黄连解毒汤靶向KIM-1/AMPK通路调控自噬对狼疮性肾炎肾小管上皮细胞损伤的保护机制研究

Renal tubular injury is an important factor in the long-term prognosis for patients with lupus nephritis(LN).Epithelial autophagy plays a critical role in this process.Our early study found that Kidney injury molecule-1(KIM-1)/AMPK signal pathway mediates tubular injury by inducing renal tubular epithelial cells(RTECs) autophagy and Huang Lian Jie Du Decoction(HLJDD) can attenuate this pathological process. However, the molecular mechanisms still remain unclear. To solve this question，we plan to use the cGVHD lupus mouse model and RTECs inflammatory damage cell model as the research objects and focus on the KIM-1/AMPK signal pathway and RTECs autophagy. The methods of in vitro and in vivo experiments such as Gene transfection, RNAi, RFP-GFP-LC3 double fluorescence autophagy indicator system, autophagy interference/induction, AMPK pathway interference, experimental zoology technology will be involoved in this project. We want to find out the biological regulation relationship between KIM-1/AMPK signal pathway and RTECs autophagy based on the level of molecules, cells and animals，as well as the protection mechanism of HLJDD on RTECs injury in LN. We expect our research may provide a scientific evidence for the clinical treatment of LN renal tubular injury based on Traditional Chinese Medical triple burner theory.

肾小管损伤是导致狼疮性肾炎（LN）肾功能远期预后不良的重要因素，自噬是肾小管上皮细胞（RTECs）损伤的关键途径。我们在前期研究发现肾脏损伤分子-1（KIM-1）/AMPK信号通路可能通过诱导RTECs自噬参与LN进展，黄连解毒汤可抑制KIM-1，但具体作用机制仍需进一步阐明。因此，本项目以前期构建的cGVHD狼疮小鼠模型、RTECs炎症损伤细胞模型为研究对象，以KIM-1/AMPK为切入点，以细胞自噬为基础，运用基因转染、RNAi、RFP-GFP-LC3双荧光自噬指示体系、自噬干扰/诱导、AMPK信号通路干扰及实验动物学等技术，采用体外与体内实验相结合的方法，从分子、细胞和动物水平动态研究LN肾小管损伤进程中KIM-1/AMPK信号通路与RTECs自噬之间存在的生物介导及效应靶向关系，阐明黄连解毒汤保护LN肾小管损伤的作用机制，以期为中医从三焦热毒伏邪论治LN理论的临床运用提供科学依据。

狼疮性肾炎（Lupus Nephritis，LN）是系统性红斑狼疮最常见的并发症，肾小管损伤是导致LN肾功能预后不良的重要因素。前期研究发现肾脏损伤分子-1（KIM-1）/AMPK信号通路诱导肾小管上皮细胞自噬性死亡可能是LN肾小管损伤重要的分子机制。中医经典方剂黄连解毒汤可抑制肾小管上皮细胞KIM-1表达。因此，本项目以cGVHD狼疮小鼠模型、RTECs炎症损伤细胞模型为研究对象，研究了LN肾小管损伤进程中KIM-1/AMPK信号通路与RTECs自噬之间存在的生物介导及效应靶向关系，阐明黄连解毒汤保护LN肾小管损伤的作用机制。主要完成以下内容：1.黄连解毒汤活性成分的网络药理学研究结果提示，黄连解毒汤及其活性成分的生物学作用主要涉及细胞增殖、凋亡、免疫炎症、过氧化损伤等方面，其作用机制与调控细胞基因表达、增殖和凋亡信号通路、炎症因子释放等相关。2.黄连解毒汤可改善cGVHD狼疮肾炎小鼠模型生存率、体重，减轻cGVHD狼疮小鼠肾脏组织肾小管上皮细胞变性、坏死脱落、间质炎症细胞浸润等病理损害，改善肾功能及免疫学指标，其作用机制与抑制肾脏STAT3炎症通路活化、下调促炎细胞因子水平、降低LC3-II相关自噬性死亡相关。3.选择性上调KIM-1可上调 p-AMPK、AMPK、ULK1 蛋白表达 水平，下调p-mTOR、p-S6蛋白表达，促进HK-2细胞自噬性死亡，抑制KIM-1可抑制细胞自噬，减少细胞死亡，结果提示KIM-1是促进cGVHD狼疮RTECs自噬性死亡的关键分子，是黄连解毒汤治疗潜在靶点。4.黄连解毒汤活性成分小檗碱可改善顺铂致AKI小鼠模型肾小管病理损伤，减少细胞凋亡及肾小管间质炎性细胞浸润，减轻顺铂诱导的肾小管上皮细胞脂质过氧化反应，增强PINK1/Parkin介导的线粒体自噬减轻细胞凋亡。本项目的实施为中医从三焦热毒伏邪理论采用黄连解毒汤治疗LN的临床运用提供科学依据。