| Objective:Resveratrol (RSV), as a PPARy (Peroxisome proliferator-activated receptor gamma) activator, has cardiovascular protective effects. Ischemic Postconditioning(IPO) is an effective means has been focus on to protect reperfusion injury from ischemia/reperfusion(I/R). Whether RSV before IPO can promote IPO effect on the myocardial cytoprotection through MAPK signal pathway has not been reported. As a downstream molecule of p38 MAPK, the role of PPARy in the IPO is not clear. The present experiment aims to study the effect of resveratrol and IPO on the average myocardial cell necrosis rate, p38 MAPK expression, p38 MAPK phosphorylation level and PPARy, PPARy downstream molecular AP-1, cyclinA2 expression in H9c2(2-1) cell. And to confirm our hypothesis:RSV as agonists of PPARy, can improve by increasing PPARy expression, to reduce ischemia-reperfusion injury of myocardial cells and promote IPO protective effect on myocardial cells through the p38 MAPK signaling pathway.Methods:Acquired H9c2 (2-1) cell line and cultured in the DMEM containing 10% FBS, do the experiment when cell growth of 80% of the dish. Cells were divided into control group (C group). I/R group, RSV+I/R group, IPO group and RSV+IPO group. C group was not treated. I/R group were treated with using ischemia and recovery of blood culture media to simulate I/R. IPO group using ischemia and recovery of blood culture media to simulate IPO. RSV+I/R group and RSV+IPO were added RSV 20 min before I/R or IPO.Different time after IPO, using AO-EB Determination of average cell necrosis rate, Western blotting detection of p38 MAPK protein expression, Semi-quantitative RT-PCR detection of p38 MAPK, cyclinA2 mRNA expression. To observe the role of RSV to reduce I/R injury of myocardial cells and induce IPO protective effect on myocardial cells.20min before the IPO, gave RSV to the medium.20min before the RSV, gave SB203580 to the medium. Used AO-EB Determination of average cell necrosis rate, Western blotting detection of p38 MAPK protein expression, Semi-quantitative RT-PCR detection of p38 MAPK, cyclinA2 mRNA expression. To observe the role of RSV to reduce I/R injury of myocardial cells and induce IPO protective effect on myocardial cells. To observe whether RSV, the PPARy agonists, can improve the the protective effect of IPO on myocardial cells by the p38 MAPK signaling pathway. Results:1. RSV with or without IPO decreased the I/R injury of H9c2 (2-1)cell At Oh,12h,24h after IPO, necrosis rate of IPO Group is lower than I/R group, statistically significant (P <0.05). Oh to 12h showed an increasing trend,12 to 24h showed an stabilize trend, little have been slashed. IPO and the RSV alone can make cell necrosis lower than I/R group, statistically significant (P<0.05).2. Influence of RSV before IPO on p38 MAPK/PPARγ/AP-1 pathway p38 MAPK protein levels:at Oh after IPO, IPO group is higher than the I/R group. When at 12h, lower than the I/R group, when at 24h, further reduced. RSV+IPO was lower than I/R group and C group, the differences were statistically significant (P<0.05). By semi-quantitative RT-PCR technique we found p38 MAPK mRNA changed same as p38 MAPK protein levels in trend.When at 12h, p38 MAPK phosphorylation levels:IPO group was significantly lower than I/R group. RSV+IPO group, respectively, lower than IPO group (P<0.05). PPARγprotein level:RSV+IPO group higher than IPO group, IPO group higher than I/R group, while I/R group and C group no difference(P<0.05). AP-1 protein level:RSV+IPO group lower than IPO group, IPO Group lower than I/R group, while I/R group was lower than C group(P<0.05). cyclinA2 mRNA expression:IPO group was higher than the I/R group and C group(P<0.05).3. Inhibition of p38 MAPK blocks RSV influence on various factors We found that::average cell necrosis rate and AP-1 protein in SB+RSV+IPO group were higher than RSV+IPO group(P<0.05), PPARy protein level was lower than RSV+IPO group(P <0.05). Inhibition of p38 MAPK did not change cyclinA2 in RSV+IPO group(P<0.05).Conclusion:1,RSV enhanced IPO-induced reducment of rate of cell necrosis phosphorylation in 0-24h. RSV reduced the I/R injury of myocardial cells and promoted the protective effect of IPO on myocardial cells.2,RSV enhanced IPO-induced reducment of the p38 MAPK, phospho-p38 MAPK and AP-1 expression, and the enhancment of the expression of PPARy and cyclinA2. RSV promoted IPO effect on the myocardial cell protection by improving the p38 MAPK signaling pathway.3,Inhibition of p38 MAPK blocks RSV influence on average cell necrosis rate and PPARy and AP-1 protein levels. Inhibit p38MAPK activation can promote the protective effect of IPO on myocardial cells. Suggested that when RSV joint IPO, p38 MAPK may play an important role to regulate the downstream molecule PPARy and AP-1 protein expression. |
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