| Ischemic preconditioning(IPC) is a process whereby a transient sublethal ischemic stress enhances the tolerance of myocardium to a subsequent lethal ischemic insult. It has been proved to be the most effective endogenous protective mechanism of protecting the heart against ischemia-reperfusion(I/R) injury up to now and thus has important significance in clinical prophylaxis and therapy of cardiovascular diseases. However, as an endogenous protective mechanism, IPC could not be used directly as a clinical therapeutic method, which limited its applications. Therefore, pharmacological agents capable of imitating IPC and protecting the heart from infarction have been the subjects for intensive research. Recently, it has been proposed that several agents could mimic the protection of IPC, which is called pharmacological preconditioning(PC). Nitric oxide(NO) is an important gas messenger molecule which is implicated in IPC process. NO donor drugs, nitrate-based vasodilating agents, are the elementary drugs for cardiac angina and have been proved safe for more than one century. However, it is not clear whether NO has a direct cardioprotective effect. The study of NO-mimicked IPC aimed at yielding novel therapeutic ways of cardioprotection and providing theoretical evidence for the development of new NO donor drugs with better stability, longer half life and less tolerance.In this study, a cell model of myocardial hypoxia preconditioning (HPC) was established and the protective effects and the mechanisms were observed. Based on the study of HPC, NO-mimicked early and late HPC and possible mechanisms were observed, respectively.1. Protective effects of HPC and it mechanisms LDH activity in the cell model of HPC was detected by colorimetry and cell viability was detected by MTT assay to evaluate injury of cardiomyocytes. Acridine orange (AO) staining and flow cytometer based on PI staining were used to detect apoptosis. Colorimetry was used to detect caspase-3 activity, and immunohistochemistry with computer image analysis was used to observe the expression of Bcl-2 protein in cardiomyocytes. The results showed that early protection of HPC may probably activate G protein and subsequently activate protein kinase C (PKC) and open ATP-sensitive potassium channel (K (ATP) channel). However, the effects of late HPC may be related to the synthesis and secretion of some agents through G protein-PKC-NF-B pathway. HPC could reduce H/R-induced apoptosis of cardiomyocytes by upregulationg the expression of anti-apoptotic protein Bcl-2 and inhibiting activity of caspase-3, the executioner of apoptosis. The inhibitory effect of apoptosis in late HPC was stronger than that in early HPC. These results provided evidence for further study of HPC.2. NO mimic early protection of HPC The influence of NO on necrosis and apoptosis of cardiomyocytes induced by H/R injury were observed in this experiment. LDH activity and cell viability were detected to evaluate injury of cardiomyocytes. AO staining and TUNEL staining were used to observe morphological changes of apoptotic cells. Flow cytometer based on Annexin V-FITC/P1 double staining was used to detect apoptotic rates. Fluo-3-based laser confocal scanning microscopy was used for measuring changes in intracellular calcium content of cultured neonatal cardiomyocytes. Colorimetry was used to detect SOD and MDA activity of cardiomyocytes. The results showed that NO could mimic the early protection of HPC by inhibiting H/R-induced necrosis and apoptosis of cardiomyocytes. The inhibitory effect on necrosis was cGMP-dependent and activation of PKC and opening of K(ATP) channels were important downstream events, while apoptosis was inhibited through reducing the caspase-3enzyme activity. The study of the underlying mechanisms revealed that NO may exert early protection by improving antioxidant capability and reducing intracellular calcium overload of cardiomyocytes, thus decreasing I/R-induced oxygen paradox and calcium paradox.3. NO mimic late pro...
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