Pioglitazonc Suppresses Oxidative Stress Via P38MAPK In Cultued Rat Glomerular Mesangial Cells

Objective To observe the effects of pioglitazone on the expression of p38mitogen-activated protein kinase (P38MAPK) and oxidative stress in cultured ratglomerular mesangial cells (MCs). To explore its renoprotective mechanism and toprovide a new thought for the prevention and cure of diabetic nephropathy.Methods MCs were cultured in the medium with normal glucose concentration (groupNG), high glucose concentration (group HG), P38MAPK special inhibitor SB203580(group S) and different concentrations of pioglitazone (P1, P2), respectively. After48hexposure, the supernatants and MCs were collected separately. The levels ofIntracellular reactive oxygen species (ROS) were determined by flow cytometry. Theprotein levels of phosphory1ated P38MAPK (p-p38), P38MAPK, p22phox andp47phox were measured by Western blot. Expressions of p22phox and p47phox mRNAwere detected by semiquantitative RT-PCR. The activities of catalese (CAT),superoxide dismutase (SOD) and the levels of maleicdialdehyde (MDA) weredetermined by coloimetry method respectively.Results1Changes of intracellular ROS production in mesangial cellsCells exposed to high glucose for48h displayed a significant increase in theintracellular level of ROS as compared with that in the group NG (P<0.05). When cellswere treated with pioglitazone or SB203580, the intracellular ROS levels decreasedsignificantly compared with that of high glucose group (P<0.05). 2Changes of the phosphorylation level of P38MAPK and P38MAPK proteinexpression in mesangial cells.Compared with group NG, the levels of phospho-P38MAPK in group HG wassignificantly increased (P<0.01). While the phospho-P38MAPK was significantlyrescued notablely in group S, group P1and group P2(P<0.05) compared with groupHG. However, the level of P38MAPK protein was unchanged in different groups.3Changes of p22phox and p47phox protein expression in mesangial cellsThe protein expression level of p22phox and p47phox in group NG was significantlylower than that in the experimental group. Compared with group NG, the level ofp22phoxand p47phoxin group HG was significantly increased(P<0.01).Treatment withpioglitazone and SB203580markedly inhibited the high glucose simulated p22phoxandp47phoxexpression in mesangial cells(P<0.05).4Changes of p22phox and p47phox mRNA expression in mesangial cellsCompared with group NG, the gene expression level of p22phox and p47phox wassignificantly upregulated after exposing to high glucose for48h. Pioglitazone marklysuppressed the upregulation in a concentration-dependent manner. SB203580play asimilar role. Moreover, a dose-dependent manner was detected among group P1andgroup P2.5Changes of the activities of SOD, CAT and MDA level in the supernatant inmesangial cellsCompared with group NG, the activities of SOD and CAT decreased in high glucosegroup, while the level of MDA greatly increased (P<0.01). When treated withpioglitazone or SB203580, those changes mentioned above were improved in aconcentration-dependent manner (P<0.05).6The correlation analysisThe application of Pearson correlation analysis revealed that the level of p-p38waspositively correlated with ROS and MDA(P <0.01), and was negatively correlated withSOD and CAT (P <0.05). Moreover, it also show ROS was positively correlation between both p22phox and p47phox expression.ConclusionAfter exposing to high glucose, the expression levels of ROS, p-p38, p22phox andp47phox were significantly increased in rat mesangial cells in vitro. The activities ofSOD and CAT decreased, while the level of MDA greatly increased. Pioglitazone caninhibit oxidative stress induced by high glucose via P38MAPK, which displayed in adose-dependent manner. This effect may contribute partly to its reno-protection.