| Myocardial infarction is the major cause of death in the world. Myocardialischemia reperfusion injury （MIRI） is a clinical relevant problem occurring withdamage to the myocardium after a critical period of coronary occlusion andreperfusion, and associated with a host of pathologic derangements, includingarrhythmias, transient mechanical dysfunction, myocardial stunning, and cell death.Reperfusion of ischemic tissue is necessary to terminate the processes of ischemicinjury, and is the major treatment for impending acute myocardial infarction （AMI） bythrombolysis or primary percutaneous coronary angioplasty. However, abruptreperfusion may results in the so-called myocardial ischemia/reperfusion injury （MIRI）that can provoke further tissue injury and myocardial cell death.There is great interestin the development of adjunct therapies, which might attenuate reperfusion injury andthereby maximize the benefits of reperfusion. Reactive oxygen species （ROS） andcardiomyocyte apoptosis play an important role in the etiology of myocardialischemia-reperfusion injury. Therefore, studies on the safe and effective newcardioprotective agents used to prevent MI/RI are still a pivotal issue.DhHP-6（Deuterohemin-AlaHisThrValGluLys） is a novel microperoxidasemimetic, designed and synthesized in our laboratory, containing deuterohemintogether with six amino acid residues （AlaHisThrValGluLys）. The in vitro peroxidaseactivity of DhHP-6is very similar to that of microperoxidase-11(MP-11), butDhHP-6has very good water solubility, stability and its chemical synthesis is muchsimpler. We have previously reported that DhHP-6exhibit high peroxidase activity,delaye and alleviate cataract induced by galactose in lens of cultured rat in vitro andgalactose induced cataract in rat in vivo. DhHP-6has been demonstrated to improvethe survival rate of Caenorhabditis elegans in the acute heat stress and rescued the C.elegans’ sensitivity to paraquat in acute oxidative stress.However, there is relatively little information about the protection potential ofDhHP-6against oxidative injury in MI/IR model of rats. Therefore, in this study, we investigate the protective effects and potential molecular mechanism of DhHP-6onoxidative injury and apoptosis in model rat of MI/RI and in hypoxia/reoxygenationinduced injury model of H9c2cardiomyocyte.1. Protective effects of DhHP-6on myocardial ischemic reperfusion injury in ratsTo investigate the protective effects of DhHP-6on myocardial ischemicreperfusion injury in rats and its underlying mechanisms, the model rats of myocardialischemia reperfusion injury （MI/RI） was produced by a40min occlusion of the leftanterior descending （LAD） branch of the coronary artery, followed by a2hreperfusion. The rats were divided randomly into six groups: sham-operated group;MI/RI model group; diltiazem hydrochloride1.0mg·kg-1group; DhHP-62.0mg·kg-1group; DhHP-66.0mg·kg-1group; DhHP-620mg·kg-1group.DhHP-6was injected intraperitoneally each of the experimental rats30minrespectively before LAD occlusion. The same volume of saline was administered intothe sham-operated group and MI/RI model group. The indicators of myocardial injurysuch as infarct size, ST elevation, serum levels of creatine kinase （CK）, lactatedehydrogenase （LDH）, aspartate transaminase （AST）, the antioxidant indicatorssuch as superoxide dismutase （SOD），CAT, GSH-Px, malondialdehyde （MDA），GSH,ROS, the apoptosis indicators such as rate of apoptosis, protein expression level ofBcl-2,Bax,Caspase-3,SIRT1、P53in rat myocardial tissue were measured at2h afteradministration.Treatment of DhHP-62.0-20mg·kg-1could significantly result in reductionsin the infarct size and decreased ST elevation in the model rats of myocardialischemia reperfusion injury （MI/RI）, compared with the MI/RI control rats （P <0.01）.the serum levels of creatine phosphokinase （CK）, lactate dehydrogenase （LDH）,aspartate transaminase（AST） and malondialdehyde （MDA） were significantlyreduced, and myocardium Na+-K+ATPase and Ca2+-Mg2+ATPase activities and theantioxidant enzyme activities （SOD, CAT, GSH-Px） in the serum and myocardiumwere markedly increased in DhHP-6-treated groups compared to the MI/RI modelgroup. Pretreatment of DhHP-6reduced significantly myocardium MDA and ROSlevel, and enhanced myocardium GSH level in MI/RI rat. DhHP-6treatment （6mg/kg）significantly increased the myocardium protein expression level of Bcl-2, Bcl-2/Bax,Caspase-3, inhibit the rate of apoptosis, and activated SIRT1expression, decreasedacetylated-P53（p-P53）expression, inhibited P53, in MI/RI rats compared to MI/RI rat control model. In conclusion, we clearly demonstrated that DhHP-6pretreatmentcan decrease oxidative injury in rats subjected to MI/RI. DhHP-6exerts significantcardioprotective effects against MI/RI in rats, likely through its