The present study was performed in accordance with the National Institutes for Food and Drug Control (http://www.nicpbp.org.cn) for the Use of Laboratory Animals and was approved by the Tongji Hospital of Tongji University (Shanghai, China) Committee on Animal Care. A total of 30 male eight-week-old Sprague-Dawley rats (Experimental Animal Center, Tongji University) weighing between 260 and 280 g were housed in diurnal lighting conditions (12 h light/12 h dark; 22–24°C) and allowed free access to food and water for 7 days prior to performing the investigation. GSRg3 (purity>98%; Fig. 1) was purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China).

The cardial MI/R surgery was performed, as described previously (5). Briefly, the rats were anaesthetized via intraperitoneal injection of pentobarbital sodium (60 mg/kg body weight; Sigma-Aldrich, St. Louis, MO, USA) and were placed on a warm board (25°C) to control the body temperature at 37°C for surgery. The neck was opened with a ventral midline incision and the animals were ventilated with room air using a rodent respirator (tidal volume 8 ml/kg body weight; 60–80 breaths/min; Shanghai Alcott Biotech Co., Ltd., Shanghai, China). An electrocardiogram in lead II was recorded through needle electrodes attached to the limbs. Following the adjustment of the respiratory rate and the tidal volume of gases, the chest was opened by a middle thoracotomy. Following pericardiotomy, a 4-0 black silk thread (Millar, Inc., Houston, TX, USA) was passed behind the left anterior descending coronary artery and was occluded by a knot for 30 min to cause ischemia. Subsequently, the knot was released and reperfusion was performed for 3 h. For the sham control group, the black silk was placed under the left anterior descending coronary artery without occlusion.

The right common carotid artery was exposed and cannulated with a Millar vessel (Millar, Inc.) into the left ventricular cavity of the rat through the ascending aorta. The heart function, including the left ventricular systolic pressure (LVSP), heart rate (HR) and first derivative (±dp/dt) of left ventricular pressure of the rats in each group (sham, I/R and I/R+Rg3) were recorded and programmed using a biotic signal collection and processing system (PowerLab; AD Instruments, New South Wales, Australia), as described previously (6).

Following 3 h reperfusion, blood samples (5 ml) were collected through the ventral aorta using a scalp vein set, and the serum was frozen at −80°C until subsequent analysis. The activities of LDH, CK and SOD were determined using an ELISA kit, according to manufacturer’s instructions (BioAssay Systems, Hayward, CA, USA).

Following reperfusion, the hearts were rapidly extracted from the rats using surgical scissors and frozen at −20°C. The left ventricular area was sliced into six 2–3 mm-thick slices perpendicular to the base-apex and incubated in 2% triphenyltetrazolium chloride (TTC; pH 7.4; Xiya Reagent, Chengdu, China) buffer for 15 min at 37°C. The viable tissues were stained dark red with TTC, while the infarcted portion remained grayish-white. The area of infraction was measured using an image analysis system [National Institutes of Health (NIH) image software, version 1.60; NIH, Bethesda, MA, USA]

Primary neonatal Sprague-Dawley rats (1–3 days-old) were purchased from the experimental Animal center of Tongji University (Shanghai, China). Cardiac myocytes were cultured from 1–3 day-old Sprague-Dawley rats with Dulbecco’s modified Eagle’s medium (DMEM; Gibco Life Technologies, Carlsbad, CA, USA) at a density of 5×10⁵ for three days, as described previously (1,2). The primary neonatal rats were anaesthetized with sodium pentobartbital and decapitated, and then immersed in 75% ethanol (20 ml) for 30 sec. The chest was opened and the heart ventricles were dissected rapidly and immersed in ice-cold Krebs-Ringer buffer containing 137 mM NaCl, 2 mM CaCl₂, 5.4 mM KCl, 1.1 mM MgCl₂·6H₂O, 0.4 mM NaH₂PO₄·2H₂O, 11.9 mM NaHCO₃ and 5.6 mM glucose (pH 7.4), and the ventricles were minced into small sections using eye scissors and digested with trypsin (0.1%; Beyotime Institute of Biotechnology, Guangzhou, China). The cardiomyocytes were cultured in DMEM containing 10% newborn calf serum (NCS; Gibco Life Technologies), in a humidified atmosphere of 95% air and 5% CO₂ at 37°C. The neonatal rat cardiomyocytes (NRCs) were used and the ginsenoside-Rg3 and solvent were preincubated with cells for 30 min prior to I/R injury.

