| 【Background】Ischemic heart disease is a serious threat to human life which causes onethird of mortality in humans. Myocardial ischemia-reperfusion injury willappear inevitably which is characteristic of oxidative stress and calciumoverloading with the development and the improvement of thrombolytic therapy,spasmolysis treatment, coronary artery bypass grafting, percutaneous coronaryintervention and some other methods treating ischemic heart disease. Themechanism of myocardial ischemia-reperfusion injury increasing myocardialcell apoptosis is in close relations with calcium overloading, oxygen radicals,microvessel damage and leukocytes. However, the exact mechanisms have notbeen fully clarified. Mitochondria are closely related to myocardial cell apoptosis induced by ischemia-reperfusion. Myocardial ischemia-reperfusioninjury can activate mitochondrial permeability transition pore in mitochondriainner membrane and then release mitochondrial pro-apoptotic proteins followedby myocardial damage with respect to ultrastructure, function, metabolism andelectrophysiology. Cell apoptosis aggravates myocardial damage with clinicalmanifestation of a series of adverse cardiovascular events including malignantreperfusion arrhythmia, cardiac dysfunction, coronary microcirculation dysfunctionand expanding myocardial infarction size and so forth. Therefore, the mainpoints of our study focus on how to prevent or ease Myocardial ischemia-reperfusion injury and protect cardiomyocytes.Adiponectin is a kind of specific protein without obvious circadian rhythmschange which is secreted by adipose tissue and can’t be affected by dining. Ithas many functions such as antioxidant stress, resistance to atherosclerosis,antiinflammatory, improving the resistance to insulin and participating inglucolipid metabolism. The globular domain of adiponectin is closely related tothe functions of adiponectin with full length. Adiponectin protect myocardialtissues and reduces the cardiomyocytes apoptosis during ischemia-reperfusioninjury, which thereby decreases the death rate. The mechanisms of Adiponectinin protecting myocardial cells during Myocardial ischemia-reperfusion injuryare not fully understood.Orphan nuclear receptor Nur77(also known as NR4A1, TR3and NGFI-B),the immediate early gene, mostly locates in myocardial nuclear under normalcircumstances. However, the nuclear receptor Nur77will directedly translocatefrom the nucleus to the mitochondria, which will lead to the release ofpro-apoptotic proteins from mitochondrial and contribute to cardiomyocyteapoptosis. NuBCP-9is a kind of small peptide derived from Nur77and can mimic the behaviors and functions of it. Mitochondrial pro-apoptotic proteinsencoded by nuclear genes, such as Omi/HtrA2protein and Smac/DIABLOprotein, are normally stored in the intermembrane space of mitochondria. Whileduring ischemia/reperfusion, Omi/HtrA2protein and Smac/DIABLO proteincan be released into the cytoplasm from the intermembrane space ofmitochondria, which in turn contributes to myocardial cell apoptosis.Currently, it has not been reported that whether adiponectin alleviates thecardiomyocyte apoptosis induced by hypoxia/reoxygenation through theinhibition of Nur77translocation to mitochondrial. This study was designed toinvestigate the expression and subcellular localization of nuclear receptor Nur77,Omi/HtrA2protein, Smac/DIABLO protein in myocardial cells and therelationship among them through exogenous adiponectin intervening thehypoxia/reoxygenation model of cardiomyocytes in vitro.【Objectives】1. To study the effect of nuclear receptor Nur77on subcellular localizationof mitochondrial proapoptotic protein Omi/HtrA2and Smac/DIABLO protein inH/R.2. In the H/R cardiomyocytes from neonatal rats, to investigate the effect ofexogenous adiponectin on subcellular localization of nuclear receptor Nur77.