The Effect And Mechanism Of Poly(ADP-Ribose) Polymerase On Rat Heart Ischemia/Reperfusion Injury

With the improvement of people's standard of living,cardiovascular disease has now become a threat to human life and health.The main cause of death of cardiovascular diseases is coronary artery disease.The best treatment of coronary heart disease is the resumption of blood perfusion of myocardium as soon as possible.However,both animal experiments and clinical studies have found that the damage of myocardial cell function and structural became worse with the restoration of blood supply.We call the pathophysiological state ischemia-reperfusion injury(I/R).In the course of I/R,a large amount of oxygen free radicals,superoxide anion,oxidative stress and the calcium overload of cells and mitochondrial which accelerated cardiomyocyte apoptosis and necrosis and expanded the scope of myocardial infarction.As a result, the heart function deteriorated rapidly.In clinical situations,reperfusion following ischemia result in a sharp drop in blood pressure,heart failure,arrhythmia and even sudden death which worsening pathogenetic condition.Reperfusion involved in the clinical practice such as reperfusion myocardial infarction after coronary artery bypass grafting,heart transplants,heart resuscitation of cardiac arrest and cardiopulmonary bypass heart surgery.It is very important of the resumption of blood perfusion of myocardium as soon as possible.At the same time,we should try to avoid the reperfusion injury.To elucidate the mechanisms of the pathophysiological state is a prerequisite for the effective prevention and treatment of reperfusion injury. Recently,a new apoptosis-related gene,poly(ADP-ribose)polymerase(PARP)was proposed.PARP is a chromatin-bound enzyme that plays a physiological role in the repair of strand breaks in DNA.PARP locates in the nuclei of cells of various tissues, including the heart and skeletal muscle.The breakage of DNA strands can activate PARP.The activation of PARP initiates an energy-consuming cycle by transferring ADP ribose units from NAD+ to nuclear proteins.As a result,the damaged DNA was repaired.However,in many pathophysiological processes such as inflammation, circulatory collapse and heart ischemia/reperfusion(I/R)injury,massive of reactive oxygen species including superoxide anions,hydrogen peroxide and hydroxyl radicals generate which cause the breakage of DNA strands and subsequently hyperactive of PARP.This process lead to the depletion of the intracellular NAD+ and ATP energetic pools which leading to cellular dysfunction and death.Extensive PARP activation has been proved to be associated with the pathogenesis of heart I/R injury.Several independent studies have confirmed that pharmacological inhibition of PARP significantly reduced the infarct size and improved the cardiac function in I/R injury, which indicated that the PARP pathway is involved in the pathogenesis of heart I/R injury.The aim of this study is to investigate the effect of PARP inhibition on heart I/R injury and try to elucidate the underlying mechanisms.We established rat heart I/R model.The activation of PARP,apoptosis and necrosis of cardiomyocytes,area of myocardial infarction and related cytokines and signaling pathway were detected. This study includes three parts:PartⅠThe effect of PARP on rat heart I/R injuryPartⅡThe effect of PARP on inflammation signaling pathway and it's related inflammatory factor in rat heart I/R injuryPartⅢThe effect of PARP on AIF and Akt signaling pathway in rat heart I/R injury PartⅠThe effect of PARP on rat heart I/R injuryObjectiveTo investigate the effect of PARP on rat heart I/R injuryMethod1.Myocardial ischemia and reperfusionThe left anterior descending branch(LAD)of the left coronary artery was occluded and loosened to establish rat heart I/R model.The following experimental groups were studied.(1)control group;(2)I/R group:LAD was occluded for 60 rain and reperfused for 180 min;(3)I/R + DPQ group:LAD was occluded for 60 min and reperfused for 180 min plus administration of 3,4-dihydro-5-[4-(1-piperidinyl)butoxy] -1(2H)-isoquinolinone[DPQ,inhibitor of PARP)[10 mg/kg i.p.2 hours before reperfusion.DPQ was dissolved in 100μL dimethylsulfoxide(DMSO)];(4)I/R + DMSO group:LAD was occluded for 60 rain and reperfused for 180 rain plus administration of DMSO(100μL DMSO were injected i.p.2 hours before reperfusion.)2.To evaluate apoptotic activity,the TUNEL technique was used.TUNEL-positive cells were determined by randomly counting 10 fields of the section and were expressed as a percentage of normal nuclei.3.Determination of myocardial infarct size:Nitro blue tetrazolium was used to evaluate the infracted and noninfarcted areas.Segments with blue staining were designated as viable,and those without staining were designated as nonviable (infarcted).Infarct size was expressed as the percentage of the area at risk.4.Electron microscope was used to detect the cell injury of heart.Results1.PARP overexpression in rat heart I/R protocol.After DPQ was used,the expression of