| Part one:The cardioprotection of morphine delayed preconditioning in the ischemia-reperfused rat heartsObjective:To investigate the cardioprotection of morphine delayed preconditioning against myocardial ischemia reperfusion injury in the rats.Methods:Sixty male adult Sprague-Dawley rats were randomly assigned to 4 groups. Group S:the sham operation group, the rats received a thoracotomy without occlusion of the left anterior descending (LAD) of coronary artery; group IR, the ischemia reperfusion group, rats treated with NS (1.0 ml/kg iv) 24 h before ischemia; group M, the morphine preconditioning group, rats treated with morphine (0.3 mg/kg iv) 24 h before ischemia; and group D, morphine preconditioning+ 5-hydroxydecanoate group, morphine-treated animals received the 5-hydroxydecanoate (5-HD,10mg/kg), an inhibitor of mitochondrial KATP channel 15 min before ischemia. Myocardial ischemia-reperfusion in groups IR, M and D were induced by 30 min of LAD occlusion, followed by 2 h of reperfusion. At the end of the reperfusion, myocardial infarct size was measured and the myocardial ultrastructure was observed under the electron microscopy. Hemodynamics, including HR, LVEDP, LVESP, etc, were recorded through the operation. Results:1. Myocardial infarct size:Compared with group IR, morphine caused a significant reduction in infarct size (23.78±4.82 vs. 37.87±5.92, P<0.05).The effect of morphine reducing infarct size was blocked by 5-HD (39.62±5.18 vs.23.78±4.82, P<0.05).5-HD did not affect the infarct size compared with group IR (P>0.05).2. Myocardial ultrastructures:Compared with group IR, the severity of myocardial injury in Group M was ameliorative under the light microscope, but group D had a similar situation with the group IR.3. Hemodynamics:The function of hearts in the group M were better than group IR (P<0.05). There were no differences between the group IR and group D (P>0.05).Conclusion:Morphine preconditioning induces a delayed cardioprotection against ischemia reperfusion injury in the rats, and which was mediated by the opening of mitochondrial KATP channel. Part two The disassociation of myocardial mitochondria and abstraction of the proteins in ratsObjective:To obtain the purified myocardial mitochondria and validate the purityMethods:Purified mitochondria were obtained by Nycodenz density gradient centrifugation, the purity were identified by western blot analysis.Results:Western blot analysis showed that the purity of myocardial mitochondria isolated by Nycodenz density gradient centrifugation were good enough for mitochondrial proteomic analysis.Conclusion:The purity of purified myocardial mitochondria was obtained.Part three Effects of morphine delayed preconditioning on myocardial mitochondria proteomic in rat heartsObjective:To investigate the changes of myocardial mitochondrial protein expression after morphine delayed pretreatment, and to search for the possible mechanisms involved in the delayed preconditioning.Methods:Proteomics analysis of myocardial mitochondria protein of morphine pretreatment. The left ventricle tissues from morphine preconditioning group (group M) or IR group (group IR) were sampled for proteomics analysis. The total proteins were extracted and separated by two dimensional gel elecrtophoresis(2-DE). The differentially expressed protein spots were analyzed with martix-assisted laser desorption/ionization time-of-flihgt mass spectrometry (MALDI-TOF-MS).Results:Analysis of 2-DE showed that 634±13 protein spots were in group IR and 651±17 protein spots in group M. Totally 30 protein spots were differentially expressed between the two groups and subjected to mass spectrometry analysis. Of the 30 protein spots,12 proteins were preliminarily identified. These proteins could be classified into four functional groups:metabolism related proteins (ATP synthase subunit beta, Elongation factor Tu), anti-oxidant related proteins (Cytochrome c oxidase, Peroxisome proliferator-activated receptor gamma), respiratory chain related proteins(Electron transfer flavoprotein subunit beta, NADH dehydrogenase) and membrane mitochondrial channel related proteins(Voltage-dependent anion-selective channel protein, Mitochondrial import inner membrane translocase subunit Tim9).Conclusions:Morphine delayed preconditioning resulted in the changes of protein expression profiles in the myocardial mitochondria. The differential proteins might function as anti-oxidant reaction and improving the energy metabolism of myocardial mitochondrial, which would be the basic mechanisms of delayed cardioprotection. Part four Effect of morphine delayed preconditioning on NADH dehydrogenase (NADH-DH) expression during myocardial ischemia-reperfusion in ratsObjective:To investigate the effect of morphine delayed preconditioning on NADH dehydrogenase (NADH-DH) expression during myocardial ischemia-reperfusion in rats.Methods:Methods:Forty male adult Sprague-Dawley rats were randomly assigned to 4 groups. Group S:the sham operation group, the rats received a thoracotomy without occlusion of the left anterior descending (LAD) of coronary artery; group IR, the ischemia reperfusion group, rats treated with NS (1.0 ml/kg iv) 24 h before ischemia; group M, the morphine preconditioning group, rats treated with morphine (0.3 mg/kg iv) 24 h before ischemia; and group D, morphine preconditioning +5-hydroxydecanoate group, morphine-treated animals received the 5-hydroxydecanoate (5-HD,10mg/kg), an inhibitor of mitochondrial KATP channel 15 min before ischemia. Myocardial ischemia-reperfusion in groups IR, M and D were induced by 30 min of LAD occlusion, followed by 2 h of reperfusion. At the end of the reperfusion, myocardial infarct size was measured. NADH-DH was measured by Western blotting.Results:1. Myocardial infarct size:Compared with group IR, morphine caused a significant reduction in infarct size (23.78±4.82 vs. 37.87±5.92, P<0.05), that was blocked by 5-HD (39.62±5.18 vs.23.78±4.82, P<0.05).5-HD did not affect the infarct size compared with group IR (P>0.05).2. Myocardial NADH-DH expression:Compared with group S, there were decreases in groups IR, M and D (P <0.05).Morphine treatment induced a higher expression of NADH-DH in hearts compared with group IR (P<0.05).5-HD abolished the effect of morphine (P<0.05), but had no differences with group IR (P>0.05).Conclusion:Morphine induced delayed preconditioning attenuating myocardial I/R injury, possibly through up-regulating myocardial NADH-DH expression in rats. |
PDF Full Text Request