The Study Of The Protective Effect Of PJ-34 On Myocardial Ischemia Reperfusion Injury And Its Mechanisms Of Anti-oxidative Stress Induced Cell Injury

Part?:The protective effect of PJ-34 on rat isolated heart involving in ischemia reperfusion injuryBackground:Ischemic heart disease is a common disease which threatens human health severely. Reperfusion therapy (including drug thrombolysis, percutaneous transluminal coronary angioplasty and stenting, coronary artery bypass grafting,et al.), which can efficiently resume the blood supplement of ischemic myocardial tissues, is a main therapy strategy of myocardial ischemia and infarction nowadays in the world. However, accompanied with the blood re-supplement, there comes a new potential threat, which is so-called ischemic-reperfusion injury. Ischemic-reperfusion injury is a special pathological process that a successful reperfusion of ischemic myocardial tissues may cause an even more severe injury than before, and the common clinical manifestations of reperfusion are reperfusion induced arrhythmia and the heart function disorder caused by myocardial stunning. As the spread of the reperfusion therapy all of the world, the influence of reperfusion injury becomes much more prominent. How to alleviate the injury of reperfusion has become the focus point of the clinical investigation.The pathogenesis of ischemic reperfusion injury is due to several factors, but the most important factors are ROS induced oxidative stress injury and the direct cell injury caused by Ca+ overloaded. Researchers have confirmed that several treatment including ischemic preconditioning, drug preconditioning, ischemic postconditioning, drug postconditioniing and remote ischemic conditioning are all beneficial to protecting cells form reperfusion injury. However, drug postconditioning is much more valuable in clinical practice on account of its simple operation process, exact interference time and specific target.Poly(ADP-ribose) Polymerase-1 (PARP-1) is a protein kinase which exists in the nucleus and is highly expressed in myocardial cells. Its function is to monitor intracellular DNA injury and to repair the injured DNA subsequently. Experiments have demonstrated that PARP-1 also play a important role in cellular energy metabolism and cell death process, and its inhibitors may have potential cytoprotective actions.Aims:The aims of our experiment is to find out whether PARP-1 inhibitor PJ-34 has the protection effect on isolated rat heart suffered from ischemic reperfusion injury and to investigate the potential mechanism. Methods:We used the Langendorff perfusion apparatus to build the experimental model of ischemic reperfusion injury of isolated rat heart. All SD rat were randomly divided into 3 groups (5 rats per group):Control group, I/R group and I/R+PJ-34 group. The isolated hearts of each group on Langendorff perfusion apparatus were given K-H buffer perfused for 30min as a stabilization period. After that period, the hearts of control group were continually perfused with K-H buffer for 150min until the end. The hearts of I/R group were broken off for K-H buffer perfusion as a ischemic period for 30min, and then continually perfused with K-H buffer again for 120min as a reperfused period. The hearts of I/R+PJ-34 group were broken off for K-H buffer perfusion as a ischemic period for 30min, and then continually perfused with K-H buffer containing with 3mg/kg/h PJ-34 for 120min to the end as a reperfused period.There are 6 endpoints for measurement.(1) LDH content of cellular leakage.(5min, 10min,20min,40min at reperfusion period)(2) Cell viability was measured by MTT assay.(3) Myocardial infarct size was measured by TTC staining.(4) Pathomorphism of injured myocardial tissues was measured by H&E staining.(5) The indexes (LVDP, LVEDP,+dp/dtmax,-dp/dtmax) of hemodynamics are measured by Medlab system.(5min, 10min,20min,40min,60min at reperfusion period)(6) The extent of oxidative stress injury was measured by MDA assay indirectly.Results:(1) PJ-34 treatment reduced LDH content of cellular leakage remarkably at each measure point during reperfusion period.(5min, 10min,20min,40min)(2) PJ-34 treatment improved cell viability dramatically compared with I/R group(3) PJ-34 treatment efficiently decreased the myocardial infarct size compared with I/R group.(4) PJ-34 treatment alleviated the reperfusion-induced change of pathomorphism of myocardial tissues, as to reduce the swelling of myocardial cells and infiltration of inflammatory cells.(5) PJ-34 treatment dramatically improved the indexes (LVDP, LVEDP,+dp/dtmax,-dp/dtmax)of hemodynamics compared with I/R group at each measure point during reperfusion period.(5min, 10min,20min,40min,60min)-.