Effects Of Pioglitazone On High Glucose Mediated Oxidative Stress And MCP-1Expression In Cultured Rat Glomerular Mesangial Cells

Objective To observe the effects of pioglitazone(PIO) on high glucose(HG) mediatedoxidative stress and monocyte chemoattractant protein-1(MCP-1) expression in culturedrat glomerular mesangial cells(MCs) and discuss its mechanisms and the relationshipwith reno-protection.Methods Rat glomerular mesangial cells were cultured in the medium with normalglucose and then grouped into sections as follows:①Group NG: the control group (glucose5.6mmol/L DMEM media with10%fetal bovine serum);②Group H3: the high glucose-stimulated group (High glucose25mmol/LDMEM media with10%fetal bovine serum)③Group PIO: the pioglatazone-interfered group,Group P1: High glucose DMEM media containing10-5mol/L pioglitazonestimulating;Group P2: High glucose DMEM media containing10-6mol/L pioglitazonestimulating;Group P3: High glucose DMEM media containing10-7mol/L pioglitazonestimulating;After48h exposure, the supernatants and RGMCs were collected. Every group cells cultured six repetitions each. Flow cytometry deternined the levels of Intracellularreactive oxygen species(ROS)，Coloimetry method was measured to detected the levelsof malondialdehyde(MDA) and superoxide dismutase(SOD). RT-PCR was used todetect the expression of MCP-1mRNA, while the levels of MCP-1protein insupernatants were determined by ELISA.Results1RGMCs could express and synthesize MCP-1.2Effect of piogilitazone on high glucose induced-reactive oxygen stress in mesangialcellsIn comparison with group NG (6.91±0.23),the levels of intracellular ROS weresignificantly increased in group H3(46.8±1.50),P<0.05.Compared with group H3, the levels of intracellular ROS were significantlydecreased in group P1（10.7±0.69）、group P2(28.3±1.84）and group P3（33.9±1.02）with a dose-dependent manner,P<0.05.3Effect of piogilitazone on high glucose induced-malondialdehyde in thesupernatantsIn comparison with group NG (1.08±0.06),the levels of MDA in the supernatantwere significantly increased in group H3(1.70±0.08),P<0.05.Compared with group H3, the levels of MDA in supernatants were significantlydecreased in group P1（1.19±0.07）、group P2(1.42±0.09）and group P3（1.54±0.08）with a dose-dependent manner,P<0.05.4Effect of piogilitazone on high glucose induced-SOD level in the supernatants In comparison with group NG(19.13±0.74),the levels of SOD in the supernatantwere significantly decreased in group H3(11.68±0.9),P<0.05.Compared with group H3, the levels of SOD in supernatants were significantlyincreased in group P1（17.19±0.93）、group P2(15.61±0.91)and group P3（13.49±0.64）with a dose-dependent manner,P<0.05.5Effect of piogilitazone on high glucose induced MCP-1mRNA in mesangial cellsIn comparison with group NG(0.3777±0.0213),the levels of intracellularMCP-1mRNA expression were significantly increased in group H3(0.5378±0.033),（P<0.01）.Compared with group H3, the levels of intracellular MCP-1mRNA expression inmesangial cells were significantly decreased in group P1（0.4068±0.0219）、group P2(0.4537±0.0203）and group P3（0.4857±0.228）with a dose-dependent manner, P<0.05.6Effect of piogilitazone on high glucose induced production of MCP-1protein in thesupernatantsIn comparison with group NG(42.58±8.21),the levels of MCP-1protein content inthe supernatants were significantly increased in group H3(228.95±37.88),P<0.05.Compared with group H3, the levels of MCP-1protein content in supernatantswere significantly decreased in group P1（85.36±19.21）、group P2(126.48±25.69）andgroup P3（162.35±32.92）, P<0.05.7The correlation analysisA significant negative correlation was detected between intracellular reactiveoxygen species and SOD activity（r=0.510,P=0.04）in the supernatant.A vocabulary positive correlation was detected between intracellular reactiveoxygen species and MDA（r=0.958,P=0.00）in the supernatant. A significant positive correlation was detected between intracellular reactive oxygenspecies and MCP-1mRNA（r=0.919,P<0.01）in mesangial cells.A vocabulary positive correlation was detected between intracellular reactiveoxygen species and MCP-1protein content（r=0.955,P<0.01）in the supernatant.Conclusion1. Glomerular mesangial cells can synthetize and express MCP-1;2. High glucose can induce the increase of intracellular ROS production and MCP-1mRNA expressions in rat mesangial cells, enhance the malondialdehyde andMCP-1protein level in the supernatants, meanwhile decrease SOD activitymarkedly;3. Increased ROS generation has correlation with the enhanced expression of MCP-1mRNA in MCs and MCP-1protein content in the supernatants;4. Pioglitazone can inhibit high glucose-induced oxidative stress and MCP-1expressions in glomerular mesangial cells in a dose-dependent manner, which maycontribute to its reno-protection partly.