miR-124靶向作用ROCK1调节早期糖尿病肾病肾小球内皮通透性和凋亡的机制研究

Nowadays, the pathogenesis, precaution and treatment of Diabetic kidney disease (DKD) are the major focus in Nephrology research. We previously reported that ROCK1 up-regulation and activation in glomerular endothelial cells (GEnCs) is one of the key mechanisms leading to DKD. Since up-regulation of ROCK1 protein is not associated with increase in its mRNA level, this indicates there should be an alternative mechanism to promote ROCK1 expression. In our preliminary study, we found that miR-124 could be an important factor that suppresses the translation of ROCK1. The evidence include: the levels of miR-124 in the glomeruli of DKD patients or diabetic mice are significantly decreased compared the results in normal controls. Moreover, high glucose significantly suppresses miR-124 expression in GEnCs. These results suggest that down-regulation of miR-124 and hence increase in ROCK1 translation, might be a crucial mechanism in the development of DKD. To prove this hypothesis, we propose below specific aims: 1) in vitro, we plan to investigate if down-regulation of miR-124 promotes ROCK1 expression in GEnCs after stimulated with high glucose. To this end, we will use several molecular techniques including miRNA mimic transfection, dual luciferase reporter assay. 2) in vivo, we will employ loxP-Cre technique to generate tamoxifen-inducible, endothelial-specific miR-124 overexpression mice, and then to examine if these mice would be resistant to develop DKD in diabetic condition induced by STZ. We will use situ hybridization, SmartFlare RNA technique and histological analysis in this study. 3) by pharmacological targeting miR-124, we will explore if pioglitazone treatment could enhance miR-124 to suppress ROCK1 in db/db mice with DKD. If our project were granted, it should provide sufficient scientific evidence to identify miR124-ROCK1 pathway as a new target to prevent early GEnCs injury in DKD.

肾小球内皮细胞（GEnCs）损伤是糖尿病肾病（DKD）产生蛋白尿和肾小球硬化的主要原因之一，但机制尚未明了。我们的前期研究提示糖尿病导致的miR-124-ROCK1通路异常可能是DKD肾小球内皮细胞损伤、蛋白尿发生的重要机制。为证实该假设，本项目拟采用小鼠GEnCs、诱导型内皮细胞特异性表达miR-124的转基因小鼠为研究对象，通过细胞转染、原位杂交、SmartFlare RNA探针、肾小球分离等技术分别从分子、细胞和动物模型层面进一步阐述糖尿病引起GEnCs内miR-124降低，后者靶向上调ROCK1导致细胞损伤、继而导致DKD进展的新机制；并观察PPAR-γ激动剂吡格列酮是否通过调节miR-124-ROCK1通路改善DKD的早期病变和蛋白尿。本项目如获资助，将为调节miR-124-ROCK1通路作为防治DKD早期内皮损伤和蛋白尿的新靶点提供充分依据。

目的 探讨高糖损伤肾小球内皮细胞（GEnCs）的机制以及吡格列酮是否通过miR-124-ROCK1信号通路改善糖尿病肾病GEnCs损伤，延缓糖尿病肾病进展。方法 不同葡萄糖浓度培养大鼠GEnCs，细胞分为正常对照组和高糖组，用ROCK1抑制剂Y-27632处理细胞，在高糖培养的GEnC中转染miR-124-3p模拟物（miR-124-3p mimic）或添加吡格列酮，检测miR-124-3p表达及ROCK1活性、细胞紧密连接蛋白、细胞凋亡蛋白的表达；观察Occludin、ZO-1蛋白的表达及分布；测量牛血清白蛋白（Bovine serum albumin，BSA）漏出率；检测细胞凋亡。构建含ROCK1种子序列的双荧光素酶报告基因，分别和miR-124-3p对照、miR-124-3p模拟物转染GEnCs，多功能酶标仪检测荧光素酶活性。32只小鼠随机分为4组：db/m生理盐水组、db/m吡格列酮组、db/db生理盐水组、db/db吡格列酮组，分别给予吡格列酮或生理盐水灌胃16周后，检测各小鼠肾脏miR-124-3p表达及相关蛋白表达，观察肾小球病理改变。结果 与对照组相比，高糖组miR-124-3p表达下降；ROCK1活性增加；细胞紧密连接蛋白ZO-1、Occludin表达减少；细胞通透性增加、细胞凋亡增加。用Y-27632处理GEnCs后减轻上述高糖诱导的GEnCs损伤。高糖培养GEnCs后，转染miR-124-3p模拟物或加入吡格列酮，高糖导致的ROCK1活性升高、紧密连接蛋白下调、细胞高通透性、细胞凋亡增加都受到了抑制（均P＜0.05）。双荧光素酶活性结果显示：miR-124-3p模拟物可靶向结合ROCK1，抑制ROCK1活性。体内实验结果显示，与db/m小鼠相比，db/db小鼠的血糖、血肌酐、尿白蛋白较db/m组显著小鼠高，肾脏miR-124-3p降低，肾小球细胞凋亡增加，肾小球增大、系膜区扩张、基底膜增厚、足突融合和肾脏纤维化加重。在吡格列酮治疗db/db小鼠16周后，上述异常指标均有改善。结论 糖尿病状态下，高糖通过激活ROCK1信号通路损伤GEnCs ；miR-124靶向抑制ROCK1活性，改善高糖诱导的肾小球内皮细胞损伤；吡格列酮通过激活miR-124-ROCK1信号通路改善糖尿病肾病肾小球内皮细胞损伤，保护肾功能，延缓糖尿病肾病的进展。