(6) PJ-34 treatment dramatically reduced the intracellular MDA content compared with I/R group and indicated to attenuate the oxidative stress injury caused by reperfusion.Conclusions:PJ-34 treatment can attenuate myocardial ischemic reperfusion injury, and its cardioprotective action may involve a reduction of oxidative stress in reperfused myocardial cells. Part?: PJ-34 Attenuates Oxidative Stress induced Cell Injury through Depressing AMPK Activity:Crosstalk between PARP-1 and AMPKBackground:Oxidative stress is known to be the chief criminal during cardiac ischemia-reperfusion injury and exerts a deleterious effect on mitochondria function and cell survival. Two crucial proteins in cellular energy metabolism, poly(ADP-ribose)polymerase-1 (PARP-1) and AMP activated protein kinase (AMPK), play important roles in this pathologic process.The protein kinase poly(ADP-ribose)polymerase-1 (PARP-1) mainly presents in the nucleus of eukaryotic cells and acts as a DNA damage sensor to participate in DNA repair and transcription. The activation of PARP-1 result in poly(ADP-ribosyl)action. By conferring negative charges to acceptor proteins, the covalent protein modification alters the physic-chemical properties of the modified target proteins and thus affects their function, subsequently may influence a diverse array of important cellular processes. AMP-activated protein kinase (AMPK) is a key regulator of energy metabolism in mammalian cells, and is highly expressed in heart. Upon activation, AMPK signaling regulates energy balance at the cellular, organ and whole-body level. Researchers have already confirmed that AMPK also plays an important role as a cellular activity regulator, which is able to coordinate not only the metabolism, but also intracellular signaling and gene expression in cells.However, as two major proteins involved in cellular activities, the connections between PARP-1 and AMPK activity as well as its relevance to reperfusion injury have not been explored indeed.Aims:In this study, we used exogenous H2O2 stimuli to mimic acute oxidative stress injury in H9c2 cells for further determination of the cytoprotective effect of PJ-34 on oxidative stress injured cells. We also aimed to investigate the relationship among PARP-1, AMPK and cell death in H9c2 cell lines in response to acute oxidative stress.Methods:Exogenous H2O2 at a certain concentration (1000?mol/L) was treated shortly (1h) to mimic acute oxidative stress injury in cultured rat H9c2 cells. In the subsequent studies, PARP-1 inhibitor PJ-34, AMPK inhibitor Compound C and AMPK activator AICAR were added into the culture media 30min prior to H2O2 stimuli as intervention conditions.The whole research includes three main parts:(1) To investigate the cytoprotective effect of PJ-34 on H2O2 induced injured cells.(2) To investigate the role of PARP-1/AMPK pathway in H2O2 induced oxidative stress injury in H9c2 cells.(3) To investigate the relationship between PARP-1 and AMPK.In the first part, we used LDH assay, MTT assay and flow cytometry analysis to evaluate the cell viability and death; NAD+/NADH assay, ATP assay and mitochondrial membrane potentials assay to estimate intracellular energy status, Western-Blot experiment to detect the expression of related proteins. In the second part, we intervened at different levels of proteins in PARP-1/AMPK pathway to investigate the influence of signal pathway activation on cell survival and death. In the third part, we used protein transfection and subsequent co-immunoprecipitation experiments to investigate the direct interaction between PARP-1 and AMPK. Results:(1) PJ-34 pretreatment exerted effective protective effect on H2O2 induced H9c2 cell injury and death, which including enhanced cell viability, reduced cell death rate, improved cellular energy metabolism and mitochondrial function, inhibited apoptosis-related protein activation, et al.(2) PARP-1/AMPK pathway activation plays an important role in H2O2 induced oxidative stress injury in H9c2 cells. Inhibition of PARP-1 and AMPK activity could attenuate H2O2 induced injury in H9c2 cells, otherwise, AMPK activator AICAR could partially reverse the beneficial effect of PJ-34.(3) Protein transfection and co-immunoprecipitation experiments demonstrated that after exogenous active PARP-1 protein was successfully transfected into target cells, the expression and activity of intracellular PARP-1 and AMPK both enhanced, and the activation of AMPK is directly coupled with the expression and activity of PARP-1.Conclusions:PJ-34 pretreatment can attenuate H2O2 induced H9c2 cell injury and death, its cytoprotective effect under oxidative stress may take place through inhibiting the activity of PARP-1 and subsequent inhibit AMPK activation. Furthermore, there is a positive feedback between PARP-1 and AMPK.