白藜芦醇通过抑制miR-138表达减少高糖状态下足细胞凋亡的机制

High glucose-induced podocyte apoptosis is one of the major causes leading to podocyte detachment and albuminuria in patients with diabetic nephropathy. The mechanism of high glucose-associated podocyte apoptosis remain largely lacking. Previously, we reported that resveratrol, a natural compound, can reduce albuminuria and improve the renal function of mice with diabetic nephropathy.Using a comparative miRNA expression array, currently, we identified miR-138 as a signature down-regulated miRNA in podocyte under high glucose condition treated with resveratrol. We demonstrated that autophagy-related gene (Atg) 7 was a direct target of miR-138. Transfection with anti-miR-138 expression construct increased the level of Atg7 protein and the autophagic vesicle protein LC3, which mimicked the phenotype of the resveratrol treatment. In addition, we found resveratrol-treatment attenuated high glucose-induced apoptosis in conditionally immortalized mouse podocytes incubated in high glucose. Thus, we hypothesize that resveratrol may protect podocyte from apoptosis resulted from high glucose via the augmentation of podocyte autophagy. To test our hypothesis, we have designed three specific aims: First，to evaluate the effects of resveratrol on the podocyte autophagy and apoptosis under high glucose condition. Second, to probe the mechanism of resveratrol on high glucose-induced podocyte autophagy and apoptosis via the inhibition of miR-138. In the end, to investigate the mechanism that enhanced autophagy protect podocyte aganist the high glucose-induced apoptosis. We will use in vivo db/db mice model with diabetic nephropathy and in vitro podocyte culture system to pursue these specific aims. A variety of novel techniques, such as gene overexpression assay, siRNA interfering and transmission electron microscope, will be administrated in the project. The study will validate resveratrol as an effective treatment agent against high glucose-induced podocyte apoptosis via the inhibition of miR-138. The proposed work in this application is also designed to fill a knowledge gap defining the possible role and mechanism of autophagy in the protection of podocyte from high glucose-induced apoptosis. Accomplishment of this project will not only provide a mechanistic explanation for the development of apoptosis in high glucose-treated podocyte, but also gain a new insight into the application of resveratrol in diabetic nephropathy.

高血糖引起的足细胞凋亡是糖尿病肾病蛋白尿形成的重要原因，目前机制尚不明确。预实验应用白藜芦醇干预高糖培养的足细胞，发现白藜芦醇能增加足细胞自噬、减少高糖状态下足细胞凋亡，miRNA芯片筛选和实时定量 PCR验证发现miR-138表达明显下调，进一步用生物信息学分析和双荧光素酶报告检测系统检测，发现自噬相关基因Atg7是其靶基因。因此，我们推测白藜芦醇可能通过抑制miR-138的表达、促进自噬来减少高糖状态下足细胞凋亡。本项目拟采用miRNA、基因过表达及抑制技术，观察miR-138对白藜芦醇干预高糖状态下足细胞自噬、凋亡的影响，以及足细胞自噬在白藜芦醇干预高糖状态下足细胞凋亡中的作用，并分析其作用机制。有关miR-138对白藜芦醇干预高糖状态下足细胞自噬、凋亡的影响及机制的研究均无文献报道，因此本研究具有源头创新性，可为高血糖引起足细胞凋亡发病机制的研究和糖尿病肾病的治疗提供新的思路。

高血糖引起的足细胞凋亡是引起足细胞脱落、导致糖尿病肾病患者蛋白尿形成的重要原因。自噬是一种溶酶体依赖的蛋白质降解途径，糖尿病肾病时足细胞自噬活性下降。白藜芦醇是一种多酚类化合物，近年研究发现其具有潜在的肾脏保护作用，但具体的作用靶点和作用机制均未完全明确。.本项目主要研究内容：①在db/db糖尿病肾病小鼠模型和体外高糖培养的小鼠足细胞中，观察白藜芦醇干预后足细胞自噬和足细胞凋亡的变化，明确白藜芦醇能否增加足细胞自噬这一针对高糖应激的保护机制，同时白藜芦醇能否减少高糖引起的足细胞凋亡；②采用基因过表达、siRNA基因沉默等方法，研究自噬对白藜芦醇干预高糖所致足细胞凋亡的影响，明确白藜芦醇是否通过诱导足细胞自噬来减轻高糖引起的足细胞凋亡；③应用microRNA表达芯片、荧光素酶报告分析和基因过表达、基因沉默等方法，研究白藜芦醇如何诱导足细胞自噬以及自噬影响高糖所致足细胞凋亡的分子机制。.基本完成研究目的，获得了一些重要的研究结果：①在db/db糖尿病肾病小鼠模型和体外高糖培养的小鼠足细胞中白藜芦醇干预，免疫荧光检测、Annexin V-FITC / PI 双染流式细胞技术、westernblot检测Cleaved caspase-3和Bax蛋白，发现白藜芦醇干预后高糖引起的足细胞凋亡明显下降；②白藜芦醇的干预能明显增加db/db糖尿病肾病小鼠和体外培养的足细胞自噬相关蛋白Atg5和LC3-Ⅱ蛋白的表达、透射电镜下自噬小体的形成、GFP-LC3绿色荧光颗粒的增加。③自噬特异性抑制剂3-甲基腺嘌呤（3-MA）和自噬相关基因Atg5基因沉默，抑制足细胞自噬，白藜芦醇促进足细胞自噬的作用减少，足细胞凋亡增加；④Affymetrix miRNA芯片分析db/db小鼠肾脏组织显示白藜芦醇抑制miR-383-5p、miR-205-5p；增加miR-18a-5p的表达，通过激活自噬相关基因5（ATG5）、ATM serine/threonine kinase基因的表达，来减轻高糖状态下足细胞的凋亡。.通过上述研究我们阐明了白藜芦醇能够通过抑制足细胞miR-383-5p、miR-205-5p的表达，激活足细胞自噬，减少高糖状态下足细胞的凋亡。这不仅为高血糖引起足细胞凋亡发病机制的研究提供新的视点，同时为临床糖尿病肾病的治疗提供新的思路。