| Objective:Constructing rat ischemia/reperfusion injury(IRI) model to investigate the protective effect of ischemic postconditioning on ischemic rat myocardium which suffered IRI. Myocardial mitochondria was purified and the mitochondrial protein line 2D-DIGE comparative was performed,to explore the effect of protein expression in mitochondria following ischemic postconditioning.Methods:1 Sprague-Dawley rats were divided randomly into 4 groups(n=6, each group), i.e. the normal (Nor), the control (Con), the ischemic postconditioning (IPO),5-hydroxydecanoate plus postconditioning group (5-HD+IPO). Langendorff apparatus were used to establish the model of myocardial ischemia reperfusion injury. Each group of rats were perfused with K-H for 20 minutes for equilibration. Group Nor:went on perfusion for another 100 minutes after equilibration. Group Con:perfusing 4℃ST.Thomas solution to make the heart stop beating after equilibration, and then underwent 40 minutes global ischemia, followed by 60 minutes reperfusion. Group IPO:after 40 minutes of global ischemia, then reperfusion for 10 secnds, and arrested for 10 secnds.The above procedures were repeated 6 times, reperfusion 58 minutes. Group 5-HD+IPO:perfused with 5-HD(100μmol/Lof K-Hsol) for 5min and then postconditioning was administered as that in IPO group, then perfuse 53 minutes. Investigating the cardiac function of each group at the point of equilibration and reperfusion end such as HR,LVDP,LVDEPand the max dp/dt.2 The myocardial mitochondrial were purified by using Nycodenz density gradient centrifugation and verified purity by western blot.3 The technique of 2D-DIGE and MALO-TOF-MS were utilised to investigate the myocardium mitochandrial protein.Results:1 The cardiac function:no obvious difference between each group after equilibrium,and group Nor,IPO were better than group Con.Intersetly,5-HD+IPO and IPO have no obvious differ.2 Using Nycodenz to purify the myocardial mitochondrial and enrich the degree of mitochondria.3 Well focused and distinct 2-DE maps with good reproducibility were obtained, means of 752±49 protein spots were detected.28 good protein maps were found. We pay close attention to five protein spots of them.4 (1) Group Nor and the Con identified:The expression level of spot554 (NDUFA10, NADH dehydrogenase Asia base)reduces 3.84 times. spot555 (rCG55630, isoform CRA, is extrapolated as the NADH dehydrogenase Asia base) expresses the level to reduce 3.76 times.(2) Group IPO and the Con compares appraises:spot554 (NDUFA10) and spot254 (SDHA, succinic acid dehydrogenase-flavine protein) expression level remarkable reduces 3.33 time,1.68 times separately. spot645 (CoQ9) express the level to reduce 3.43 times. spot648 (CoQ9) expresses the level to elevate 2.42 times.(3) 5-HD+IPO and the IPO group compares appraises:The spot254 (SDHA) expression level elevates 1.65 times.Conclusion:1 Ischemic postconditioning can improve rat myocardium;whereas this protective effects can be antagonistic by 5-hydroxy decanoate.2 Nycodenz density gradient centrifugation is a useful technique to isolate and purify mitochondria.3(1) Ischemia reperfusion injury can affect the NDUFA10.(2) Ischemic postconditioning can maintain the stability of mitochondrial NADH respiratory chain.(3) There is familiar relationship between SDHA and mitoKATP;Both of them may together participate in the protection mechanism of endogenous IPO.(4) CoQ9 may be sheared or modified of Ischemic postconditioning. |
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