| The most effective method of limiting acute myocardial ischemia necrosis is reperfusion.The effect of reperfusion itself may be associated with polymophonuclear leucocyte (PMN) accumulation, a burst of oxygeon free radical production.activation of inflammation, introcelular calcium overload.which may result in paradoxical cardiomyocyte dysfunction a phenomenion termed reperfution injury. Studies on the new agents used to treat myocardial ischemia reperfusion are still a pivotal issue.There is still no effect methods to provent or attenuate the MIRI, The research of efforts to protect myocardium in the current era of reperfusion by thrombolysis or percutaneous cronary intervention has focus on the limitation of irreversible reperfusion injury. Preconditioning (PC) is presently considered the most effctive endogenous protect mechnism of reperfusion injury.Studies using different method suggested that ischemia precondition (IPC) could protect ischemic myocardium and attenuate reperfusion injury,and improve the myocardial function.and decrease the ratio of apoptosis.In all of the methods of PC,the use of IPC is limited because of the ethics and the difficulty of practice.At the otherside, pharmacological preconditioning is easy to use in clinical,especialy the traditional chinese medicine has shown the effects and practicability in protection of reperfusion injury.AstragalosideⅣ(AsⅣ) is the extract of monomer Astragaloside. Astragaloside has multiple physiological and pharmacological functions: Astragaloside plays an important role in increasing contents of cAMP to protect myocardium from ischemial injury.which could clear the oxygen free radical, decrease blood viscidity. The protective effects of AsⅣon cardiovascular disease have been paid little attention by people, so developing the new preparation of AsⅣon cardiovascular disease has good prospect.This study includs two parts:study of ischemia in vivo and cultured cell in H2O2 in vitro.We used Wistar rat as the model of reperfusion and H2O2 cultured neonatal cells to further investigate the effects of AsⅣ's preconditioning on ischemia reperfusion injury and apoptosis,and the change of correlated protein.The objectives of the present study are to develepe the model of myocardial ischemia reperfusion injury in vivo and oxidative stress in vitro to invesgate the protective effects and possible mechanisms of AsⅣon the cardiovascular disease and to provide a theoretical basis for better, safer use and targeting application in clinical practice.Myocardium ishemia reperfution injury model in rats by ligating the descending anterior branch of the coronary artery for 30 min and relaxing 2h was developed, the effects of AsⅣon the serum level of lactate dehydrogenase (LDH), superoxide dismutase (SOD), malondialdehyde (MDA), nitrix oxyde (NO) were measured and myocardium injury area was detected by nitroterrazolium blue chloride (NBT) staining. At the same time, samples were taken to fix in neutral fulmarlin for HE staining, immunohistochemica and TUNEL performance.another samples were store in the liquid nitrogen for the measure of western-blot. Ultramicrostructure in myocardium tissue was detected by electron microscope. The protein expression of Bcl-2, PKCεand iNOS were analyzed by Western-blottingLigating the left anterior descending coronary artery and relaxing in rat simulated the patho-physiological process-the increasing of free radicals in myocardial ischemia. Experiments results in vivo showed that AsⅣcould significantly reduce content of MDA, and increase the activity of SOD in serum, and reduce LDH and increase NO releasing from myocardial cell. AsⅣcould decrease the positive percent of cardiac myocyte apoptosis, and reduce the injury area. In addition, AsⅣcould protect the integrity of mitochondrial memberane and prevent over proliferation of mitochondria. It is demonstrated that AsⅣis effective on the therapy of myocardium ischemia reperfution injury in animals. It was presumed that AsⅣprotected the cellular membrane, inhibited the cardiac myocyte enzymes release, reducing myocardium apoptosis percentage and reducing the infarction area probably by increasing the enzyme activity of anti-oxidation and the ability of removing free radicals.The primary cultured cardiac myocytes in neonatal rats damaged by 0.2mmol/L hydrogen peroxide (H2O2) simulated the oxidative stress model. The survival ability were detected by Methyl thiazolyl tetrazolium(MTT) method. The content of LDH in cultured conditions, SOD and MDA in myocytes were measured by the instruments. The percentage of apoptosis rate were measured by flowcytometry, mitochondrial memberane potential(△Ψm) and intracellular calcium change by Fluo-3/AM staining before and after H2O2 damage was observed by laser-confocal-microscopy system. Meanwhile, the protein expression of Bcl-2, PKCεand iNOS were analyzed by Western-blotting. The results in vitro experiments showed that AsⅣcould increase the survival rate of cardiac myocytes, reduce the release of LDH from the myocytes and the generation of MDA, increase the activity of SOD and the content of NO in myocytes, reduce apoptosis percentage, stabilize the mitochondrial membrane potential, reduce the intracellular calcium overload, increase the protein expression of Bcl-2, PKCεand iNOS.Oxidative stress in myocytes was simulated by adding the final concentration of 0.2mol/L H2O2 to the cultured conditions, resulting in calcium overload and the damage of motrochondria. These three factors may work separately or interact with each other to cause cellular structure, function and metabolism obstruction, and eventual cell death. AsⅣreducing the the generation of MDA and increasing the activity of SOD in myocytes damaged by H2O2 indirectly demonstrated that AsⅣcould protect the activities of antioxidase and inhibit the formation of lipid peroxidation production, this accorded with the finding of researchers abroad and also hinted that one of probably mechanisms of AsⅣon protecting myocardium is inhibiting the production of oxygen free radicals. AsⅣreducing the change of intracellular Ca2+ fluorescent intensity before the myocytes damaged by H2O2 indicated that AsⅣattenuated calcium overload. In addition, It indicated that AsⅣinhibited the opening of mitochondrial permeability transition pores to stabilize mitochondria membrane potential. AsⅣinhibit myocytes apoptosis by several ways①AsⅣinhibit the initiation of apoptosis by reducing the formation of oxygen free radicals and intracellular calcium overload.②AsⅣinhibited the opening of mitochondrial transition pores, thus reducing the release of apoptosis-inducing proteins, such as Cyt C,Smac,AIF.③AsⅣmodulated the protein in signal transduction of apoptosis, such as upregulating anti-apoptosis Bcl-2 protein family.In conclusion, AsⅣprotected the myocyte from acute myocardium reperfusion injury by ligating the left anterior descending coronary artery, and oxidative stress damaged by H2O2. Based on the above experiments in vivo and in vitro, it has been proven AsⅣprotected the cardiac myocyte from hypoxia damage probably owing to reducing free radical production, increasing the NO content, decreasing intracellular calcium overload, protecting the mitochondrion and inhibiting cardiomyocyte apoptosis. CHE and 5-HD abolish the protective effect of AsⅣ, So PKCεand mitoKATP is the down stream of NO which is involved in the transduction of AsⅣ. However, CHE but not 5-HD abolish the translocation of PKCεfrom cytosol to membrane, which shows that mitoKATP is the down stream of PKCε.From the above result,we can take a conclusion that the protective effectiveness of AsⅣProbably owning to inducing the production of NO,the trigger of preconditioning,then up-regulation the protein of expression of PKCεand iNOS in damaged myocytes, activating the channel of mitoKATP . NO-PKCε-mitoKATP signal transduction may play a pivotal role in IPC, so AsⅣmimicry endogenous protect mechanism -induce the pharmacological preconditioning to protective the myocardium.
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