antioxidant andantiapoptotic properties, related to activation of SIRT1and inhibition of P53.2. Protective effects of DhHP-6on hypoxia/reoxygenation（H/R） induced H9c2cardiomyocytes injuryIn this study, to examine the cardioprotective effects of DhHP-6on injury ofH9c2cardiomyocyte induced by hypoxia-reoxygenation（H/R）, the model of H/Rinjury（a simulated ischemia/reperfusion injury condition） was established byinducing hypoxia for24hours and reoxygenation for1hours in cultured H9c2cardiomyocyte, then the cell viability, cell death and apoptosis, lactate dehydrogenase（LDH） activity, malondialdehyde （MDA） level, H2O2production, Ca2+-ATPaseactivity, superoxide dismutase （SOD） activity were detected. The apoptosis wasobserved by staining of Hoechst DPI and AO/EB.Cell viability was assessed by the Trypan blue exclusion and3-（4,5-dimethyl-thiazol-2yl）-2,5-diphenyl tetrazolium bromide（MTT） uptake assay，Lactate dehydrogenase （LDH） malondialdehyde （MDA）, superoxide dismutase （SOD）,and Ca2+-ATPase activity were measured using commercial kits. cardiomyocyteapoptosis features were examined by chromatin morphology under fluorescentmicroscope. The increase in cell death was observed when the cells were exposedto hypoxia/reoxygenation. Pretreatment with DhHP-6（0.1-8.0μM） significantlyincreased the cell viability of hypoxia/reoxygenation-induced H9c2cardiomyocytesby70%in the injury group compared with the control injury group （P<0.01）, and in adose-dependent manner. DhHP-6（4μM） increased the viability of cells by20%innormal cardiomyocyte. Hypoxia followed by reoxygenation caused a significantincrease in LDH release, DhHP-6significantly lowered LDH release in adose-dependent manner, with4.0μmol·L-1DhHP-6reducing LDH release to50%ofcontrol. pretreatment with DhHP-6significantly,decreased the MDA level,increasedSOD activity and improved Ca2+-ATPase activity. Besides, the alleviation ofDhHP-6on the morphologic changes of myocardial cells, swelled, shrank and fell offin model injury group, was observed. The flurorescence microscope observation byAO/EB staining showed that the rate of apoptosis and necrosis cells in H/R modelinjury group， DhHP-6obviously reduced apoptotic body formation. In the present study, the protective effects of DhHP-6on H/R induced H9c2cardiomyocytes injury were evaluated. The results showed that DhHP-6（0.1-4.0μM）pretreatment significantly decreased H/R-induced cardiomyocyte death, lactatedehydrogenase （LDH） release, malondialdehyde （MDA） level, ROS（H2O2）production, and Ca2+-ATPase activity, increased the cell viability, superoxidedismutase （SOD） activity in H/R treated H9c2cardiomyocytes. Morphologicalobservation showed that DhHP-6（4.0μM） treatment reduced the injuriedcardiomyocytes, cell death and apoptosis. Therefore, the present results suggested thatDhHP-6exhibited significant protective effects on H/R induced H9c2cardiomyocytesinjury.3. mechanism of effects of DhHP-6on apoptosis and injury of H9c2cardiomyocyte induced by H/RIn this study, to examine the possible mechanism of the cardioprotective effectsof DhHP-6on injury of H9c2cardiomyocyte induced by H/R, the model of H/Rinjury（a simulated ischemia/reperfusion injury condition） was established byinducing hypoxia for24hours and reoxygenation for1hours in cultured H9c2cardiomyocyte. DhHP-6, Nicotinamide （Nic, inhibitor for SIRT1）, Resveratrol（Rev,activator for SIRT1）, JNK inhibitor （SP600125）, ERK inhibitor PD98059, P38inhibitor SB203580, PI3K/AKT inhibitor LY294002,were used in the study, then theexpression level of SIRT1, p-p53, Bcl-2, Bax，P-JNK, P-ERK, P-P38, P-AKT inH9c2cardiomyocyte was investigated by western blotting method. the cell viability,cell death and apoptosis, lactate dehydrogenase （LDH） activity, malondialdehyde（MDA） level, ROS（H2O2）production,were detected, The apoptosis was observed bystaining of Hoechst33342.1. Protective effect and antiapoptotic effect of DhHP-6againsthypoxia/reoxygenation-induced H9c2cardiomyocyte injury by up-regulation ofSIRT1/p53pathwayWestern blot analysis showed that SIRT1protein expression was significantlydecreased in the H/R treated cells compared with that in the control group cell （P <0.001）; DhHP-6（0.4,1.0,4.0μM） treatment significantly increased SIRT1proteinexpression in H/R injuried H9c2cardiomyocytes in concentration-dependent manner.However, the effect that nicotinamide（Nic, inhibitor of SIRT1）10mM significantlydecreased SIRT1protein expression, nicotinamide was reversed by DhHP-6, the effect that Resveratrol（Rev, activator for SIRT1）10μM significantly increased SIRT1protein expression was enhanced by DhHP-6. It was indicated that DhHP-6activating SIRT1involved in DhHP-6protecting against H/R induced H9c2cardiomyocytes injury.To further verify that DhHP-6activating SIRT1in H/R injuried H9c2cardiomyocytes, we analyzed deacetylation