基于PI3K/AKT/mTOR及AMPK/mTOR/ULK1信号通路调控自噬的交互作用探讨加味黄芪赤风汤对足细胞损伤的保护机制

Chronic kidney disease (CKD) is a major disease that seriously endangers human health. Podocyte injury is an important factor in CKD proteinuria and is closely related to the mechanism of autophagy. Previous studies have shown that activating PI3K/AKT/mTOR signaling pathway and decreasing podocyte autophagy are the mechanisms of Huangqi Chifeng Decoction to reduce proteinuria and renal damage. In the podocyte autophagy,activate PI3K/AKT/mTOR and AMPK/mTOR/ULK1 signaling pathway can inhibit or promote cell autophagy, the two signaling pathway opposing constraints, regulating autophagy and cellular homeostasis. But the mechanism of Modified Huangqi Chifeng Decoction to adjust the two signal pathways in order to regulate and maintain the level of autophagy, need to be further elucidated. This research objective is from the two signal pathways in the interaction process of podocyte autophagy and regulation of autophagy，use adriamycin to establish rat and cell model and detect protein content of kidney and Sertoli cell and gene levels by immunohistochemistry, Western blot and RT-PCR，which can prove that regulating the two signaling pathways regulating podocyte autophagy and maintaining cell homeostasis is the potential target of Astragalus Chifeng decoction, revealing the molecular mechanism of interaction between these two signaling pathways during autophagy.

慢性肾脏病(CKD)是严重危害人类健康的重大疾病。足细胞损伤是导致CKD蛋白尿的重要因素，与自噬机制密切相关。前期研究表明：激活PI3K/AKT/mTOR信号通路，减少足细胞过度自噬是加味黄芪赤风汤降低蛋白尿，减轻肾损伤的作用机制。在足细胞自噬中，PI3K/AKT/mTOR及AMPK/mTOR/ULK1信号通路的激活可抑制或促进细胞自噬水平，二者对立制约，共同调节自噬维持细胞稳态，而加味黄芪赤风汤如何调节此两条信号通路以达到调控自噬、维持细胞自噬水平的机制需进一步阐明。本研究拟从上述两条信号通路在足细胞自噬过程中的交互作用及调控自噬的角度，利用阿霉素建立大鼠及足细胞实验模型，通过免疫组化、免疫印迹、RT-PCR法检测肾组织和足细胞相关蛋白含量和基因水平，证明通过调节此两条信号通路调控足细胞自噬、维持细胞内稳态是加味黄芪赤风汤的作用靶点，揭示在自噬过程中此两条信号通路存在交互作用的分子机制。

慢性肾脏病主要表现为肾小球滤过率下降和/或尿蛋白排泄增加，呈不可逆性、进行性进展。蛋白尿是慢性肾脏病进展的重要标志以及危险因素，足细胞包裹在肾小球滤过屏障毛细血管周围，足细胞足突的融合或消失是出现蛋白尿的重要原因。足细胞显示出较高的基础自噬水平，自噬失调参与多种肾脏疾病进程。前期研究表明，加味黄芪赤风汤能够减少阿霉素肾病大鼠尿蛋白，减少足细胞内自噬小体，下调LC3以及beclin-1的表达，抑制肾脏过度自噬。自噬负调控信号通路PI3K/AKT/mTOR以及自噬正调控信号通路AMPK/mTOR对自噬的双向调控作用，与中医阴阳平衡、阴阳互根理论有一定的契合之处。基于这种认识，本研究通过体内、外实验相结合，进一步探讨加味黄芪赤风汤调控自噬对肾脏的保护作用机制。研究结果表明：（1）加味黄芪赤风汤能够减少阿霉素肾病大鼠尿蛋白，改善肾脏病理损伤。（2）3MA能够减少阿霉素肾病大鼠尿蛋白，改善肾脏病理损伤，表明过度自噬是导致肾脏损伤的重要机制。加味黄芪赤风汤通过激活自噬负调控信号通路PI3K/AKT/mTOR，同时抑制自噬正调控信号通路AMPK/mTOR，抑制过度自噬，发挥肾脏保护作用。（3）体外培养足细胞，分别通过抑制PI3K的表达，拮抗加味黄芪赤风汤对PI3K/AKT/mTOR信号通路的激活作用，以及提高AMPK的表达，拮抗加味黄芪赤风汤对AMPK/mTOR信号通路的抑制作用，进一步证明加味黄芪赤风汤能够激活PI3K/AKT/mTOR信号通路，同时抑制AMPK/mTOR信号通路，调控二者维持自噬平衡的交互作用，抑制过度自噬，发挥足细胞保护作用。本研究为加味黄芪赤风汤的肾脏保护作用提供了机制基础，为加味黄芪赤风汤的进一步开发研究成院内制剂或新药奠定了基础，也为中医药的肾脏保护机制研究提供了模式参考。