| 0bjective To observe the effects of spironolactone(SPI) in inhibiting aldosterone(ALD) mediated oxidative stress and plasminogen activator inhibitor-1(PAI-1) expression of rat glomerular mesangial cells in Vitro in order to explore the reno-protection mechanisms of spironolactone. Methods The rat glomerular mesangial cells were cultured in the medium with nomal glucose and then divided into groups as follows:①Group Con: the control group (Low glucose 5.6 mmol/L DMEM media);②Group ALD: the aldosterone-stimulated group, Group ALD 1:Low glucose DMEM media containing 10-5 mol/L aldosterone stimulating; Group ALD 2:Low glucose DMEM media containing 10-7 mol/L aldosterone stimulating; Group ALD 3:Low glucose DMEM media containing 10-9 mol/L aldosterone stimulating;③Group SPI: the spironolactone-interfered group, Group SPI 1:10-7 mol/L spironolactone plus 10-7 mol/L aldosterone; Group SPI 2:10-8 mol/L spironolactone plus 10-7 mol/L aldosterone; Group SPI 3:10-9 mol/L spironolactone plus 10-7 mol/L aldosterone;The Intracellular reactive oxygen species(ROS) level was detected by flow cytometry,which use 2',7'- dichlorofluorescein diacetate(DCFH-DA) as a reactive oxygen species fluorescent dye. RT-PCR was used to detect the expression of plasminogen activator inhibitor-1(PAI-1), aldosterone synthase CYP11B2, mineralocorticoid receptor(MR) and 11β-hydroxysteroid dehydrogenase(11β-HSD2) mRNAs. The level of PAI-1 in the supernatants was measured by ELISA.Results1. CYP11B2,MR and 11β-HSD2 mRNA expressions were detectable in glomerular mesangial cells.2. Effect of aldosterone-stimulated reactive oxygen species generationMesangial cells stimulated with aldosterone for 48h showed substantial fluorescence after the addition of DCFH-DA. Group Con: cells which erupt fluorescence, 4.28%. Group ALD 1: cells which erupt fluorescence, 21.77%. Group ALD 2: cells which erupt fluorescence, 17.33%. Group ALD 3: cells which erupt fluorescence, 15.25%. The intracellular reactive oxygen species content increased significantly when exposed to aldosterone as compared with the control group(P<0.01), which was concentration-dependent(P<0.01).3. Effect of aldosterone-stimulated production of PAI-1 mRNAAldosterone enhanced the effect of PAI-1 mRNA expression. The results showed: Group Con: 1.11±0.07,Group ALD 1: 1.95±0.06,Group ALD 2: 1.71±0.09,Group ALD 3: 1.63±0.07.PAI-1/GAPDH mRNA ratio was significantly increased from the aldosterone-stimulated group compared with the control group(P<0.01), which was concentration-dependent(P<0.05).4. Effect of aldosterone-stimulated production of PAI-1 proteinWe found that baseline concentrations of PAI-1 protein were 0.35±0.02 ng/ml for mesangial cells grown in the control group. Aldosterone induced a significant increase in its synthesis,Group ALD 1: 2.01±0.06 ng/ml,Group ALD 2: 1.52±0.11 ng/ml,Group ALD 3: 1.31±0.08 ng/ml. Compared with the control group, PAI-1 protein expression of mesangial cells in the supernatants increased significantly in the aldosterone-stimulated group(P<0.01), which was in a dose-dependent manner(P<0.05).5. Effect of spironolactone on aldosterone-induced reactive oxygen species generationWhen adding different concentrations of spironolactone to interfere aldosterone,the stimulatory action of aldosterone was blocked. Group SPI 1:cells which erupt fluorescence, 7.34%. Group SPI 2:cells which erupt fluorescence, 9.25%. Group SPI 3:cells which erupt fluorescence, 10.9%. The intracellular reactive oxygen species content was lower when adding spironolactone as compared with the aldosterone group(P<0.01), which was concentration-dependent(P<0.01).6. Effect of spironolactone on aldosterone-induced production of PAI-1 mRNAWhen the mesangial cells were cultured in the presence of spironolactone,there was a highly significant reduction in PAI-1 mRNA expression. Group SPI 1:1.18±0.10, Group SPI 2:1.38±0.10, Group SPI 3:1.50±0.11. PAI-1/GAPDH mRNA ratio was significantly decreased from the spironolactone-interfered group compared with the aldosterone-stimulated group(P<0.05), which was in a dose-dependent manner(P<0.05).7. Effect of spironolactone on aldosterone-induced production of PAI-1 proteinWe also found that incubation of aldosterone-induced mesangial cells with spironolactone for 48 h suppress production of PAI-1 protein level in the supernatants. The results revealed that,Group SPI 1:0.41±0.03 ng/ml,Group SPI 2:0.72±0.05 ng/ml,Group SPI 3:1.08±0.08 ng/ml. Compared with the aldosterone group, PAI-1 protein expression of mesangial cells in the supernatants decreased significantly in the spironolactone group(P<0.01), which was in a dose-dependent manner(P<0.05).8. Linear correlation between reactive oxygen species and PAI-1 mRNA in mesangial cellsA significant positive correlation was observed between reactive oxygen species and PAI-1 mRNA(r=0.931,P<0.01)in mesangial cells.9. Linear correlation between reactive oxygen species and PAI-1 protein in mesangial cellsA significant positive correlation was observed between reactive oxygen species and PAI-1 protein(r=0.976,P<0.01)in mesangial cells. Conclusions Aldosterone can bind to MR specifically,thus enhances PAI-1 mRNA expression and protein formation in rat mesangial cells. These effects were accompanied by increases in oxidative stress. Spironolactone can inhibit the detrimental effects of aldosterone-induced PAI-1 expression in rat mesangial cells through a ROS-dependent pathway, which may contribute to its reno-protection partly.
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