加味黄风汤调控SIRT1/PGC-1α信号通路对糖尿病肾病足细胞损伤的保护机制研究

In the occurrence and development of diabetic nephropathy, podocyte injury plays an important role. Mitochondria are organelles that provide podocyte with energy. Mitochondrial dysfunction can directly lead to podocyte injury. The SIRT1/PGC-1α signal pathway can regulate the body's energy supply in oxidative stress state. Our previous work showed that modified Huang Feng decoction can reduce proteinuria in patients with chronic kidney disease, can cure urinary protein in diabetic nephropathy rats, can play a protective role of podocyte. We speculate that the modified Huang Feng decoction can regulate the SIRT1/PGC-1 α signaling pathway to protect podocyte injury in diabetic nephropathy. This study is divided into two parts.In vitro cultured podocytes, they are grouped with the high concentration of sugar, SIRT1 agonists, inhibitors, modified Huangfeng Decoction serum and PGC-1αshRNA stimulation. We will detecte SIRT1, PGC-1α and Nephrin molecular expression, cellular energy metabolism index. In vivo, treated with the modified Huangfeng Decoction in diabetic nephropathy rats, we will detect clinical indexes and the renal damage. We will observe the expression of mRNA and protein of PGC-1α and SIRT1 in renal tissue. This study aims to investigate the regulatory effect of SIRT1/PGC-1α signal pathway on podocyte and mitochondrial injury induced by high glucose. We try to observe the protective effect of modified Huangfeng Decoction on diabetic nephropathy podocyte injury. We are searching for the probable SIRT1/PGC-1α signal pathway regulation in the treatment of the modified Huangfeng Decoction in diabetic nephropathy. We hope to provide a new target for the treatment of diabetic nephropathy with traditional Chinese medicine, but also provide new ideas for research on the mechanism of traditional Chinese medicine for the treatment of various podocyte diseases.

线粒体为足细胞提供能量，其功能缺陷可直接导致糖尿病肾病足细胞损伤。SIRT1/PGC-1α信号通路可调节机体在氧化应激状态下的能量供应。课题组前期工作显示：加味黄风汤可减少慢性肾病患者及糖尿病肾病大鼠蛋白尿，保护足细胞。我们推测加味黄风汤可调控SIRT1/PGC-1α信号通路以保护糖尿病肾病的足细胞损伤。本研究分两部分进行。体外培养足细胞，分组予高糖、SIRT1激动剂、抑制剂、加味黄风汤含药血清及PGC-1αshRNA干预刺激，检测足细胞能量代谢等指标。体内实验部分，予加味黄风汤喂养糖尿病肾病大鼠，检测肾组织各相关信号分子的表达水平。本课题旨在探讨SIRT1/PGC-1α信号通路对高糖诱导足细胞及线粒体损伤的调控作用，研究加味黄风汤治疗糖尿病肾病可能的SIRT1/PGC-1α信号通路调控作用。我们希望为中医药治疗糖尿病肾病提供新靶点，同时也为中医药治疗各种足细胞病的机制研究提供新思路。

糖尿病肾病发生发展过程中与足细胞损伤密切相关。SIRT1/PGC-1α信号通路与细胞能量代谢紧密相连，在高糖刺激及氧化应激过程中SIRT1/PGC-1α表达下调后，线粒体供能减少，可导致足细胞损伤，引起蛋白尿。我们的研究通过体内及体外实验均证实加味黄风汤可通过调控SIRT1/PGC-1α信号通路，保护足细胞。体内实验研究结果提示：糖尿病肾病模型组大鼠可见微量白蛋白尿增加，肾脏病理可见肾小球肥大，系膜基质增生，基底膜增厚及足细胞足突融合，肾组织中SIRT1及PGC-1α蛋白及mRNA表达水平明显下降。加味黄风汤治疗8周后糖尿病肾病大鼠24h尿微量白蛋白水平明显下降；光镜下肾小球肥大、系膜基质增生减轻；电镜下足细胞足突融合、基底膜增厚情况有所改善；肾组织中SIRT1及PGC-1α蛋白及mRNA表达水平较模型组均明显上升。体外实验研究结果提示:高糖刺激后可见足细胞SIRT1及PGC-1αmRNA表达显著减少，足细胞内ROS表达明显增高，足细胞凋亡率增加。加味黄风汤含药血清作用后可见足细胞SIRT1及PGC-1αmRNA表达均增加，同时足细胞内ROS表达减少，足细胞凋亡率下降。SIRT1抑制剂NAM可致足细胞SIRT1及PGC-1αmRNA表达减少，ROS表达增加，凋亡率上升；含药血清作用于足细胞后，可拮抗NAM部分作用，增加足细胞SIRT1及PGC-1αmRNA表达，降低ROS水平，减少细胞凋亡率，保护足细胞。加味黄风汤中重要单体毛蕊异黄酮亦进一步证实可通过调控SIRT1/PGC-1α信号通路起到足细胞保护的作用。项目已资助发表中文论文2篇，培养硕士研究生2名，均已经取得硕士学位。项目直接经费18万元，支出15.4724万元，各项支出基本与预算相符。剩余经费2.5276万元，剩余经费计划用于本项目研究后续支出。