【作者】 胡圆圆；

【导师】 叶山东；

【作者基本信息】 安徽医科大学 ， 内科学， 2011， 硕士

【摘要】 目的观察螺内酯(Spironolactone,SPI)对醛固酮(Aldosterone,ALD)诱导的大鼠肾小球系膜细胞(Mesangial cells,MCs)氧化应激和转化生长因子β-1(Transforming growth factor-β1,TGF-β1)表达的影响,探讨ALD致糖尿病肾病(Diabetic nephropathy, DN)及SPI肾保护作用的可能机制。方法体外培养大鼠MCs,随机分组如下:①NC组:正常对照组(低糖DMEM培养液,葡萄糖浓度为5.6 mmol/L);②ALD组:醛固酮刺激组, ALD 1组:低糖DMEM培养液中加入醛固酮(10-5 mol/L)刺激; ALD 2组:低糖DMEM培养液中加入醛固酮(10-7 mol/L)刺激; ALD 3组:低糖DMEM培养液中加入醛固酮(10-9 mol/L)刺激。③SPI组:螺内酯干预组, SPI 1组:醛固酮(10-7 mol/L)刺激的同时加入螺内酯(10-7 mol/L)干预; SPI 2组:醛固酮(10-7 mol/L)刺激的同时加入螺内酯(10-8 mol/L)干预; SPI 3组:醛固酮(10-7 mol/L)刺激的同时加入螺内酯(10-9 mol/L)干预。48小时后分别收集细胞及上清液。应用流式细胞术检测细胞内活性氧(Reactive oxygen species,ROS)水平; RT-PCR(Reverse transcription polymerase chain reaction,RT-RCR)方法检测细胞TGFβ-1、醛固酮合成酶(CYP11B2)、醛固酮受体( Mineralocorticoid receptor , MR )和11β-羟类固醇脱氢酶2(11β-hydroxysteroid dehydrogenase 2,11β-HSD2)mRNA表达;ELISA(Enzyme linked immunosorbent assay,ELISA)法检测细胞培养上清液中TGF-β1蛋白浓度。结果1 RT-PCR法检测结果显示MCs表达CYP11B2、MR和11β-HSD2 mRNA;2 ALD对肾小球MCs内ROS产生的影响ALD刺激MCs48h后,细胞内ROS水平显著增加,ALD 1,2,3组分别为(21.77±0.87)、(17.33±0.49)、(15.25±0.63),组间差异显著,P<0.01;与NC组(4.28±0.50)相比,差异显著(P均<0.01)。3 ALD对肾小球MCs TGFβ-1 mRNA表达及蛋白分泌的影响半定量RT-PCR检测发现,NC组系膜细胞可低水平表达TGFβ-1 mRNA(1.22±0.14)及蛋白(0.78±0.12 ng/ml)。ALD作用48 h后,TGFβ-1 mRNA表达及蛋白分泌显著增加,两指标在不同浓度ALD组之间分别比较,组间差异显著(P均<0.05)。4螺内酯对醛固酮诱导的肾小球MCs内ROS产生的影响醛固酮(10-7 mol/L)刺激的同时加入螺内酯干预后,SPI 1,2,3组细胞内ROS水平显著降低,分别为(7.34±0.89)、(9.25±0.65)、(10.9±0.97),组间差异显著,P<0.01;与ALD 2组(17.33±0.49)相比,差异显著(P均<0.05)。SPI 1组ROS水平稍高于NC组,差异无显著性(P>0.05)。5 SPI对ALD诱导的肾小球MCs TGFβ-1 mRNA表达及蛋白分泌的影响醛固酮(10-7 mol/L)刺激的同时加入螺内酯干预后,SPI 1,2,3组MCs TGFβ-1 mRNA表达及蛋白分泌均较ALD2组显著下降(P <0.01或0.05),两指标分别进行组间比较,差异具有显著性(P均<0.0 5)。6肾小球MCs ROS产生和TGFβ-1 mRNA表达及蛋白分泌的相关性分析MCs产生的ROS和TGFβ-1 mRNA表达及蛋白分泌分别呈正相关(r=0.866,P<0.01;r=0.823,P<0.01)。结论系膜细胞表达CYP11B2、MR和保护其配体特异性的11β-HSD2mRNA;醛固酮可与系膜细胞的MR特异性结合,促进细胞内ROS生成增多、TGF-β1mRNA和蛋白表达增多,参与肾脏损伤;螺内酯与醛固酮竞争性结合MR,拮抗醛固酮介导的肾小球系膜细胞内ROS生成及TGF-β1 mRNA及蛋白的表达,其可能是螺内酯参与DN时肾脏保护作用机制之一。更多还原

【Abstract】 Objective To observe the effects of spironolactone on aldosterone mediated oxidative stress and transforming growth factorβ-1 (TGF-β1) expression in glomerular mesangial cells and explore its renoprotective mechanism.Methods Rat glomerular mesangial cells were cultured in the medium with nomal glucose (5.6 mmol/l) and then treated with medium containing 10-5mol/L (group ALD1), 10-7mol/L (group ALD2) and 10-9mol/L (group ALD3) aldosterone, respectively. Another, different concentrations of spironolactone (group SPI1 10-7 mol/L, group SPI2 10-8 mol/L and group SPI3 10-9 mol/L) were added into group ALD2, respectively. Cells and supernatants were collected after 48h separately. The levels of intracellular reactive oxygen species (ROS) were measured by flow cytometry. The expressions of TGF-β1, CYP11B2, MR and 11β-HSD2 mRNA were detected by semi-quantitative RT-PCR, while the levels of TGF-β1 protein in supernatants were measured by ELISA.Results (1) CYP11B2, MR and 11β-HSD2 mRNA were detected in cultured rat glomerular mesangial cells. (2) Compared with group NC, the levels of ROS and the expression of TGF-β1 mRNA and protein were all significantly increased in group ALD (P<0.05 or P<0.01), moreover, a dose-dependent manner was detected among group ALD1, group ALD2 and group ALD3 (P<0.05 or P<0.01). (3) Compared with group ALD2 (17.33±0.49), the levels of ROS in group SPI1 (7.34±0.89), SPI2 (9.25±0.65) and SPI3 (10.9±0.97) were all decreased significantly (P<0.05). Concomitantly, the expressions of TGF-β1 mRNA and protein were also significantly inhibited by spironolactone. (4) The changes in ROS were positively correlated with the change in TGFβ-1 mRNA and protein, respectively (r=0.866, P<0.01; r=0.823, P<0.01, respectively.).Conclusions CYP11B2, MR and 11β-HSD2 mRNA were detected in cultured glomerular mesangial cells by RT-PCR. Spironolactone can competitively binding with MR, then suppress aldosterone mediated oxidative stress and TGF-β1 expression in a dose-dependent manner in rat glomerular mesangial cells, which may partly contribute to its reno-protection.更多还原