Mechanisms Of Neu-P11 Attenuates Cardiomyocyte Ischemia Reperfusion Injury

Within the past decade,the view of medical technology has changed substantially.Percutaneous transluminal coronary angioplasty(PTCA),which is often performed after acute myocardial infarction(AMI),may obviously improve the patient survival rate.Although restoration of blood flow to an ischaemic organ is essential to prevent irreversible tissue injury,reperfusion per se may augment tissue injury in excess of that produced by ischaemia alone.Cellular damage after reperfusion of previously viable ischaemic tissues is defined as ischaemia reperfusion(IR)injury.IR injuries are characterized by oxidant production,complement activation,leucocyte-endothelial cell adhesion,platelet-leucocyte aggregation,increased microvascular permeability and decreased endothelium-dependent relaxation.Although our understanding of the pathophysiology of IR injury has advanced significantly in the last decade,such experimentally derived concepts have yet to be fully integrated into clinical practice.Therefore,studies on the modes of myocardial cell death after IR are of great significance.Previous studies suggested that myocardial cell death following myocardial IR injury were mainly necrosis and apoptosis.However,in recent years,a number of studies have suggested that,another procedural manner of death-autophagy,also known as type II programmed cell death,plays a critical role in IR injury.The study of this death pathway may provide a new effective way to block myocardial ischemia/reperfusion injury.Autophagy is the transportation and degradation of damaged,denatured or aged proteins.It is a common cellular physiological process which can maintain cell homeostasis.Autophagy is an evolutionary conserved pathway of self-digestion that occurs in process of cell differentiation,survival and homeostasis.Many studies suggest that autophagy has a dual role in the heart depending on the stimulus.It has been speculated that too much autophagy can cause cell death by excessive degradation of essential proteins and organelles.Autophagy provides protection during ischemia,whereas it may be detrimental during reperfusion.Thus,manipulation of autophagy may represent a potential future therapeutic target to treat or prevent development of heart disease.Some previous studies reported that melatonin has significant protective action against the cardiac damage and altered physiology that occur during ischemia-reperfusion injury.Neu-P11(piromelatine,N-(2-(5-methoxy-1H-indol-3-yl)ethyl)-4-oxo-4H-pyran-2-carboxamide)is a novel melatonin(MT1/MT2)receptor agonist may serve as a novel agent for the treatment of psychophysiological insomnia,Alzheimer'disease,and hypertensive.But whether Neu-P11 has a possible beneficial effect on IR injury and the mechanism of protective effect of Neu-P11 is still unknown.Objectives The purpose of this study was to explore the potential protective effect of Neu-P11 on ischemia-reperfusion injury in vitro and in vivo.Firstly,the effect of Neu-P11 on hypoxia-reoxygenation(HR)induced H9c2 cells injury were evaluated by assay of cell vitality,and the release of lactate dehydrogenase(LDH),Creatine Kinase(CK),Creatine Kinase-MB(CK-MB)and the number of acidic vesicular organelles(AVO)in cells by flow cytometry analysis,and the changes of autophagy signaling pathway related proteins expression by western blot.Secondly,male Sprague-Dawley(SD)rats model of IR injury were subjected to ligation of left anterior descending artery for 30 min and then reperfusion for 120 min.The effect of Neu-P11 on rats model of IR injury were also measured by the release of LDH,CK and CK-MB from heart tissue,and the proper myocardial infarct delineation by TTC staining,and the changes of autophagy signaling pathway related proteins expression by western blot.Finally,the role of mTOR involved in protective effect of Neu-P11 was assessed in HR H9c2 cell model.Methods and resultsPart one: the protective effect of Neu-P11 on HR injury on H9c2 myocardial cellsHR injury model was established with H9c2 cells.The H9c2 cells in the logarithmic growth phase were randomly divided into six groups: the control group,HR group,Neu-P11 1n M+HR group,Neu-P11 10 n M+HR group,Neu-P11 100 n M+HR group and CQ+HR group.Cell vitality was measured by using MTT assay.And the cells damage induced by HR was determined by leakage of LDH,CK,CK-MB into cell culture medium.And the formations of acidic vesicular organelles(AVO)in cells were observed by flow cytometry.And the changes of Beclin-1,LC3 and p62 protein expression were measured by western blot.We found that Neu-P11 and CQ could significantly decrease HR induced cell death,and release of LDH,CK,CK-MB and the formations of AVO.In addition,the over-expressions of Beclin-1,LC3 and p62 protein induced by HR injury were also alleviated.The results suggest that the protective effect of Neu-P11 against HR injury was correlated with a reduction of excessive autophagy.Part two: the effects of Neu-P11 on IR injury in rat heartA rat model of myocardial IR injury was established by the ligation of left anterior descending coronary artery for 30 min followed by 120 min perfusion.Before 30 min of IR,the rats were treated with Neu-P11 at doses of 100 n M and CQ at doses of 10mg/kg respectively.And the myocardium damage induced by IR was determined by leakage of LDH,CK,CK-MB into serum.The assessment of the infarct size was measured by triphenyl tetrazolium chloride(TTC)following 120 min of reperfusion.And the changes of Beclin-1,LC3 and p62 protein expression were measured by western blot.We found that Neu-P11 and CQ pretreatment significantly reduced infarct size and the level of serum LDH,CK and CK-MB.Neu-P11 alleviated the over-expressions of Beclin-1,LC3 and p62 protein induced by IR injury.The results suggest that Neu-P11 pretreatment could provide cardioprotection in a rat model of myocardial IR Injury and reduce the high level of autophagy.Part three: Neu-P11 down-regulating excessive autophagy through activating mTORHR injury model was also established with H9c2 cells.The H9c2 cells in the logarithmic growth phase were randomly divided into five groups: control group,HR group,Neu-P11 100 n M+HR group,rapamycin25mmol/L+HR group,rapamycin 25mmol/L+Neu-P11 100 n M+HR group.The changes of Beclin-1,LC3,p62,p-mTOR,p-p70s6 k,p-4EBP1 protein expression were measured by western blot.We found that Treatment with 100 n M Neu-P11 could down-regulate the over-expression of Beclin-1,LC3,and p62 protein,but up-regulate the expression of p-mTOR,p-p70s6 k,p-4EBP1 protein.The results revealed that Neu-P11 could down-regulate excessive autophagy induced by HR through activating mTOR.Conclusion1.The protective effect of Neu-P11 against HR injury was correlated with a reduction of excessive autophagy.2.Neu-P11 pretreatment could provide cardioprotection in a rat model of myocardial IR Injury and reduce the high level of autophagy.3.Neu-P11 could down-regulate excessive autophagy induced by HR through activating mTOR.