微小RNA-124通过抑制rho相关蛋白激酶1激活减轻高糖导致的肾小球内皮细胞损伤

MicroRNA-124 inhibits rho associated kinase 1 activity to ameliorate the damages of glomerular endothelial cells caused by high glucose

目的探讨微小RNA-124（miR-124）和rho相关蛋白激酶（ROCK）1信号通路在高糖诱导的肾小球内皮细胞（GEnC）损伤中的作用。方法不同葡萄糖浓度培养大鼠GEnC，细胞分为正常对照组（5．5mmol／L）和高糖组（30．0mmol／L），用ROCKl抑制剂Y27632处理细胞，转染miR-124-3p模拟物（miR-124-3pmimic）或miR-124-3p抑制剂。实时定量PCR法检测miR-124表达；Western印迹法检测ROCKI活性、细胞凋亡及细胞紧密连接相关蛋白的表达；激光共聚焦显微镜观察细胞紧密连接蛋白ZO-1的表达。结果与对照组比较，高糖组miR-124表达明显下降（P〈0．01）；ROCKl活性相关蛋白肌球蛋白磷酸酶靶向亚单位（P-MYPTl／MYPTl）表达增加；细胞凋亡相关蛋白（Cleaved-Caspase3／pro-Caspase3）增加；细胞紧密连接蛋白ZO．1、Occludin表达减少（均P〈0．05或〈0．01）。用Y27632预处理细胞后可明显减轻上述损伤。细胞转染miR-124．3p模拟物后，P-MYPTl／MYPT蛋白表达下调，转染miR-124-3p抑制剂后，p-MYPTl／MYPTl蛋白表达上调（均P〈0．05），提示miR-124可直接抑制ROCKl活性。高糖组GEnC转染miR-124-3p模拟物后，高糖导致的ROCKl活性升高、凋亡增加及紧密连接蛋白的下凋都受到了明显抑制（均P〈0．05）。结论高糖引起肾小球内皮细胞miR-124表达下降，激活ROCKl介导肾小球内皮细胞损伤，过表达miR-124可减轻高糖导致的上述损伤，提示miR-124可成为防治糖尿病肾病肾小球内皮细胞损伤的新靶点。

Objective To explore the effects of miR- 124- ROCK1 signal pathway in the damages of glomerular endothelial cells （GEnCs） induced by high glucose. Methods Rat glomerular endothelial cells were cultured in different glucose concentrations： normal control group （NG： 5.5 mmol/L）, high glucose group （HG： 30.0 mmol/L）, and cells were treated with ROCK1 inhibitor Y27632, miR- 124 - 3p mimic, miR - 124 - 3p inhibitor. The expressions of ROCK1 activity, cell apotosis and tight junction proteins were detected by Western blot. The cell tight junction protein ZO- 1 in those groups were assessed by laser scanning confocal microscope. Results High glucose significantly decreased miR- 124 expression （P 〈 0.01）, ROCK1 activity （P- MYPT1/MYPT1）, and cell apoptosis （Cleaved-Caspase3/pro-Caspase3） were found increased while the tight junction proteins ZO-land Occludin were found decreased in these cells （P 〈 0.05 all P 〈 0.01）, However, when pretreated cells with ROCK1 inhibitor Y27632, these injuries were significantly reversed. In cells transfected with miR- 124- 3p mimic, p-MYPT1/MYPT1 was decreased, p-MYPTI/MYPT1 was however increased in cells transfected with miR-124-3p inhibitor （P 〈 0.05）, indicating that miR-124 could directly inhibit ROCK1 activity. The increased ROCK1 activity and apoptosis, as well as the decreased tight junction proteins induced by high glucose were significantly suppressed as miR- 124- 3p mimic transfected in GEnCs. Conelusions According to our experiments, high glucose suppressed miR- 124 in glomerular endothelial cells, consequenctly activating ROCK1 activity to damage endothelial ceils. MiR- 124 overexpression could ameliorate these damages induced by high glucose, suggesting that miR- 124 might be a new therapeutic target to prevent glomerular endothelial cells injuries in diabetic nephropathy.