miR-122低表达对糖尿病肾病足细胞损伤和IKK/NF-κB信号通路的抑制作用研究  

Inhibitory Effect of miR-122 Low Expression on Podocyte Injury and IKK/NF-κB Signaling Pathway in Diabetic Nephropathy

作　　者：王平 陈晓红[1] 郭娅琼 薛艳[1] WANG Ping;CHEN Xiao-hong;GUO Ya-qiong;XUE Yan(Department of Adult Internal Medicine,Hubei Maternal and Child Health Hospital,Wuhan 430070,China)

机构地区：[1]湖北省妇幼保健院成人内科,武汉430070

出　　处：《微循环学杂志》2023年第2期16-23,共8页Chinese Journal of Microcirculation

基　　金：湖北省卫生和计划生育委员会联合基金项目(WJ2018H0134)。

摘　　要：目的:探究微小RNA-122(miR-122)低表达对糖尿病肾病(DN)足细胞炎性因子水平、增殖、凋亡、迁移和IκB激酶(IKK)/核因子κB(NF-κB)信号通路的影响。方法:体外培养人肾小球足细胞HGPC,取对数生长期细胞,分为正常糖组(5mmol/L D-葡萄糖)、高糖组(30mmol/L D-葡萄糖)、inhibitor NC组(转染inhibitor NC片段至细胞,后加入30mmol/L D-葡萄糖)、miR-122 inhibitor组(转染miR-122 inhibitor片段至细胞,后加入30mmol/L D-葡萄糖),每组设置3个重复。24h后,实时荧光定量PCR(qPCR)测定各组细胞中miR-122表达水平,酶联免疫法(ELISA)测定细胞上清液中白介素(IL)-6、IL-1β及IL-10水平,细胞计数试剂盒8(CCK-8)法、5-乙炔基-2’脱氧尿嘧啶核苷(EdU)法、Hoechst 33258染色法及划痕法分别测定细胞活力、增殖率、凋亡率及迁移率,蛋白免疫印迹法(WB)测定细胞中IKKα、IκBα、磷酸化(p-)IκBα、NF-κB p65、p-NF-κB p65、细胞周期蛋白(Cyclin D1)及半胱氨酸天冬氨酸蛋白酶-3(Caspase-3)蛋白表达水平。结果:与正常糖组比较,高糖组细胞miR-122表达水平、IL-6水平、IL-1β水平、迁移率、凋亡率以及IKKα、p-IκBα、p-NF-κB p65、Caspase-3蛋白表达水平升高(P<0.05),细胞IL-10水平、活力、增殖率以及Cyclin D1蛋白表达水平降低(P<0.05);与inhibitor NC组比较,miR-122 inhibitor组细胞miR-122表达水平、IL-6水平、IL-1β水平、迁移率、凋亡率以及IKKα、p-IκBα、p-NF-κB p65、Caspase-3蛋白表达水平降低(P<0.05),细胞IL-10水平、活力、增殖率以及Cyclin D1蛋白表达水平升高(P<0.05)。结论:miR-122低表达可促进DN足细胞增殖,减轻细胞炎症损伤,抑制细胞凋亡、迁移和IKK/NF-κB信号通路。Objective:To investigate the effects of low expression of microRNA-122(miR-122)on inflammatory cytokines,proliferation,apoptosis,migration and iκB kinase(IKK)/nuclear factorκB(IKK)/NF-κB signaling pathway in diabetic nephropathy(DN)podocytes.Method:Human glomerular podocytes(HGPC)were cultured in vitro,and the cells growing in logarithmic stages were divided into normal glucose group(5mmol/L D-glucose),high glucose group(30mmol/L D-glucose),inhibitor NC group(transfection with inhibitor NC fragment into cells,then 30mmol/L D-glucose was added)and miR-122 inhibitor group(transfected with miR-122 inhibitor fragment into cells,then 30mmol/L D-glucose was added),with three replicates in each group.After 24h,the expression level of miR-122 in cells of each group was measured by real-time fluorescence quantitative PCR(qPCR),and the levels of interleukin-6(IL)-6,IL-1βand IL-10 in cell supernatant were measured by enzyme-linked immunosorbent assay(ELISA).Cell viability,proliferation rate,apoptosis rate and migration rate were determined by CCK-8 assay,EdU assay,Hoechst 33258 staining and scratch assay.Western blotting(WB)was used to detect IκB kinase(IKKα),IκBα,phosphorylated(p-)IκBα,nuclear factorκB(NF-κB p65),p-NF-κB p65 and Cyclin D1)and Caspase-3 protein expression levels.Results:Compared with the normal glucose group,the expression levels of miR-122,IL-6,IL-1β,migration rate,apoptosis rate and the protein expression levels of IKKα,p-IκBα,p-NF-κB p65 and Caspase-3 were increased in the high glucose group(P<0.05),the level of IL-10,cell viability,proliferation rate and Cyclin D1 protein expression were significantly decreased(P<0.05).Compared with inhibitor NC group,the expression levels of miR-122,IL-6,IL-1β,migration rate,apoptosis rate and the protein expression levels of IKKα,p-IκBα,p-Nf-κB p65 and Caspase-3 were decreased,while the level of IL-10,cell viability,proliferation rate and Cyclin D1 protein expression were significantly increased in the miR-122 inhibitor group(P<0.05).Conclusion:

关 键 词：糖尿病肾病 微小RNA-122 IΚB激酶 核因子κB 炎症因子 增殖 凋亡 迁移 

分 类 号：R587.2[医药卫生—内分泌]