清热消癥法调控线粒体自噬AMPK-ULK1关键节点修复足细胞损伤延缓糖尿病肾病进展的作用机制

Diabetic nephropathy (DN) is the main cause of end-stage nephropathy, and podocyte injury is the key factor for its development, while the injury of podocyte is closely related to mitochondrial autophagy. We believe that the core pathogenesis of diabetic nephropathy in traditional Chinese medicine is "ReXieZhiZhen ". Under the guidance of this theory, the method of clearing away heat and eliminating symptoms can regulate autophagy and delay the progression of diabetic nephropathy. On the basis that regulating mitochondrial autophagy may delay DN, we proposed the hypothesis that “QingReXiaoZheng Fa” can delay DN progression by activating mitochondrial autophagy, protecting mitochondrial function, and alleviating renal podocyte injury and apoptosis". This research by the high sugar stimulating kidney podocytes model and knocking down ULK1 model as the research object, explore “QingReXiaoZheng Fa”whether can regulate the expression of ULK1, intervention AMPK - ULK1 pathways, activate the mitochondria play a role of protecting mitochondrial autophagy, clarify QingReXiaoZheng Fa treatment of DN and activate the correlation of mitochondrial autophagy, verify the hypothesis is scientific and objective, to explore the exact effect of heat dissipation disease party in the treatment of DN targets and effect mechanism, to "ShenLouZhengJia " theory as the instruction of TCM prevention and treatment of diabetic nephropathy research and provide the theoretical basis and experimental basis of traditional Chinese medicine.

糖尿病肾病（DN）作为终末期肾病的主要原因，足细胞损伤是其发展的关键因素，而足细胞的损伤和线粒体自噬密切相关。我们认为糖尿病肾病的中医核心病机是“热邪致癥”，在此理论指导下的清热消癥法可以调控自噬延缓糖尿病肾病进展。基于调控线粒体自噬可能延缓DN的基础上，我们提出“清热消癥法可以通过激活线粒体自噬，保护线粒体功能，减轻肾脏足细胞的损伤和凋亡，从而延缓DN进展”的假说。本研究以高糖刺激的肾脏足细胞模型以及沉默ULK1的足细胞模型为研究对象，探讨清热消癥方是否可以通过调控ULK1的表达，干预AMPK-ULK1通路，激活线粒体自噬发挥保护线粒体作用，阐明清热消癥方治疗DN与激活线粒体自噬的相关性，验证假说的科学性与客观性，探讨清热消癥方治疗DN的确切作用靶点及效应机制，为以“肾络癥瘕”理论为指导的中医药防治糖尿病肾病研究提供中医理论基础和实验依据。

该课题聚焦AMPK-ULK1信号通路与线粒体自噬在足细胞发生发展中的作用及机制，同时探讨清热消癥方是否基于以上机制治疗糖尿病肾病。研究证实:（1）体内研究：以单侧肾切除+STZ诱导糖尿病肾病大鼠模型为研究对象，发现清热消癥方可以降低糖尿病肾病大鼠24h尿蛋白定量；降低尿素氮（BUN）水平以保护肾功能；改善糖脂代谢紊乱状态；改善糖尿病肾病肾小球硬化、系膜细胞增生和肾间质纤维化等病理损伤。通过实时荧光PCR、蛋白免疫印迹分析进一步证实，清热消癥方可促进线粒体自噬，改善线粒体功能，保护足细胞。（2）体外实验：以高糖刺激足细胞建立糖尿病肾病细胞模型为研究对象，发现清热消癥方有保护足细胞功能，上调Nephrin蛋白表达，促进足细胞增殖的作用；经免疫组织荧光化学染色和荧光示踪技术证实，清热消癥方可促进自噬滤泡包裹受损线粒体并将其降解，维持足细胞内环境稳态，保护足细胞过滤屏障。通过实时荧光PCR及蛋白免疫印迹分析等证实清热消癥方可调控AMPK-ULK1信号通路，促进足细胞线粒体自噬。（3）复方成分分析：经超高效液相质谱分析，明确了清热消癥方中多种有效组分，为深入研究建立基础。因而，本研究明确AMPK-ULK1-线粒体自噬是介导足细胞参与糖尿病肾病发生发展的重要分子机制；清热消癥方可调控AMPK-ULK1线粒体自噬关键节点修复足细胞损伤延缓糖尿病肾病进展。