二甲双胍对高糖诱导的肾小管上皮细胞TGF-β/ERK/MMP-9通路及上皮间质转化的影响  

Effects of metformin on TGF-β/ERK/MMP-9 pathway and epithelial mesenchymal transition induced by high glucose in renal tubular epithelial cells

作　　者：常毅娜[1] 宋白利[1] 张文博[1] 付留俊[1] CHANG Yi-na;SONG Bai-li;ZHANG Wen-bo;FU Liu-jun(Department of Endocrinology,the First Affiliated Hospital of Henan University of Science and Technology,Luoyang 471000,China)

机构地区：[1]河南科技大学第一附属医院内分泌科,河南洛阳471000

出　　处：《现代药物与临床》2021年第4期659-664,共6页Drugs & Clinic

基　　金：河南省医学科技攻关计划项目(2018020300)。

摘　　要：目的探讨二甲双胍对高糖诱导的肾小管上皮HK-2细胞上皮间质转化(EMT)的影响及其机制。方法以0、7.5、15.0、30.0、60.0、120.0mmol/L二甲双胍作用48h后,采用噻唑蓝(MTT)法检测HK-2细胞活力以筛选合适的二甲双胍作用浓度;将体外培养的HK-2细胞分为对照组(5.5 mmol/L D-葡萄糖)、高渗组(24.5 mmol/L甘露醇和5.5 mmol/L D-葡萄糖)、高糖组(30 mmol/L D-葡萄糖)、高糖+7.5 mmol/L二甲双胍组(30 mmol/L D-葡萄糖+7.5 mmol/L二甲双胍)和高糖+15 mmol/L二甲双胍组(30 mmol/L D-葡萄糖+15 mmol/L二甲双胍),倒置显微镜观察各组HK-2细胞形态,免疫印迹法(Western blotting)检测各组HK-2细胞中α-平滑肌肌动蛋白(α-SMA)、E-钙黏附蛋白(E-cadherin)、转化生长因子-β(TGF-β)、细胞外信号调节激酶(ERK)、磷酸化(p)-ERK、基质金属蛋白酶9(MMP-9)蛋白表达水平,实时荧光定量PCR(qRT-PCR)检测α-SMA、E-cadherin、TGF-β、ERK、MMP-9 mRNA表达水平。结果与对照组相比,30.0、60.0mmol/L二甲双胍作用后HK-2细胞活力明显升高,120.0mmol/L二甲双胍作用后HK-2细胞活力明显降低(P<0.05),而7.5、15.0mmol/L二甲双胍作用后HK-2细胞活力差异无统计学意义(P>0.05);与对照组比较,高渗组细胞形态无明显改变,且细胞中α-SMA、E-cadherin、TGF-β、MMP-9蛋白和mRNA表达水平以及p-ERK/ERK蛋白、ERK mRNA表达水平差异均无统计学意义(P>0.05),但高糖组细胞失去原有形态变为长梭形,且细胞中α-SMA、TGF-β蛋白和mRNA表达水平以及p-ERK/ERK蛋白、ERK mRNA表达水平明显升高,而MMP-9、E-cadherin蛋白和mRNA表达水平明显降低(P<0.05);与高糖组比较,高糖+7.5mmol/L二甲双胍组、高糖+15.0mmol/L二甲双胍组细胞形态由长梭形逐渐变成圆形或椭圆形,且α-SMA、TGF-β蛋白和mRNA表达水平以及p-ERK/ERK蛋白、ERK mRNA表达水平明显降低,而MMP-9、E-cadherin蛋白和mRNA表达水平明显升高(P<0.05),且高糖+15.0 mmol/L二甲双胍组细胞上述指�Objective To investigate the effect of metformin on high glucose-induced epithelial mesenchymal transition(EMT)in HK-2 cells in renal tubular epithelium.Methods After treated with 0,7.5,15.0,30.0,60.0 and 120.0 mmol/L metformin for 48 h,the viability of HK-2 cells was detected by MTT method to select the appropriate concentration of metformin.HK-2 cells cultured in vitro were divided into control group(5.5 mmol/L D-glucose),hypertonic group(24.5 mmol/L mannitol and 5.5 mmol/L D-glucose),high glucose group(30 mmol/L D-glucose),high glucose+7.5 mmol/L metformin group(30 mmol/L D-glucose+7.5 mmol/L metformin)and high glucose+15.0 mmol/L metformin group(30 mmol/L D-glucose+15.0 mmol/L metformin).The morphology of HK-2 cells was observed under inverted microscope.Western blotting method was used to detect the protein expression levels ofα-smooth muscle actin(α-SMA),E-cadherin,transforming growth factor-β(TGF-β),extracellular signal regulated kinase(ERK),phosphorylation(p)-ERK and matrix metalloproteinase-9(MMP-9)in HK-2 cells,and real time fluorescent quantitative PCR(qRT-PCR)was used to detect the mRNA expression levels ofα-SMA,E-cadherin,TGF-β,ERK and MMP-9.Results Compared with 0 mmol/L,30 and 60 mmol/L metformin increased the viability of HK-2 cells significantly.After treated with 120.0 mmol/L metformin,the viability of HK-2 cells decreased significantly(P<0.05).Compared with those in the control group,there was no significant difference in HK-2 cell viability between the treat of 7.5 mmol/L and 15.0 mmol/L metformin(P>0.05).Compared with those in the control group,there was no significant change in cell morphology in the hypertonic group,the expression levels ofα-SMA,E-cadherin,TGF-βand MMP-9 protein and mRNA,and p-ERK/ERK protein and ERK mRNA were not significantly different(P>0.05).However,the cells in high glucose group lost their original shape and became long spindle shape,the expression levels ofα-SMA and TGF-βprotein and mRNA,p-ERK/ERK protein and ERK mRNA were significantly higher,and the expre

关 键 词：肾小管上皮细胞 高糖 二甲双胍 上皮间质转化 转化生长因子-β/细胞外信号调节激酶/基质金属蛋白酶9通路 

分 类 号：R966[医药卫生—药理学]