关指标caspase-3、Bcl-2、p-JNK1/JNK1，氧化应激相关指标NOX4、p22phox及p47phox蛋白在肾组织的表达。结果表明，金匮肾气汤通过AMPK-mTOR途径促进足细胞自噬，保护足细胞，延缓DN的进展；同时，金匮肾气汤可能通过对JNK1/Bcl-2信号途径来减少足细胞凋亡，从而延缓DN进展；金匮肾气汤对足细胞自噬与凋亡的调节可能与其改善足细胞氧化应激有关。这些发现为进一步了解金匮肾气汤在DN中肾保护作用的分子机制提供了新的思路，为金匮肾气汤在临床治疗糖尿病肾病时提供了理论支持。