| Objective:Constructing rat ischemia/reperfusion injury (IRI) model to investigate the protective effect of diazoxide postconditioning on ischemic rat myocardium which suffered IRI, and to profile protein expression in mitochondria following the treatment of diazoxide.Methods:Langendorff apparatus were used to establish the model of myocardial ischemia reperfusion injury.24 male Sprague-Dawley rats were chosen randomly and divided into 4 groups (n=6, each group), i.e. the normal group (Group Nor), the control group(Group Con), the diazoxide postconditioning group (Group DZ),5-hydroxydecanoate plus diazoxide group (Group 5HD-DZ).Group Nor:went on perfusion Kerbs-Henseleit (K-H) solution 120 minutes without ischemia-reperfusion injury. The other three groups of rats were perfused with K-H soloution about 20 minutes for equilibration. ST. Thomas solution was purfused to stop the heart beating after equilibration, and the heart then underwent 40 minutes global ischemia. Group Con:perfusion of the K-H solution for 60 minutes. Group DZ:after 40 minutes of global ischemia, diazoxide (50μmol/L) was perfused 5 minutes, and then perfused K-H solution for 55 minutes. Group 5HD-DZ was perfused with 5-HD (100μmol/L) for 5 minutes, then perfused diazoxide 5 minutes, and perfused K-H solution 50 minutes finally. Cardiac function of the equilibration and reperfusion were examined on the condition of telophase and the left heart ventricle tissue was collected at the end of reperfusion. To detect the myocardium difference protein in mitochondria, we purified mitochondria by differential centrifugation and Nycodenz density gradient centrifugation and proteomics were analyzed by the technique of 2D-DIGE and MALDI-TOF MS.Results:(1) The purity and integrity of purified myocardial mitochondria isolated by Nycodenz density gradient centrifugation were proved quite well through western blot analysis.(2) The cardiac function:After reperfusion, Group DZ were obviously better than Group Con and 5HD-DZ(P<0.05), while Group Nor were better than Group DZ(P<0.05). There is no significant difference between Group Con and Group 5HD-DZ. (3) Increased proteins include pyruvate dehydrogenase E1 component subunit alpha (PDHA1), acyl-Coenzyme A dehydrogenase, short/branched chain (ACADSB), NADH dehydrogenase (ubiquinone) 1 alpha subcomplex 10 (NDUFA10) and rCG55630 in Group Con, Group DZ, and Group 5HD-DZ, while the level of NDUFA10 and rCG55630 in Group DZ is lower than that in Group Con and Group 5HD-DZ. PDHA1 and ACADSB did present different among Group Con, Group DZ and Group 5HD-DZ.Conclusion:(1) The upregulation of PDHA1, ACADSB, NDUFA10 and rCG55630 in the early period of ischemia-reperfusion may be induced by hypoxia.(2) Diazoxide may partially inhibit the upregulation of NDUFA10 and rCG55630 in the early period of ischemia-reperfusion.(3) Diazoxide can decrease cardiac ischemia-reperfusion injury, protect injuried myocardium and improve the injuried heart fuction.(4) Nycodenz density gradient centrifugation is a very useful technique to isolate and purify mitochondria. |
PDF Full Text Request