PARP decreased.2.Inhibition of PARP activity reduced myocardial infarct size:The infarct size of the heart was 43.48±3.89%in I/R group.Administration of DPQ decreased myocardial infarction size from 43.48±3.89%to 25.47±5.88%.3.Inhibition of PARP reduced the apoptosis of cells of the heart:No TUNEL positive cells were found in the control group.The number of TUNEL positive cells were significantly increased in I/R group(35±5.3%).After DPQ was used,the TUNEL positive cells reduced to 20±4.1%(P＜0.05 vs I/R group).4.It can be found via electron microscope that inhibition of PARP reduced the injury of cardiomyocytes in rat heart I/R.Conclusion1.The overexpression of PARP in rat heart I/R protocol can aggravate the heart damage.2.Inhibition of PARP have the cardiacprotective effect via reduced the apoptosis of cardiomyocytes and infarct size.PartⅡThe regulation of PARP on NF-κB and inflammatory factor in rat heart I/R injuryObjectiveTo investigate the effect of PARP on NF-κB and inflammatory factor in rat heart I/R injury.Method1.Myocardial ischemia and reperfusionThe left anterior descending branch(LAD)of the left coronary artery was occluded and loosened to establish rat heart I/R model.The following experimental groups were studied.(1)control group;(2)I/R group;(3)I/R + DPQ group and(4)I/R + DMSO group2.Electrophoretic mobility shift assay(EMSA)was used to detect the activation of nuclear factor-kappaB(NF-κB).The NFκB oligonucleotides(5'-AGTTGAGGGGACTTTCCCAGGC-3') were end-labeled with[γ-32P]ATP.After binding reactions,polyacrylamide gel electrophoresis was performed.The bands were quantified by image analyzer.3.Reverse Transcriptase-Polymerase Chain Reaction and immunohistochemistry were used to detect the expression of intercellular adhesion molecule-1(ICAM-1), cyclooxygenase-2(COX-2)and matrix metalloproteinase-9(MMP-9).Results1.Inhibition of PARP activity reduced the activation of NF-κB:Little activation of NF-κB in control group.The activation of NF-rd3 increased after reperfusion in I/R group.DPQ significantly attenuated the activation of NF-κB in I/R+DPQ group.2.Inhibition of PARP activity reduced the expression of ICAM-1,COX-2 and MMP-9:Very weak expression of ICAM-1,COX-2 and MMP-9 mRNA was apparent in the ventricles of control group.These mRNA levels increased significantly in I/R group.After DPQ was used,these mRNA levels reduced.We further used immunohistochemistriey to evaluate the expression of ICAM-1,COX-2 and MMP-9. There is little immunoreactivity in the control group.However,the weighted score were significantly increased in I/R group.The weighted score were significantly reduced in the I/R+DPQ group.ConclusionInhibition of PARP activity can reduce the inflammation signaling pathway mediated by NF-κB and it's related inflammatory factor in rat heart I/R injuryPartⅢThe regulation of PARP on AIF and Akt signaling pathway in rat heart I/R injuryObjectiveTo investigate the effect of PARP on AIF and Akt signaling pathway in rat heart I/R injuryMethod1.Myocardial ischemia and reperfusionThe left anterior descending branch(LAD)of the left coronary artery was occluded and loosened to establish rat heart I/R model.The following experimental groups were studied.(1)control group;(2)I/R group;(3)I/R + DPQ group;(4)I/R + DPQ +LY294002 group(LY294002,inhibitor of Akt,10 mg/kg i.p.2 hours before reperfusion.LY294002 was dissolved in 100μL DMSO);(5)I/R +SP600125 group (SP600125,inhibitor of JNK,6 mg/kg i.p.2 hours before reperfusion.SP600125 was dissolved in 100M1 DMSO)and(6)I/R + DMSO group2.Western Blot analysis was used to detect the expression of PARP,JNK,AIF,Akt, GSK-3βand FOXO3a in rat heart.Results1.Inhibition of PARP activity reduced the activation of JNK:phospho-JNK increased after reperfusion in I/R group rat.The activation of phospho-JNK was significantly attenuated in I/R + DPQ group.2.Inhibition of PARP or JNK activity reduces the AIF translocation:AIF increased after reperfusion in nuclear fractions.The activation of AIF was signifiCarLtly attenuated by inhibition of PARP or JNK activity.On the contrary,AIF decreased,in mitochondria fractions after reperfusion.Inhibition of PARP or JNK activity could increase the activation of AIF in mitochondria fractions.These data suggest that inhibition of PARP or JNK activity was able to reduce the mitochondrial-nuclear translocation of AIF.3.DPQ facilitated Akt activation and decreased the activity of GSK-3βand FOXO3a: In I/R group,the expression of phosphor-Akt,phosphor-GSK-3βand phosphorFOXO3a increase.DPQ further induced the increase of them.Then we administrated DPQ in combination with LY294002 before heart I/R.We found that LY294002 prominently reversed DPQ mediated Akt,GSK-3βand FOXO3a phosphorylation.Conclusion1.JNK may be downstream of PARP activation and JNK may be required for PARP mediates AIF translocation in a rat model of myocardial ischemia and reperfusion in vivo.PARP/JNK/AIF pathway may be a new pathway of cardiac myocyte injury in vivo.2.PARP can regulate the survival signaling pathway mediated by Akt in rat heart I/R injury.