Simulated I/R (SI/R) was performed, as described previously (1,2). Briefly, simulated ischemia buffer, containing 98.5 mM NaCl, 1.2 mM MgSO₄, 10 mM KCl, 1 mM CaCl₂, 40 mM sodium lactate and 20 mM HEPES (pH 6.8), and simulated reoxygenation buffer, containing 20 mM HCO₃, 0.9 mM NaH₂PO₄, 1 mM CaCl₂, 1.2 mM MgSO₄, 20 mM HEPES, 5 mM KCl, 129.5 mM NaCl and 5.5 mM glucose (pH 7.4), were prepared in advance. The medium of the NRCs was replaced with 1 ml simulated ischemia buffer, incubated in a hypoxic chamber (humidified atmosphere 5% CO₂/0% O₂ balanced with N₂ at 37°C) for 3 h, and then reoxygenated in a standard incubator for 2 h with reoxygenation buffer. The cells subjected to control conditions were cultured with normal Tyrode solution (pH 7.4; Beijing Leagene Biotech Co., Ltd., Beijing, China) in a humidified atmosphere of 5% CO₂/21% O₂ balanced with N₂ at 37°C for 5 h (4). The cells were divided randomly into four groups: Control group, incubated with Tyrode solution during the entire experimental period; SI/R group, incubated with simulated ischemia buffer for 3 h hypoxia, followed by 2 h re-oxygenation; Vehicle group, subjected to 0.2% (v/v) dimethyl sulfoxide administration 30 min prior to SI/R; SI/R+GSRg3 group, subjected to GSRg3 (10 mM) administration 30 min prior to SI/R (5).

The cell viability was assessed using a Cell Counting Assay kit-8 (CCK-8, Beyotime Institute of Biotechnology) according to the manufacturer’s instructions. The NRCs (100 μl) were plated into 96-well plates at a density of 1×10⁵ cells/well, followed by 30 min pre-incubation with different concentrations of GSRg3 (0.1–100 μM). Following treatment, the cells were exposed to 10 μl CCK-8 solution for a futher 2 h and the absorbance at 450 nm was measured using a microplate reader (ELx808; Bio-Tek Instruments, Winooski, VT, USA) (1).

The apoptotic rate of the NRCs was determined by flow cytometry using annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) staining according to the manufacturer’s instructions. Briefly, the NRCs were pretreated with 10 μM GSRg3 for 30 min followed by SI/R treatment. The cells were digested with trypsin (0.25%) and were then resuspended in phosphate-buffered saline (PBS) containing 10% NCS. The cells were centrifuged at 200 × g for 10 min at 4°C and then washed twice with cold PBS. The cells were then treated with 5 μl annexin V-FITC (1:80) and 10 μl PI (1:40) (Bioworld Technology Co., Ltd., Nanjing, China), and incubated in the dark at room temperature for 15 min. Each sample was analyzed using a Beckton-Dickinson flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA).

The NRCs were lysed in lysis buffer (Beyotime Institute of Biotechnology) containing Protease Inhibitor Cocktail (Merck Millipore, Billerica, MA, USA) for 30 min on ice. The cellular proteins were collected using a cell scraper. Following centrifugation for 15 min at 12,000 rpm, a Bicinchoninic acid Protein Assay kit (Beyotime Institute of Biotechnology) was used to determine the protein concentrations. Equal quantities of protein were separated using 10% SDS-PAGE gels and transferred onto polyvinylidene fluoride membranes (EMD Millipore, Billerica, MA, USA). The membranes were blocked using 5% non-fat milk in Tris-buffered saline (8 g NaCl and 6 g Tris), containing 1% Tween-20 (TBST), for 1 h at room temperature. The membranes were then incubated with primary antibodies against eNOS (610297; IgG1; polyclonal; 1:3,000; BD Biosciences), p-Akt (2920; mouse monoclonal; 1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA), Akt (4691; rabbit monoclonal; 1:1,000; Cell Signaling Technology, Inc.) Bcl-2 (2870; mouse poly-clonal; 1:10000; Cell Signaling Technology, Inc.), Bax (2772; mouse polyclonal; 1:2,000; Cell Signaling Technology, Inc.) and PARP (5625; rabbit monoclonal; 1:1,000; Cell Signaling Technology, Inc.) overnight at 4°C. Following washing three times for 5 min with TBST, the membranes were incubated with secondary antibodies, including alkaline phosphatase-linked anti-mouse (7056; IgG; 1:5,000; Cell Signaling Technology, Inc.) or horseradish peroxidase-linked anti-rabbit antibodies at room temperature for 1 h. β-actin (7074; IgG; 1:5,000; Cell Signaling Technology, Inc.) was used as an internal control. The protein bands were visualized using a Chemiluminescence Electophoretic Mobility Shift Assay kit (Beyotime Institute of Biotechnology) and X-ray film (Kodak BioMax MS Film; Kodak. Corp., Rochester, NY, USA). The band density was statistically analyzed using Image J software (National Institutes of Health, Bethesda, MD, USA).

