| Reperfusion is an important method of treatment for ischemic heart disease, however,ischemic/reperfusion(I/R) injury always attenuated its earning.Ischemia preconditioning(IPC) become a effective method to relieve I/R injury because of its protective effects against myocardial cell damage.Nowadays,Pharmacological preconditioning(PPC) is gradually recognized after some experiments have confirmed that some drugs may relieve I/R myocardial injury during past few years.Previous studies showed that the protective of some preconditionings was due to release of adenosine,bradykinin,which actived pathways of protein kinase C(PKC), external-signal regulated kinase(ERK),and phosphoinositide kinase-3(PI3K),and opened mitochondrial ATP-sensitive K+ channels(motiKATP).Previous studies showed that the protective effect of man-made peroxisome proliferator activated receptorsγ(PPARγ) ligands--Thiazolidinediones(TZDs),just as rosiglitazone, troglitazone and pioglitazone,was due to its simulating for IPC.Our previous study also proved that pioglitazone,as a man-made PPARγagonist,might protect rat myocardial cell from I/R injury by opening mitochondrial ATP-sensitive K+(mitoKATP) channel.However,this study employs primary culture neonate rat myocardial cells try to made a further investigation for the pioglitazone's protective effects and its mechanism against I/R injury under the basement of previous studies.1.Objective1.1.To observe the effects of pioglitazone on expression of protein kinase C(PKC) in mitochondria of neonate rat myocardial cells experiencing hypoxia/reoxygenation process.1.2.To observe the effects of pioglitazone on mitochondrial membrane potential in neonate rat myocardial cells experiencing hypoxia/reoxygenation process. 2.Methods2.1.Primary cultureed of cardiac myocytes,which were prepared from the ventricles of neonatal SD rats,were divided into four groups:Solvent Control group,H-r+ Solvent Control group,H-r+ 2μM pioglitazone group,H-r+ 2μM pioglitazone+GW9662 group,then hypoxia-reoxygenation injury model were established at 24 hours after pretreatment.The cells stained with Hoechst33258 which was used to detect the apoptosis of cardial myocyte;Western-blot was employed to detect the expression of PKC.2.2.Primary cultures of cardiac myocytes,which were prepared from the ventricles of neonatal SD rats,were divided into seven groups:Solvent Control group,H-r+ Solvent Control group,H-r+0.1μM pioglitazone group,H-r+1μM pioglitazone group,H-r+2μM pioglitazone group,H-r+ 2μM pioglitazone+chelerythrine group,H-r+ 2μM pioglitazone+GW9662 group,and hypoxia-reoxygenation injury model were established at 24 hours after pretreatment,then stain with JC-1 Staining solutions,at last,detect mitochondria membrane potential with confocal laser scanning microscopy.3.Results3.1.Myocardial cells were induced to apoptosis by hypoxia-reoxygenation.The percentage of cells early apoptosis increased from 0.20%±0.03%of control group to 12.22±1.45%of hypoxia-reoxygenation group(p＜0.05).Pioglitazone decreased myocardial cells apoptosis(12.46±1.62%,p＜0.05),and this action could be abolished by PPARγreceptor specific antagonist GW96623.2.Compared with simple hypoxia-reoxygenation group,pioglitazone group did not increase the expression of PKC in myocardial cells.3.3.Compared with H-r+Solvent Control group,pioglitazone group might inhibit degression of mitochondrial membrane potential,and was concentration dependent.But this protective action was attenuated in both groups in which chelerythrine of GW9662 was added(p＜0.01).4.Conclusion4.1.Pioglitazone pretreatment can reduce myocardial cells apoptosis induced by hypoxia-reoxygenation via partly by activation of PPARγ.4.2.Pioglitazone pretreatment did not increase the expression of PKC in myocardial cells.4.3.Pioglitazone can protect myocardial cells from hypoxia-reoxygenation injury by means of inhibiting degression of mitochondrial membrane potential.This effect is associated with activation of PPARγ,and may related to PKC pathway. |
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