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

作　　者：杨莹[1] 范秋灵[1] 李露露 汪旭[2] 文思 徐莉[3] Yang Ying;Fan Qiuling;Li Lulu;Wang Xu;Wen Si;Xu Li(Department of Nephrology,the First Hospital of China Medical University,Shenyang 110001,China;Department of Digestive Medicine,the First Hospital of China Medical University,Shenyang 110001, China;Department of Clinical Laboratory,the First Hospital of China Medical University,Shenyang 110001,China)

机构地区：[1]中国医科大学附属第一医院肾内科,沈阳110001 [2]中国医科大学附属第一医院消化内科,沈阳110001 [3]中国医科大学附属第一医院检验科,沈阳110001

出　　处：《中华肾脏病杂志》2019年第1期43-47,共5页Chinese Journal of Nephrology

基　　金：国家自然科学基金(81770724、81270808);辽宁省自然科学基金(201602821);辽宁省高等学校重大科技平台免疫皮肤病学重点实验室自主创新课题基金(201303);沈阳市科技计划项目(F16-205-1-40).

摘　　要：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.

关 键 词：糖尿病肾病 氧化应激 肾小管上皮细胞 miRNA-148b AMPKα1 

分 类 号：R587.2[医药卫生—内分泌] R692.9[医药卫生—内科学]