螺内酯对高糖、醛固酮诱导的大鼠肾小球系膜细胞单核细胞趋化蛋白-1表达的影响
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摘要
目的探讨螺内酯(spironolactone, Spi)对高糖或醛固酮(aldosterone, Ald)诱导的大鼠肾小球系膜细胞(rat glomerular mesangial cells, RMCs)表达单核细胞趋化蛋白-1(monocyte chemoattractant protein-1, MCP-1)的影响
方法①体外培养RMCs并按如下分组:正糖组(葡萄糖浓度5.6 mmol/l,normal glucose, NG),高糖组(葡萄糖浓度30 mmol/l,high glucose, HG),Spi 1组(螺内酯10-7 mol/l + HG),Spi 2组(螺内酯10-8 mol/l + HG),Spi 3组(螺内酯10-9 mol/l + HG)。②体外培养RMCs并按如下分组:正糖组(葡萄糖浓度5.6 mmol/l,NG),醛固酮组(醛固酮10-7 mol/l,Ald),Spi A组(螺内酯10-7 mol/l + Ald),Spi B组(螺内酯10-8 mol/l + Ald),Spi C组(螺内酯10-9 mol/l + Ald)。48小时后收集各组细胞和上清液,以RT-PCR检测MCP-1、醛固酮受体(mineralocorticoid receptor,MR)和保护其配体特异性结合的11β-羟类固醇脱氢酶2(l1β-hydroxysteroid dehydrogenase,11β-HSD2)的mRNA表达,采用ELISA检测培养细胞上清中MCP-1蛋白的浓度。
结果(1)RT-PCR结果显示系膜细胞表达MCP-1、MR和11β-HSD2的mRNA。(2)30 mmol/l葡萄糖条件下培养48小时后,大鼠系膜细胞MCP-1 mRNA和上清液MCP-1蛋白水平较正糖组显著升高[1.44±0.29 vs 1.03±0.22, P<0.01; 194.04±50.62 pg/ml vs 90.03±10.77 pg/ml, P<0.01 ],而螺内酯(10-7 mol/l, 10-8 mol/l, 10-9 mol/l)干预可有效抑制高糖的促MCP-1表达作用。(3)与正糖对照组比较(P<0.01),10-7 mol/l醛固酮可以有效上调大鼠系膜细胞MCP-1 mRNA和蛋白表达(1.84±0.29和261.23±58.87),而10-7 mol/l和10-8 mol/l浓度的螺内酯干预可有效抑制醛固酮(10-7 mol/l)的促MCP-1表达作用,但10-9 mol/l螺内酯并不能抑制醛固酮诱导的系膜细胞MCP-1 mRNA表达。
结论螺内酯可与大鼠肾小球系膜细胞上的盐皮质激素受体特异性结合,并有效抑制高糖和醛固酮诱导的大鼠系膜细胞中MCP-1 mRNA的表达和蛋白合成。
Objective To investigate the effects of spironolactone (Spi) on expression of monocyte chemoattractant protein-1 (MCP-1) induced by high glucose (HG) or aldosterone (Ald) in rat mesangial cells.
Methods①The rat glomerular mesangial cells(RMCs) were cultured in the medium with nomal glucose (5.6 mmol/l, NG) and then divided into five group: NG group, HG group(30 mmol/l, HG), Spi 1 group (Spi 10-7mol/l was added in HG group), Spi 2 group (Spi 10-8mol/l was added in HG group), Spi 3 group (Spi 10-9mol/l was added in HG group). After 48 hours' incubation, the MsC and its supernatant were collected.②Another RMCs were cultured and then divided into five group: NG group, Ald group (Ald 10-7mol/l was added), Spi A group (Spi 10-7mol/l and Ald 10-7mol/l were added), Spi B group (Spi 10-8 mol/l and Ald 10-7 mol/l were added), Spi C group (Spi 10-9 mol/l and Ald 10-7 mol/l were added). RT-PCR was used to measure the expression of Mineralocorticoid receptor (MR) and l1β-hydroxysteroid dehydrogenase (11β-HSD2, an enzyme required for MR ligand specificity) and MCP-1 mRNA.The levels of MCP-1 in the supernatants were determined by enzyme-linked immunosorbant assay(ELISA).
Results (1) MR, 11β-HSD2 and MCP-1 mRNA expressions were detectable in RMCs by RT-PCR. (2) After 48h exprosure of RMCs to HG, the levels of MCP-1 mRNA and protein were increased significantly compared to that in NG group [1.44±0.29 vs 1.03±0.22, P<0.01; 194.04±50.62 pg/ml vs 90.03±10.77 pg/ml, P<0.01 ], and the antagonist of MR spironolactone (10-7 mol/l, 10-8 mol/l, 10-9 mol/l) treatment could inhibit these effects (P<0.01). (3) After treatment of 10-7mol/l Ald for 48 hours, the levels of MCP-1 mRNA and protein were 1.84±0.29 and 261.23±58.87 pg/ml, significantly higher than the baseline levels (P<0.01), and spironolactone (10-7 mol/l, 10-8 mol/l) treatment could inhibit the increased MCP-1 expressions stimulated by Ald (P<0.01). And there was no statistical diference in the levels of MCP-1 mRNA expression between Spi A group and NG group(P>0.05).