【Methods】Experiment1Primary culture of neonatal rat myocardial cells and theestablishment of hypoxia/reoxygenation (H/R) model:Neonatal rats were killed by cervical dislocation to take the hearts withaseptic manipulation, and0.1%collagenase II was used to digest myocardialtissue for several times, and myocardial cells cultured for72h were randomly divided into four groups:1) control groups: normal cultured cells, with nointervention factors;2) hypoxia/reoxygenation groups (H/R groups): withhypoxia for3h and reoxygenation for4h;3): Nur77groups: normal culturedcells were incubated for36hours with15μmol/l NuBCP-9diluted by low sugarmedium DMEM containing10%fetal bovine serum.4) adiponectin+hypoxia/reoxygenation groups (gAd+H/R groups): neonatal rat myocardial cells wereincubated for24hours with6ug/ml gAd, followed with hypoxia for3h andreoxygenation for4h.The survival rates of myocardial cells were detected by trypan blue stainingcell viability assay kit; neonatal rat myocardial cells were identified byimmunoperoxidase staining technique with rat striated muscle actin antibody;the activities of Caspase-3in myocardial cells were detected using Caspase-3assay kit; myocardial cell apoptosis was detected with TUNEL staining methodand apoptosis index was calculated.Experiment2Targeting translocation of nuclear receptor Nur77tomitochondria could lead to that Omi/HtrA2protein and Smac/DIABLO proteinreleased from mitochondria into the cytoplasm.1. The separation of subcellular organellesThe separation of subcellular organelles in each group was performed instrict accordance with the instruction of KeyGEN mitochondrial/nucleipreparation kit (Nanjing KeyGEN), to extract nuclear proteins, cytoplasmicproteins and mitochondrial proteins from the cells in experimental groups;wherein, nuclear proteins and mitochondrial proteins required ultrasound withcell ultrasonic disrupter on ice.2. Of control group and Nur77group, mitochondrial proteins andcytoplasmic proteins were extracted in accordance with the subcellular organelle separation method and with ultrasonic disrupter on ice, and the expression levelsof Omi/HtrA2and Smac/DIABLO proteins in mitochondria and cytoplasm ofthe two groups were determined by Western-blot.Experiment3Adiponectin reduced myocardial apoptosis induced byhypoxia/reoxygenation by inhibiting mitochondrial translocation of Nur77.1. Of control group and H/R group, total proteins of myocardial cells wereextracted, and the expression levels of Nur77protein in cells of the two groupswere detected by Western-blot;2. Extract nuclear proteins, mitochondrial proteins and cytoplasmicproteins of control group, H/R group and gAd+H/R group in accordance withthe method for separating subcellular organelle mentioned above. After thetreatment of cell ultrasonic grinder on ice, the expression levels of Nur77proteinin nucleus and mitochondria of each group were detected by Western-blot.【Results】1. The cardiomyocytes of SD suckling mouse were successfully isolatedand primarily cultured in vitro and the model of cardiomyocyte apoptosisinduced by hypoxia/reoxygenation was established successfully as well.Compared with the control group, the caspase-3activity of H/R groupsignificantly increased (p<0.05), myocardial apoptosis index obviouslyincreased (p<0.05). While after offering the intervention by exogenousadiponectin globular domain gAd, the caspase-3activity significantly decreased(p<0.05) and apoptotic index decreased to a large extent (p<0.05).2. The nuclear proteins, cytoplasmic proteins and mitochondrial proteins ofthe cells in each group were extracted successfully. Compared with the controlgroup, the content of Omi/HtrA2protein and Smac/DIABLO protein in mitochondria of Nur77group significantly reduced (p<0.05) while those in thecytoplasm obviously increased (p<0.05).3. The expression level of whole-cellular Nur77protein did not changedistinctly in the control group and H/R group (p>0.05); compared with thecontrol group, the expression level of nuclear Nur77protein in H/R groupsignificantly reduced (p<0.05); compared with H/R group, the expression levelof nuclear Nur77protein in gAd+H/R group was higher than H/R group (p<0.05);compared with the control group, the expression level of mitochondrial Nur77protein in H/R group significantly increased (p<0.05), and compared with H/Rgroup, the expression level of mitochondrial Nur77protein in gAd+H/R groupwas lower than that in H/R group (p<0.05).【Conclusions】1. The H/R insult increases cardiomyocytes apoptosis index and caspase-3activity. gAd intervention for24hours before hypoxia reduces the caspase-3activity and cardiomyocytes apoptosis index.2. The translocation of Nur77causes the release of mitochondrialpro-apoptotic protein, Omi/HtrA2protein and Smac/DIABLO protein, intocytoplasm from mitochondria.3. By offering exogenetic gAd intervention in cardiocytes for24hoursbefore hypoxia, we demonstrate that the translocation of Nur77to mitochondriasignificantly decreased. These data suggest that adiponectin inhibits translocationof Nur77nucleus to mitochondria and alleviates cardiomyocytes apoptosisinduced by hypoxia/reoxygenation. |
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