of p53, a downstream target of SIRT1.SIRT1activation was measured by a decrease in acetylated p53protein expression.The results showed that H/R induced a significant increase in the protein expressionratio of acetylated-p53（A-p53）/total p53（T-p53），enhanced P53acetylation, in H9c2cardiomyocytes compared to control cells. DhHP-6（0.4-4.0μM） treatmentsignificantly decreased the ratio of A-p53/T-p53in concentration-dependent mannerin H/R injuried H9c2cardiomyocytes. However, The effect that nicotinamideincreased p53acetylation was prevented by DhHP-6-dependent SIRT1activation, theeffect that Resveratrol decreased p53acetylation was enhanced by DhHP-6-dependentSIRT1activation, in H/R injuried cardiomyocytes. It was indicated that DhHP-6directly activate SIRT1, decrease A-p53protein expression, decrease p53acetylation,increase deacetylation of p53in H/R injuried cardiomyocytes..To further verify that DhHP-6activating SIRT1negatively regulated p53in H/Rinjuried H9c2cardiomyocytes, we analyzed p21, a downstream target of p53. Theresults showed that H/R induced a significant increase in P21protein expression inH9c2cardiomyocytes compared to control cells. DhHP-6（0.4-4.0μM） treatmentsignificantly decreased the P21protein expression in concentration-dependent mannerin H/R injuried H9c2cardiomyocytes. However, The effect that nicotinamideincreased P21protein expression was prevented by DhHP-6-dependent SIRT1activation, the effect that Resveratrol decreased P21protein expression was enhancedby DhHP-6-dependent SIRT1activation, in H/R injuried cardiomyocytes. It wasindicated that DhHP-6activate SIRT1, decrease A-p53protein expression, decreasep53acetylation, decrease P21protein expression in H/R injuried cardiomyocytes..The effects of DhHP-6antagonizing nicotinamide on apoptosis and injury inhypoxia/reoxygenation（H/R） injured H9c2cardiomyocytes were evaluated. Theresults showed that pretreatment of DhHP-6（0.4μM） with nicotinamide significantlydecreased the rate of cardiomyocyte apoptosis, significantly increased Bcl-2proteinexpression, and Bcl-2/Bax expression, reduced Caspase-3expression in H/R injuried H9c2cardiomyocytes. Furthermore, increased the cell viability, decreased lactatedehydrogenase （LDH） release, reduced ROS（H2O2）production level. It was indicatedthat DhHP-6exhibited significant protective effects and antiapoptotic on H/R inducedH9c2cardiomyocyte injury by directly activating SIRT1.From the above results, we demonstrated that DhHP-6exhibited significantprotective effects and antiapoptotic on H/R induced H9c2cardiomyocyte injurythrough SIRT1/p53signal pathway: DhHP-6directly activate SIRT1, increasedeacetylation of p53, decrease A-p53protein expression, decrease P21proteinexpression, then positively regulate Bcl-2/Bax expression, and negatively regulateCaspase-3expression, therefore DhHP-6reduce apoptosis and injury in H/Rinduced H9c2cardiomyocyte.2. Protective effect and Antiapoptotic effect of DhHP-6againsthypoxia/reoxygenation-induced H9c2cardiomyocyte injury by up-regulation of SIRT1/AKTpathwayThe effects of DhHP-6antagonizing nicotinamide（Nic, inhibitor of SIRT1）10mM and LY294002（AKT inhibitor）on apoptosis and injury in hypoxia/reoxygenation（H/R） injured H9c2cardiomyocytes were evaluated. The results showed thatpretreatment of DhHP-6（0.4μM） with nicotinamide10mM and LY294002（AKTinhibitor） directly activated SIRT1, furthermore activated AKT signal pathway,significantly decreased the rate of cardiomyocyte apoptosis, increased Bcl-2proteinexpression, and Bcl-2/Bax expression, reduced Caspase-3expression in H/R injuriedH9c2cardiomyocytes. Furthermore, increased the cell viability, decreased lactatedehydrogenase （LDH） release, reduced ROS（H2O2）production level. From the aboveresults, we demonstrated that DhHP-6exhibited significant protective effects andantiapoptotic on H/R induced H9c2cardiomyocyte injury through SIRT1/AKT signalpathway: DhHP-6directly activate SIRT1, furthermore activate AKT signal, thenpositively regulate Bcl-2/Bax expression, and negatively regulate Caspase-3expression, therefore DhHP-6reduce apoptosis and injury in H/R induced H9c2cardiomyocyte.3. DhHP-6inhibit SIRT1/JNK signal pathway: DhHP-6directly activate SIRT1,furthermore inhibit JNK signal, by antagonizing nicotinamide（Nic, inhibitor of SIRT1）and SP600125（JNK inhibitor） in H/R injuried H9c2cardiomyocyte.4. DhHP-6activated ERK signal, inhibit P38signal, not through SIRT1, in H/R injuried H9c2cardiomyocyte.In summary, DhHP-6exhibited significant protective effects on H/R inducedH9c2cardiomyocytes injury and myocardial ischemia reperfusion injury（MI/IR） inrats through activating SIRT1/p53signal pathway and SIRT1/AKT signal pathway.DhHP-6may have potential in developing a novel cadioprotective agent. |
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