摘要
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.
出处
《中华肾脏病杂志》
CAS
CSCD
北大核心
2017年第1期30-36,共7页
Chinese Journal of Nephrology
基金
国家自然科学基金(81470955)
广东省科技计划项目(2014A020212074)