Data are expressed as the mean ± standard error of the mean. The statistical comparisons between groups were performed using one-way analysis of variance. SPSS version 19.0 was used to perform all statistical analyses (SPSS Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

GSRg3 had no effect on blood glucose, cardiac function or blood pressure normality and no significant differences were observed between the groups at the baseline conditions. Pretreatment with GSRg3 increased LVSP and +dp/dt max, and decreased LVEDP and −dp/dt max following 3 h reperfusion, compared with the MI/R group (P<0.05; Fig. 2). Treatment with GSRd markedly increased the mean heart rate compared with the MI/R group (P<0.05; Fig. 3). The hemodynamic data demonstrated that GSRg3 improved rat cardiac systolic and diastolic function following MI/R.

The infacted areas and areas at risk are shown in Fig. 4. No MI was observed in the hearts from the sham group. Pretreatment with GSRg3 significantly decreased the infarct size compared with the MI/R group (P<0.05). To determine whether GSRg3 attenuated MI/R induced cardio-myocyte necrosis, the plasma levels of CK, LDH and SOD were measured following reperfusion. GSRg3 treatment markedly decreased the levels of CK and LDH, and increased the levels of SOD compared with the MI/R group (P<0.05). These data demonstrated that GSRg3 reduced myocardial necrosis following MI/R.

The NRCs were treated with different concentrations of GSRg3 (0.1–100 mM) to determine the effects of GSRg3 alone. Treatment with these concentrations of GSRg3 for 24 h were not cytotoxic, as demonstrated by the CCK-8 assay (Fig. 5A). As shown in Fig. 5B, the concentration response curves determined cellular viability, and this was observed at dosage of 10 mM GSRg3.

Flow cytometric analysis was perfomed to assess the cellular apoptosis (Fig. 5C). Annexin V/PI double staining revealed a significant increase in apoptosis in the sham group compared with the control post-SI/R (8.6±0.3%, vs. 3.1±0.2%; P<0.05), and treatment with 10 mM GSRg3 markedly decreased cellular apoptosis (4.6±0.1%; P<0.01; Fig. 5D). Overall, these in vitro results suggested that GSRg3 protected cardiomyocytes, which was in accordance with the in vivo data.

The present study aimed to determine whether GSRg3 inhibited the apoptosis of NRCs induced by SI/R by modulating the Bcl-2 family proteins. SI/R treatment reduced the expression of Bcl-2 and increased the expression of Bax, therefore, downregulating the Bcl-2/Bax ratio (Fig. 6A). Pretreating NRCs with 10 mM GsRg3 prior to SI/R induced the expression of Bcl-2 and inhibited the expression of Bax, therefore, increasing the Bcl-2/Bax ratio (Fig. 6A).

The caspase family of proteins regulate cellular apoptosis. Caspase-9 is activated by cytochrome c, which activates caspase-3, causing cell apoptosis (4). SI/R significantly increased the protein expression levels of cleaved caspase-9 and caspase-3, however, pretreating NRCs with 10 mM GsRg3 significantly attenuated the expression levels of cleaved caspase-9 and caspase-3 (Fig. 6B).

To further investigate the molecular mechanism underlying GsRg3-mediated cardioprotection, western blot analysis was performed to determine the protein expression levels of phosphorylated (p)-Akt/Akt and p-eNOS in NRCs following SI/R. No significant differences were observed in the expression levels of Akt and eNOS between the treatment groups at the baseline (Fig. 7A and B). Consistent with previous reports, pretreatment with 10 mM GSRg3 significantly increased the expression levels of p-Akt and p-eNOS, and consequently increased the ratios of p-Akt/Akt and p-eNOS/eNOS (P<0.01). Treatment with the phosphoinositide 3-kinase inhibitor, LY294002, inhibited the GSRg3-mediated phosphorylation of Akt (Fig. 7B).