Effects Of Spironolactone On Aldosterone Mediated Oxidative Stress And Nf-kB、MCP-1Expressions In Cultured Rat Glomerular Mesangial Cells

Objective To observe the effects of spironolactone(SPI) on aldosterone(ALD)mediated oxidative stress, nuclear factor-κB(NF-κB) and monocyte chemoattractantprotein-1(MCP-1) expressions in cultured rat glomerular mesangial cells(MCs) in orderto explore its mechanisms of reno-protection.Methods The MCs were cultured in the medium with nomal glucose and then dividedinto groups as follows:①Control Group: the control group (Low glucose5.6mmol/L DMEM media);②Group ALD: the aldosterone-stimulated group,Group ALD1：Low glucose DMEM media containing10-5mol/L aldosteronestimulating;Group ALD2：Low glucose DMEM media containing10-7mol/L aldosteronestimulating;Group ALD3：Low glucose DMEM media containing10-9mol/L aldosteronestimulating;③Group SPI: the spironolactone-interfered group,Group SPI1：10-7mol/L spironolactone plus10-7mol/L aldosterone;Group SPI2：10-8mol/L spironolactone plus10-7mol/L aldosterone;Group SPI3：10-9mol/L spironolactone plus10-7mol/L aldosterone;The cells and the culture supernatants were collected after48hours separately.Every group cells cultured three repetitions each. The levels of intracellular reactive oxygen species(ROS)were measured by flow cytometry. The expressions of NF-κB,MCP-1,CYP11B2,MR and11β-HSD2mRNA were detected by semi-quantitativeRT-PCR.Results1CYP11B2, MR and11β-HSD2mRNA expressions were detectable in glomerularmesangial cells.2Effect of aldosterone-stimulated reactive oxygen species generationControl Group: cells which erupt fluorescence,4.28%. Group ALD1: cells whicherupt fluorescence,21.77%. Group ALD2: cells which erupt fluorescence,17.33%.Group ALD3: cells which erupt fluorescence,15.25%. The intracellular reactiveoxygen species content increased significantly when exposed to aldosterone ascompared with the control group（P<0.01）, which was concentration-dependent（P<0.01）.3Effect of aldosterone-stimulated production of NF-κB and MCP-1mRNAAldosterone enhanced the effect of NF-κB mRNA expression of glomerularmesangial cells. The results showed: Control Group:0.44±0.11，Group ALD1:0.93±0.09，Group ALD2:0.72±0.10，Group ALD3:0.63±0.13. NF-κB/GAPDH mRNAratio was significantly increased from the aldosterone-stimulated group compared withthe control group（P<0.01）, which was concentration-dependen（tP<0.05）. Aldosteronealso enhanced the effect of MCP-1mRNA expression. The results showed as: ControlGroup: follow:0.38±0.14，Group ALD1:0.95±0.14，Group ALD2:0.77±0.17，GroupALD3:0.65±0.07. MCP-1/GAPDH mRNA ratio was significantly increased from thealdosterone-stimulated group compared with the control group（P<0.01）, which wasconcentration-dependent（P<0.05）. 4Effect 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 SPI1：cells which eruptfluorescence,7.34%. Group SPI2：cells which erupt fluorescence,9.25%. Group SPI3：cells which erupt fluorescence,10.9%. The intracellular reactive oxygen species contentwas lower when adding spironolactone as compared with the aldosterone group（P<0.01）, which was concentration-dependent（P<0.01）.5Effect of spironolactone on aldosterone-induced expression of NF-κB and MCP-1mRNAWhen the mesangial cells were cultured in the presence of spironolactone，therewas significant reduction in NF-κB mRNA expression. Group SPI1：0.41±0.05, GroupSPI2：0.45±0.09, Group SPI3：0.58±0.14. NF-κB/GAPDH mRNA ratio wassignificantly decreased from the spirono-lactone-interfered group compared with thealdosterone-stimulated group（P<0.05）, which was in a dose-dependent manner（P<0.05）.There was a highly significant reduction in MCP-1mRNA expression too.Group SPI1：0.50±0.18, Group SPI2：0.52±0.20, Group SPI3：0.69±0.16.MCP-1/GAPDH mRNA ratio was significantly decreased fromthespironolactone-interfered group compared with the aldosterone-stimulated group（P<0.05）, which was in a dose-dependent manner（P<0.05）.6Linear correlations between reactive oxygen species and NF-κB mRNA in mesangialcellsA significant positive correlation maybe observed between reactive oxygen speciesand NF-κB mRNA（r=0.929,P<0.01）in mesangial cells.7Linear correlations between NF-κB and MCP-1mRNA in mesangial cells A significant positive correlation maybe observed between NF-κB and MCP-1mRNA（r=0.682,P<0.01）in mesangial cells.Conclusions CYP11B2, MR and11β-HSD2mRNA were detected in culturedglomerular mesangial cells by RT-PCR; Aldosterone can bind to MR specifically,thusenhance the generation of ROS and NF-κB and MCP-1mRNA expressions in ratmesangial cells; Increased ROS generation maybe related to the enhanced expressionsof NF-κB and MCP-1mRNA in MCs; Spironolactone can inhibit the detrimental effectsof aldosterone-induced NF-κB and MCP-1expressions in rat mesangial cells through aROS-dependent pathway, which may contribute to its reno-protection partly.