| BackgroundOxidative stress is a significant component of myocardial ischemia/reperfusion (MI/R)injury. Toxic reactive oxygen species (ROS) generated during MI/R both directly andindirectly affect cardiomyocyte function, promoting apoptosis and necrosis. Mitochondriaare both a major endogenous source and target of ROS, including superoxide anions, hydrogen peroxide, peroxynitrite, and hydroxyl radicals. Mitochondrial dysfunctionincreases ROS production, exacerbating oxidant-induced apoptosis. During earlyreperfusion, ROS burst alters intracellular redox state, modifies the inner mitochondrialmembrane potential (MMP), and releases mitochondrial cytochrome c into the cytosol,ultimately activating caspase-3in the final apoptotic pathway. Preventing ROS productionand preserving mitochondrial integrity are therefore protective against MI/R injury.Ginsenoside Rd (GSRd) is a biologically active agent extracted from the Chinesemedicinal plant Panax Ginseng. Evidence suggests that GSRd is highly lipophilic withunique pharmacological effects. It is able to easily pass through bio-membranes anddecrease reactive oxygen species (ROS) formation to exert antioxidant effect. It has beendemonstrated that GSRd has neuroprotective effect against transient focal cerebralischemia via anti-oxidative stress, anti-inflammation and anti-apoptosis. We hypothesizethat GSRd may exert cardioprotective effect against MI/R injury. The present study aimsto investigate the role of GSRd in cardioprotection and the underlying mechanisms.Aims1. To determine whether GSRd exerts cardioprotective effect against MI/R injury2. To determine whether GSRd may decrease oxidative stress following MI/R3. To investigate the responsible underlying mechanismsMethods1. Adult male Sprague-Dawley rats were subjected to myocardial ischemia for30minand reperfusion for3h. Rats were randomly assigned to one of the following groups(n=8/group):1) Sham group, receiving vehicle IP injection (10ml/kg saline) andoperative procedures without coronary slipknot;2) MI/R group, receiving vehicle IPinjection (10ml/kg saline)30minutes prior to coronary I/R; and3) MI/R+GSRd group, receiving IP injection of GSRd (50mg/kg)30minutes prior to coronary I/R, adose established from prior investigations. MI/R-induced cardiac dysfunction wasdetermined by invasive hemodynamic evaluation methods. Infarct size was measuredby Evans blue/TTC double staining. Myocardial apoptosis was determined by acommercially available terminal deoxynucleotidyl nick-end labeling (TUNEL).Myocardial cellular damage and necrosis were evaluated by measuring plasma levelslactate dehydrogenase (LDH) and creatine kinase (CK).2. In vitro study was performed on cultured neonatal rat cardiomyocytes (NRCs)subjected to simulated ischemia/reperfusion (SI/R,3h/2h). Four separate NRCgroups were tested:1）Control group, incubated with Tyrode solution for the entireexperimental period;2）SI/R group, incubated with simulated ischemia buffer for3hours hypoxia, followed by2hours re-oxygenation;3）Vehicle group, subjected to0.2%(v/v) DMSO administration30minutes prior to SI/R;4）SI/R+GSRd group,subjected to GSRd (10μM) administration30minutes prior to SI/R, a dose selectedbased upon dose-response experiments and previous investigations. NRC injury wasdetermined by MTT and lactate dehydrogenase (LDH) leakage assay. ROSaccumulation and apoptosis were assessed by flow cytometry. Mitochondrialmembrane potential (MMP) was determined by5,5’,6,6’-tetrachloro-1,1’,3,3’-tetrathylbenzimidazol carbocyanine iodide (JC-1). Cytosolic translocation ofmitochondrial cytochrome c and expression of caspase-9, caspase-3, Bcl-2familyproteins, and phosphorylated Akt and GSK-3β were determined by western blot.Results1. No significant hemodynamic differences existed between groups at baselineconditions. Additionally, there were no significant differences in heart rate (HR) andmean arterial pressure (MAP) between ant groups during MI/R. Pretreatment with GSRd enhanced±LVdP/dtmax, and markedly decreased LVEDP post-I/R comparedto MI/R