SIRT3对糖尿病肾病氧化应激的影响及分子机制的研究

Diabetic kidney disease, the most common complication of diabetes, has been the leading cause of end-stage renal disease. Recent researches demonstrate that oxidative stress caused by mitochondria may play a key role in the development of diabetic kidney disease. Sirt3 protects the mitochondria against oxidative stress via regulating the acetylation level and activity of a diverse range of key mitochondrial enzymes involved in a growing body of cellular processes such as antioxidant defences, mitochondrial respiratory chain activity, fatty acid metabolism, tricarboxylic acid cycle and apoptosis. It is logical to hypothesize that Sirt3 may have the ability to modulate the cellular oxidative stress level and the development of diabetic kidney disease. This topic detects the influence of high glucose on the expression and distribution of Sirt3 in diabetic rats and HK-2 cells. We also study the influence of Sirt3 on cellular oxidative stress, mitochondrial dynamic and apoptosis in db/db mouse and HK-2 cells by Western blotting, Realtime PCR, immunohistochemistry, immunofluorescence, flow cytology ,confocal imaging and so on. In addition, we also detect whether Sirt3 influnce the cell apoptosis through mitochondria dynamics and PI3k/Akt/FoxO3 cell signaling pathyway.

糖尿病肾病是糖尿病最常见的微血管并发症。研究发现线粒体诱导的氧化应激可能在糖尿病肾病的发病中起着重要的作用。SIRT3 可以通过调控线粒体相关蛋白的乙酰化来调节线粒体氧化呼吸链的功能，从而改善线粒体的氧化应激状态。本课题主要运用db/db鼠、转基因鼠、人肾小管上皮细胞，在整体水平和细胞水平观察去乙酰化酶SIRT3对肾脏氧化损伤的影响及可能的分子机制。利用转基因技术在动物水平过表达Sirt3、质粒转染方法在细胞水平过表达SIRT3、siRNA技术在细胞水平敲低Sirt3，运用Western blotting、Real time PCR、免疫组化、免疫荧光、流式细胞术、共聚焦成像等实验技术观察研究线粒体SIRT3对糖尿病动物和人肾小管上皮细胞线粒体氧化应激和凋亡的影响以及SIRT3是否通过PI3K-Akt-FoxO信号通路及线粒体动力学介导对细胞凋亡的影响。

糖尿病肾病是糖尿病最常见的微血管并发症。研究发现线粒体诱导的氧化应激可能在糖尿病肾病的发病中起着重要的作用。SIRT3 可以通过调控线粒体相关蛋白的乙酰化来调节线粒体氧化呼吸链的功能，从而改善线粒体的氧化应激状态。本课题应用转基因技术在动物水平过表达SIRT3、质粒转染方法在细胞水平过表达SIRT3、siRNA技术在细胞水平敲低SIRT3，运用Western blotting、Real time PCR、免疫组化、免疫荧光、流式细胞术、共聚焦成像等实验技术观察研究线粒体SIRT3对糖尿病动物和人肾小管上皮细胞线粒体氧化应激和凋亡的影响，结果提示过表达SIRT3可降低氧化应激（如SIRT3过表达降低高糖刺激导致的线粒体ROS聚集）及凋亡水平（TUNEL细胞染色实验说明SIRT3过表达降低了高糖导致的HK-2细胞凋亡），敲减SIRT3后促进氧化应激及凋亡表达。同时SIRT3过表达可以显著增高p-Akt、p-Fox O1和p-Fox O3a蛋白表达，降低细胞核内Fox O1和Fox O3a的表达，提示其可通过PI3K-Akt-FoxO信号通路介导对细胞凋亡的影响。