miRNA-148b靶向AMPKα1通过氧化应激介导高糖诱导的人肾小管上皮细胞凋亡

MiRNA-148b targeted AMPKα1 mediates high glucose-induced apoptosis in human renal tubular epithelial cells via oxidative stress

目的探讨微小RNA-148b(miRNA-148b)在高糖诱导肾小管损伤中表达变化及其作用机制。方法体外培养人肾小管上皮细胞(HK-2细胞),分为正常糖组、甘露醇高渗对照组、高糖组,培养48 h后,实时定量PCR法检测miRNA-148b表达;采用2′,7′-二氯二氢荧光素二乙酸酯(DCFH-DA)在荧光显微镜下检测细胞内活性氧(ROS)水平;Western印迹检测HK-2细胞腺苷单磷酸活化蛋白激酶α1(AMPKα1)、NOX2、NOX4、Bcl-2、cleaved-caspase3的蛋白表达。结果培养48 h后,与正常糖组相比,高糖组、高渗组HK-2细胞内miRNA-148b表达上调(P<0.01),ROS产生增多(P<0.01),NOX2、NOX4蛋白表达增多(均P<0.01),AMPKα1蛋白和抗凋亡蛋白Bcl-2蛋白表达减少(均P<0.01),线粒体凋亡通路相关蛋白cleaved-caspase3蛋白表达增加(P<0.01),差异均有统计学意义。结论高糖上调体外培养HK-2细胞miRNA-148b的表达,靶向抑制AMPKα1的表达,促进NOX2、NOX4表达,活性氧产生增多,活化线粒体凋亡途径,激活caspase酶,诱导HK-2细胞凋亡。高糖的肾小管毒性部分是渗透压的影响。miRNA-148b可能参与了糖尿病肾病病理损伤的发生,有望成为糖尿病肾病新的治疗靶点。

Objective To investigate the expression and mechanism of microRNA-148b (miRNA-148b) in high glucose-induced renal tubular injury.Method HK-2 cells cultured in vitro were divided into normal glucose group, mannitol hypertonic control group and high glucose group. After 48 hours of culture, the expression of miRNA-148b was detected by real-time quantitative PCR. 2′,7′-Dichlorodihydrofluorescein diacetate (DCFH-DA) was used for detecting production of ROS and observed under fluorescence microscope for analysis;The expression of AMPKα1, Bcl-2, NOX2, NOX4, activated caspase3 (cleaved-caspase3) were detected by Western blotting.Results Compared with the normal glucose group, the expression of miRNA-148b was up-regulated in HK-2 cells in high glucose group and hypertonic group (P<0.01), and the production of ROS increased (P<0.01). The expression of NOX2 and NOX4 was increased, AMPKα1 and Bcl-2 decreased, and cleaved caspase-3 was increased (all P<0.01).Conclusions HG up-regulated miRNA-148b expression and down-regulated its target gene AMPKα1 which promotes the expression of NOX2 and NOX4 in HK-2 cells. MiRNA-148b promotes apoptosis of HK-2 cells via increasing production of ROS and enhancing cleaved-caspase3 for Bcl-2 insufficiency. The tubular toxicity of high glucose is partly due to osmotic pressure. MiRNA-148b may be involved in the pathological injury of diabetic nephropathy and is expected to become a new therapeutic target for diabetic nephropathy.