group. Hemodynamic data showed that GSRd improved rat cardiac systolicand diastolic function after MI/R.2. Thirty minutes MI followed by3hours R resulted in significant infarction, increasedplasma CK and LDH levels, significant increase of TUNEL-positive cells andcaspase-3activity in MI/R group compared to sham. GSRd treatment significantlydecreased infarct size (20.9%±2.3%versus36.0%±1.5%, P<0.01), CK and LDHlevels (2,238±160and1,320±109U/L versus3,324±228and2,327±143U/Lrespectively, P<0.01), and markedly reduced TUNEL-positive staining cells(11%±2.3%versus16.3%±1.8%, P<0.01), caspase-3activity (1.9±0.3versus3.0±0.2,P<0.05) compared to the MI/R-group. Together, these data suggest that GSRddecreased post-MI/R myocardial necrosis and apoptosis in vivo.3. Cells were treated with varying concentrations of GSRd (0.1-50μM). GSRd alone atthese concentrations for24hours was not cytotoxic by MTT and LDH leakage assay.After SI/R（3h/2h）, cellular viability in the vehicle group was significantly reducedto41%±0.6%and LDH leakage increased to16.33%±2.3%compared to the control(all P<0.01). Annexin V/PI double staining showed significant apoptotic increase invehicle group (19.9%±1.1%versus3.1%±0.2%, P<0.01). GSRd (0.1,1, and10μM)markedly reduced SI/R-induced cell death, increasing viability rate to59%±1.8%,63%±3.9%, and69%±3.7%and decreasing LDH leakage to11%±1.7%,10.3%±0.9%, and7.3%±0.9%(P<0.01) respectively. GSRd at10μM markedlydecreased apoptosis (6.3%±0.7%, P<0.01). Taken together, these in vitro resultssupport GSRd as a potent cardioprotective agent and significantly preserved cellularviability following in a dose-dependent manner (at concentrations up to10μM).4. SI/R (3h/2h) induced a rapid and significantly increase of intracellular ROS levels,decrease of Bcl-2(an anti-apoptotic protein) expression, increase of Bax (a pro-apoptotic protein) expression, and decrease of the Bcl-2/Bax ratio (P<0.01). SI/Rsubstantially decreased mitochondrial depolarization compared to the control(P<0.01). Mitochondrial depolarization causes release of several apoptogenicproteins, most notably cytochrome c into the cytosol which is an important earlydeterminant of the mitochondrial apoptotic pathway. Pretreatment with GSRdsignificantly reduced ROS generation, increased the Bcl-2/Bax ratio, stabilized theMMP, decreased cytochrome c release (0.9±0.03versus0.7±0.02, P<0.05), andattenuated expression of both cleaved caspase-9and caspase-3during SI/R in NRCs.These results suggest that GSRd may attenuate apoptosis partly through themitochondrial apoptotic pathway. To further investigate the molecular mechanismunderlying GSRd-mediated cardioprotection, we determined P-Akt/Akt andP-GSK-3β/GSK-3β in NRCs post SI/R by western blot. There was no significantdifference in Akt and GSK-3β expression between treatment groups at the baseline.Consistent with previous reports, SI/R alone increased phosphorylation of Akt andGSK-3β. Pretreatment with GSRd significantly increased phosphorylation of Akt andGSK-3β (and consequently increased P-Akt/Akt and P-GSK-3β/GSK-3β ratios(P<0.01). Pretreatment with PI3K inhibitor LY294002blocked GSRd-mediatedphosphorylation of Akt and GSK-3β.ConclusionsThe major findings of the present study are as follows. Firstly, pretreatment with GSRdattenuated MI/R injury in a rat model, evidenced by improved cardiac function, reducedinfarct size, and reduced myocardial apoptosis after MI/R; GSRd reduced SI/R injury incultured NRCs, evidenced by increased cardiomyocyte viability, decreased cardiomyocyteLDH activity and reduced cardiomyocyte caspase-3and-9cleavage. Secondly, GSRdreduced intracellular ROS generation in cardiomyocytes, and inhibited myocardialapoptosis induced by SI/R via the mitochondrial-dependent apoptotic pathway. Finally,our findings demonstrated that Akt/GSK-3β signaling pathway activation significantly contributed to the anti-apoptotic effect of GSRd. The traditional herbal medicine GSRdmay have therapeutic potential attenuating MI/R injury. |
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