The Protective Effects And Mechanisms Of New Compound Methylamine Irisolidone On Acute Myocardial Ischemia Injury

Objective:Acute myocardial ischemia(AMI) is one of the worst diseases harming the health of middle and elder people in our country,which is also one of the commonest reasons leading to sudden death.The reparative process and prognosis of acute myocardial infarction is correlated to functional status of vassular in myocardial tissue.Therefore,forming and opening of coronary artery collateral circulation, ameliorating blood supply and myocardial survival of infarction zone by rapid angiogenesis or revascularization is extremely considerable significance.Nowadays, interventional therapy such as percutaneous tranluminal coronary angioplasty(PTCA) and intracoronary stent are the commonly used methods of revascularization after AMI.Interventional therapy is only suitable for arterial diameter above 2 mm, however,which is no use for capillary and arterial diameter below 2 mm.Furthermore, interventional therapy exist re-narrow(25%～50%) resulting in considerable acute patient who can not achieve completely revascularization and influence prognosis. Recently,besides opening of coronary artery collateral circulation,inducing angiogenesis to reestablish revascularization have developed to a new strategy for curing myocardial ischemia after AMI.The new strategy is also called "therapeutic angiogenesis".There are major foundation and clinical research related to angiogenesis and therapeutic angiogenesis,and a number of vascular growth factors and transgenic therapy have been approved to promote angiogenesis.However,the local and tedious application of vascular growth factors,and transgenic therapy existing many problems,all that confine its clinical application.Therefore,we need search more convenient,effect and pragmatic drugs to replace growth factors.Our previous research showed that kakklide extracting from Puerariae Flos could counteract hypoxia and myocardial ischemia significantly,and also significantly counteract thrombosis and improve blood viscosity.We modified the structure of the compound owing to its worse water-solubility,and obtained a new compound—methylamine irisolidone.The toxicity testing of mice and cells showed that this new compound had less toxicity.On the contrary,the new compound could increase the survival time of standing hypoxia in mouse.Accordingly,on the basis of our previous study,we want to identify the protection and mechanisms in myocardial ischemia of methylamine irisolidone,including the improvement of microcirculation,apoptosis and angiogenesis of ischemic myocardium.,which may offer a new strategy and medicine for curing myocardial ischemia.Methods:There are two parts of our study.The first part researched the effects and mechanism of methyalmine irisolidone on acute myocardial infarction and myocardial cells cultured under ischemia and hypoxia.The second part investigated the mechanism of methylamine irisolidone on angiogenesis induced by acute myocardial infarction.Part 1:In vivo experiment,the survival test of hypoxic mice,and electrical stimulation of thrombosis model in rats were used to evaluate the pharmacy activity of methylamine irisolidone.Isoprenaline and coronary artery ligation were used to induce the acute myocardial infarction model,several experiments including the degree of myocardial ischemia(∑-ST),the range of myocardial ischemia(N-ST) and the infarct area,myocardial zymogram(AST,LDH,CK,CK-MB),heart function,the serum levels of CGRP and ET-1 were investigated.In vivro experiment,we investigated the effect of methylamine irisolidone on hypoxia injury in cultured rat cardial myocytes.Neonatal rat cardiac myocytes in primary culture were exposed to hypoxia for 3 hours and subsequently reoxygenated for 6 hour.Myocytes injury was determined by the release of lactate dehydrogenase (LDH),and the activity of SOD,MDA and ROS levels,the change of mitochondrial membrane potential(MMP),myocardial cells cytosolic Ca²⁺ content were measured to investigate the effects and mechanism of methylamine irisolidone.Part 2:Coronary artery ligation was used to induce the acute myocardial ischemia model.After 30 d administration of methylamine irisolidone,the gene and protein expression of HIF-1α,VEGF,eNOS in ischemia border zones were detected and the serum level of VEGF,CGRP,ET-1,NO and NOS activity were measured by ELISA,radioimmunity,nitroreductase methods,respectively.At the same time,the multiplication and migration and tube structure formation of HUVECs were detected.Results:1.Methylamine irisolidone administration markedly prolonged the living time of the hypoxia experimetal mice at the doses of 300mg and 150mg/kg,reduced the formation time of carotid artery thrombosis,decreased blood