醛固酮自分泌环在高糖致人系膜细胞损伤中的作用

Effect of aldosterone autocrine loop on the injury of human mesangial cells induced by high glucose

目的研究高糖环境下人系膜细胞(HMC)中醛固酮自分泌环及活性氧(ROS)、癌胚纤连蛋白(FN)表达水平的变化,进一步探讨醛固酮自分泌环与ROS、癌胚FN mRNA表达的关系。方法常规培养HMC,取对数生长期细胞分为5组:正糖组(5mmol/L D-葡萄糖),渗透压对照组(5mmol/L D-葡萄糖+20mmol/L L-葡萄糖),正糖+依普利酮组(5mmol/L D-葡萄糖+10μmol/L依普利酮),高糖组(25mmol/L D-葡萄糖),高糖+依普利酮组(25mmol/L D-葡萄糖+10μmol/L依普利酮)。采用RT-PCR法检测醛固酮合酶(CYP11B2)、11β-羟基类固醇脱氢酶Ⅱ(11βHSD2)、癌胚FN mRNA表达水平,Western blotting检测CYP11B2蛋白表达水平,放射免疫法分析细胞培养液中醛固酮水平,通过激光共聚焦显微镜观察盐皮质激素受体(MR)蛋白表达及转位情况,荧光显微镜和荧光酶标仪检测细胞内ROS水平。结果与正糖组比较,高糖组HMC细胞CYP11B2 mRNA及蛋白表达上调,ROS和癌胚FN mRNA表达亦上调,培养液中醛固酮浓度升高(P<0.05)。高糖可促进MR蛋白发生核转位,定量分析显示高糖组胞质/胞核荧光强度比值较正糖组降低30%(P<0.05)。与高糖组比较,高糖+依普利酮组ROS及癌胚FN mRNA表达下调(P<0.05)。结论高糖负荷可通过激活HMC局部醛固酮自分泌环而导致HMC损伤。

Objective To investigate the formation of aldosterone autocrine loop,fluctuation of reactive oxygen species（ROS） and expression of oncofetal fibronectin（oncofetal FN） mRNA in human mesangial cells（HMCs） exposed to high glucose,and explore the relationship of aldosterone autocrine loop with ROS production and expression of oncofetal FN mRNA.Methods HMCs were divided into ve groups： normal glucose group（D-glucose 5mmol/L）,osmotic control group（D-glucose 5mmol/L＋L-glucose 20mmol/L）,normal glucose＋eplerenone group（D-glucose 5mmol/L＋eplerenone 10μmol/L）,high glucose group（D-glucose 25mmol/L）,and high glucose＋eplerenone group（D-glucose 25mmol/L＋eplerenone 10μmol/L）.The mRNA expression of aldosterone synthase（CYP11B2）,11β-hydroxysteroid dehydrogenase type 2（11βHSD2） and oncofetal FN were determined with RT-PCR.The protein expression of CYP11B2 was detected by Western blotting.Aldosterone level in cells culture supernatant was assessed with radioimmunoassay.The expression and translocation of mineralocorticoid receptor（MR） protein were observed by confocal laser scanning microscopy.ROS levels were determined by fluorescence microscopy and fluorescence microplate reader.Results（1） High glucose increased the expressions of HMCs CYP11B2 mRNA and protein by 3.45 and 2.09 folds,respectively,of the level in normal glucose group（P0.05）.The aldosterone level of HMCs culture supernatant in high glucose group was increased by 2.01-fold compared with that in normal glucose group（P0.05）.High glucose induced MR translocation from cytosol to nucleus,and quantitative analysis showed the ratio of plasma/nuclear fluorescence intensity in high glucose group decreased by 30% compared with that in normal glucose group（P0.05）.（2） ROS levels and oncofetal FN mRNA in high glucose group were up-regulated by 1.99 and 2.23 folds,respectively（P0.05）,compared with that in normal glucose group.Compared with high glucose group,the ROS level and oncofetal FN mRNA expression in high glucose＋eplerenone group showed a decrease of 37% and 53%,respectively（P0.05）.Conclusions High glucose leads to injury of HMCs via activation of local aldosterone autocrine loop.