Conclusions Spironolactone can acts by specificly binding with MR on RMCs, and significantly down-regulate the MCP-1 mRNA and protein expressions induced by HG or Ald in RMCs.
方法①体外培养RMCs并按如下分组:正糖组(葡萄糖浓度5.6 mmol/l,normal glucose, NG),高糖组(葡萄糖浓度30 mmol/l,high glucose, HG),Spi 1组(螺内酯10-7 mol/l + HG),Spi 2组(螺内酯10-8 mol/l + HG),Spi 3组(螺内酯10-9 mol/l + HG)。②体外培养RMCs并按如下分组:正糖组(葡萄糖浓度5.6 mmol/l,NG),醛固酮组(醛固酮10-7 mol/l,Ald),Spi A组(螺内酯10-7 mol/l + Ald),Spi B组(螺内酯10-8 mol/l + Ald),Spi C组(螺内酯10-9 mol/l + Ald)。48小时后收集各组细胞和上清液,以RT-PCR检测MCP-1、醛固酮受体(mineralocorticoid receptor,MR)和保护其配体特异性结合的11β-羟类固醇脱氢酶2(l1β-hydroxysteroid dehydrogenase,11β-HSD2)的mRNA表达,采用ELISA检测培养细胞上清中MCP-1蛋白的浓度。
结果(1)RT-PCR结果显示系膜细胞表达MCP-1、MR和11β-HSD2的mRNA。(2)30 mmol/l葡萄糖条件下培养48小时后,大鼠系膜细胞MCP-1 mRNA和上清液MCP-1蛋白水平较正糖组显著升高[1.44±0.29 vs 1.03±0.22, P<0.01; 194.04±50.62 pg/ml vs 90.03±10.77 pg/ml, P<0.01 ],而螺内酯(10-7 mol/l, 10-8 mol/l, 10-9 mol/l)干预可有效抑制高糖的促MCP-1表达作用。(3)与正糖对照组比较(P<0.01),10-7 mol/l醛固酮可以有效上调大鼠系膜细胞MCP-1 mRNA和蛋白表达(1.84±0.29和261.23±58.87),而10-7 mol/l和10-8 mol/l浓度的螺内酯干预可有效抑制醛固酮(10-7 mol/l)的促MCP-1表达作用,但10-9 mol/l螺内酯并不能抑制醛固酮诱导的系膜细胞MCP-1 mRNA表达。
结论螺内酯可与大鼠肾小球系膜细胞上的盐皮质激素受体特异性结合,并有效抑制高糖和醛固酮诱导的大鼠系膜细胞中MCP-1 mRNA的表达和蛋白合成。
Objective To investigate the effects of spironolactone (Spi) on expression of monocyte chemoattractant protein-1 (MCP-1) induced by high glucose (HG) or aldosterone (Ald) in rat mesangial cells.
Methods①The rat glomerular mesangial cells(RMCs) were cultured in the medium with nomal glucose (5.6 mmol/l, NG) and then divided into five group: NG group, HG group(30 mmol/l, HG), Spi 1 group (Spi 10-7mol/l was added in HG group), Spi 2 group (Spi 10-8mol/l was added in HG group), Spi 3 group (Spi 10-9mol/l was added in HG group). After 48 hours' incubation, the MsC and its supernatant were collected.②Another RMCs were cultured and then divided into five group: NG group, Ald group (Ald 10-7mol/l was added), Spi A group (Spi 10-7mol/l and Ald 10-7mol/l were added), Spi B group (Spi 10-8 mol/l and Ald 10-7 mol/l were added), Spi C group (Spi 10-9 mol/l and Ald 10-7 mol/l were added). RT-PCR was used to measure the expression of Mineralocorticoid receptor (MR) and l1β-hydroxysteroid dehydrogenase (11β-HSD2, an enzyme required for MR ligand specificity) and MCP-1 mRNA.The levels of MCP-1 in the supernatants were determined by enzyme-linked immunosorbant assay(ELISA).
Results (1) MR, 11β-HSD2 and MCP-1 mRNA expressions were detectable in RMCs by RT-PCR. (2) After 48h exprosure of RMCs to HG, the levels of MCP-1 mRNA and protein were increased significantly compared to that in NG group [1.44±0.29 vs 1.03±0.22, P<0.01; 194.04±50.62 pg/ml vs 90.03±10.77 pg/ml, P<0.01 ], and the antagonist of MR spironolactone (10-7 mol/l, 10-8 mol/l, 10-9 mol/l) treatment could inhibit these effects (P<0.01). (3) After treatment of 10-7mol/l Ald for 48 hours, the levels of MCP-1 mRNA and protein were 1.84±0.29 and 261.23±58.87 pg/ml, significantly higher than the baseline levels (P<0.01), and spironolactone (10-7 mol/l, 10-8 mol/l) treatment could inhibit the increased MCP-1 expressions stimulated by Ald (P<0.01). And there was no statistical diference in the levels of MCP-1 mRNA expression between Spi A group and NG group(P>0.05).
Conclusions Spironolactone can acts by specificly binding with MR on RMCs, and significantly down-regulate the MCP-1 mRNA and protein expressions induced by HG or Ald in RMCs.
引文
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