| Ischemic heart disease (IHD) is one of the diseases which severely impact human life. The pathogenesis and prevention and treatment methods for it has been a hot research area for decades. Recent research shows that precondition stimulates (activates) internal cardioprotective mechanism and improve its capability of anti-ischemia. This kind of endogenetic protective mechanism is related with mitochondria and mitochondrial ATP-sensitive K(mitoKATp) channels. Diazoxide is a typical mitoKAip opener and has the myocardioprotective effects of anti-ischemia and anti-hypoxia . In this study, we explore the probable mechanism of cardioprotection by diazoxide, detect the pharmacological characteristic of diazoxide for myocardium mitochondrial function, and find the new compounds with higher tissue selectivity, better myocardium protection function and less side-effect. 1. The characteristic of respiratory function of myocardium mitochondriaThe structures of mitochondria of isolated rat heart, liver and brain were observed by transmission electron micrograph. Respiratory function of mitochondria was measured by oxygen electrode method. The respiratory function of mitochondria is represented as respiration control ratio (RCR), state 3 respiration (R3), state r respiration (R4) and ADP/O. R3 is the oxygen consumption rate with ADP. R4 is the oxygen consumption without ADP or after ADP was consumed totally. RCR is the ratio of R3 and R4. ADP/O is the ratio of mol of produced ATP while one gram atom oxygen is consumed within mitochondria. The substratesuccinate is oxidated through the succinate oxidation chain, while pyruvate, malate and glutamic acid were oxidated respectively by NADH oxidation chain.2. The effect of diazoxide to cardiac mitochondria functionDiazoxide can decrease R3 and R4 of succinate chain within rat myocardium mitochondria, but don't decrease RCR. It has little effect to R3, R4 and RCR of the compounds which take malate and pyruvate as substrate.The effect of diazoxide to the mitochondria of liver and brain is similar as it to the mitochondria of myocardium. It has no tissue selectivity.Pinacidil has little effect to the respiratory function parameters of rat myocardium and liver, but it can increase R4 of brain mitochondria which take malate and pyruvate as substrates. There are pharmacological difference between diazoxide and pinacidil.3. The anti-hypoxia function of myocardium by diazoxideBased on the hypoxia/reoxygen injured model of cardiac mitochondria, Oxygen Electrode Method was used to measure RCR and Electron micrograph was used to observe the structure of mitochondria. It showed that diazoxide could protect the integrality of cardiac mitochondria and reduce the decrease of RCR.Based on oxidative stress model of the neonatal rat cardiomyocytes, we measured LDH by auto-biochemical detection, detected mitochondrial membrane potential and cell livability on flow cytometry by dual labelling with rhodamine-123 (Rh-123)and propidium iodide(PI). Neonatal rat cardiomyocytes were loaded with Fluo-3M,a Ca2+marker,and observed under a confocal laser scanning microscope.The result was shown that the activity of LDH in the cell culture decreased with the pretreatment by diazoxide than by H2H2, the livability of cell increased. It can also decrease the lose of membrane potential and over loading of calcium, bothof which were caused by oxidation stress. 4. Preliminary study of new ramificationsWe observed the effect to the respiratory function of cardiac mitochondria of 20 ramifications of diazoxide, which were the new compounds synthesized from diazoxide. The result was shown that compounds 2012, 2015 and 2016 have the similar effects to the respiratory function parameter of cardiac mitochondria as diazoxide. These ramifications have no effects to the hemodynamics of rat.In general, diazoxide can modulate the functions of rat myocardial mitochondria, and protect myocytoes and mitochondria on the hypoxia/reoxygen and oxygen stress injured model. As an effective means, to measur... |
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