viscosity shear rate.2.For in vivo experiments,compared with model group,pretreatment with methylamine irisolidone at dose of 200 and 100mg/kg could improve the myocardiac injury induced by isoprenaline,and decrease the release of cardiac creatase in rats.3.Methylamine irisolidone(80 mg/kg,i.v) could reduce the myocardial infarct areas in dogs with myocardial infarction.The serum lactate dehydrogenase(LDH) activity and MB isoenzyme of creatine kinase(CK-MB) were suppressed by methylamine irisolidone after 2 and 4 h of administration.Methylamine irisolidone (80 mg/kg) can lessen the degree of myocardial ischemia obviously,manifestly reduce the range of myocardial ischemia(N-ST) in contrast with the myocardial ischemia control group.Myocardial infarct area displayed by the N-BT staining is roughly similar to the results measured by the epicardial electrogram.Methylamine irisolidone (80 mg/kg) exhibited obvious effects of lessening injury in myocardial ischemia,and the infarct area is conspicuously decreased as compared with the ischemia control group.4.Methylamine irisolidone could improve the heart function of rats in AMI injury,increased the systolic pressure and diastolic pressure,decreased the heart rate. Methylamine irisolidone could increase the level of calcitonin gene related protein (CGRP) and decrease the level of endothelin-1(ET-1) in the blood plasma and achieve the dynamic balance.5.The cell viability of cardiomyocytes treated with methylamine irisolidone during H/R injury significantly increased compared with methylamine irisolidone-untreated cells.Similar results were shown by LDH release.Methylamine irisolidone enhanced the mitochondrial membrane potential and decrease intracelluar calcium associated with the attenuated reactive oxygen species(ROS) generation,reduced levels of malondialdehyde(MDA) and increased activity of superoxide dismutase (SOD) after H/R in a dose-dependent manner.6.Methylamine irisolidone induced angiogenesis in the ischemic myocardium, myocardial vessel density was significantly increased in ischemic zones.The gene expression or activation of vascular endothelial growth factor(VEGF),and endothelial nitric oxide synthase(eNOS) which correlated with angiogenesis were also induced by pretreated with methylamine irisolidone.On the other hand, methylamine irisolidone could improve the ultrastructural organization of ischemic myocardial zones,myofibrillae well-arranged,mitochondria structure integrated, nuclear chromatin well-distributed and a few blood capillary appeared in mesenchymal.For cultured serial subcultivation HUVECs,pretreated with methylamine irisolidone could speed up the endothelial cell proliferation(cells in S and G2 period increased),cell migrate and tube structure formation.Conclutions:1.The present study demonstrated that methylamine irisolidone could lessen the degree of myocardial ischemia obviously(∑-ST) and the range of myocardial ischemia(N-ST) and the infarct area in acute myocardial ischemia dogs,and could improve the myocardial injury induced by isoprenaline.These results suggested that methylamine irisolidone exert the protective effects on myocardial ischemia injury, which may be due to its function of inhibiting LDH and CK-MB releasing,stabilizing myocardial cell membrane and improving myocardial microcirculation and metabolism.2.The present study demonstrated that methylamine irisolidone could directly protect cardiomyocyte against hypoxia injury,primarily as a result of reduction of the intracellular Ca²⁺ overload coincident with an attenuation of ROS generation and ROS-mediated lipid peroxidation which may contribute to the preservation of mitochondrion function and antioxidant against hypoxic injury.3.Under ischemic and hypoxic connditions,there is no change of HIF-1αmRNA expression,however,HIF-1αprotein expression enhanced.At the same time,the activity of NOS increased and NO level enhanced.That is to say,reduce HIF-1αdegradation,improve its activity,priming HIF-1α—VEGF—NO signal pathway,can induce the compensation protective effects.But with time extended,the compensation is attenuated,the concentration of VEGF and NO were decreased markedly.So, improving the HIF-1α—VEGF—NO transduction is one of the targets of treatment of ischemic heart diseases.4.Under ischemic and hypoxic connditions,pretreated with methylamine irisolidone could improve the formation of capillary in ischemic zone.It is indicated that improve the transduction of HIF-1α—VEGF—NO sinal pathway could induce "drug bridge" effect for treating ischemic disease.5.For cultured serial subcultivation HUVECs,pretreated with methylamine irisolidone could speed up the endothelial cell proliferation,cell migrate and tube structure formation,which may be one of the mechanisms of its therapeutic angiogenesis.