Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco-BRL (Grand Island, NY, USA). The kits used for the determination of serum lactate dehydrogenase (LDH) and creatine kinase isoenzyme-MB (CK-MB) content were obtained from Jiancheng Bioengineering Institute (Nanjing, China). MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide], TTC (2,3,5-triphenyltetrazolium chloride), Evans blue, and compound C [AMP-activated protein kinase (AMPK) inhibitor] were purchased from Sigma-Aldrich (St. Louis, MO, USA). The primary antibodies were as follows: anti-AMPKα (Thr172; #2532), anti-phosphorylated AMPKα (p-AMPKα) (Thr172; #2531), anti-caspase-3 (#9662), anti-Bcl-2 associated X protein (Bax; #2772), anti-cytochrome c (#11940), anti-acetyl CoA carboxylase (ACC; #3676), anti-p-ACC (#11818), β-actin (#4970), and anti-Bcl-2 (#2870; all from Cell Signaling Technologies, Beverly, MA, USA). All these are rabbit monoclonal antibodies. The secondary antibody (anti-rabbit IgG-B; sc-53804) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The caspase-3 assay kit was purchased from Chemicon International, Inc. (Temecula, CA, USA). The fluorescent kit for Hoechst 33258 was obtained from Roche Diagnostics (Mannheim, Germany).

The root bark of Aralia taibaiensis Z. Z. Wang & H. C. Zheng was collected at Mountain Taibai, Shaanxi, China, in September 2008, and botanically identified by Dr Haifeng Tang (Department of Pharmacy, Xijing Hospital, and Fourth Military Medical University, Shaanxi, China). A voucher specimen (FMMUDP-Voucher No. SAP012) was deposited in the Herbarium of the Department of Pharmacy, Xijing Hospital, and the Fourth Military Medical University, China.

A crude extract (total saponins) of Aralia taibaiensis (sAT) was prepared as previously described, with slight modifications (16). Dry, powdered root bark (20 g) was extracted three times with 80% (v/v) ethanol (herb:ethanol, 1:10, w/v) under reflux (80°C) for 60 min. The alcohol extract was concentrated, suspended in distilled water, and then partitioned successively with chloroform (ratio 1:3, v/v) and n-butanol saturated with water (ratio 1:3, v/v, three times). The n-butanol extracts were combined and evaporated using a rotary evaporator at 60°C to produce a powdered residue. The yield was 0.78% (w/w). Oleanolic acid was used as a reference standard, and the total saponins content was expressed as oleanolic acid equivalents (i.e., 459.30 µg oleanolic acid equivalents/ mg extract). The content of total saponins in sAT was >90%.

sAT was dissolved in physiological saline (0.9% NaCl) prior to use. After a 1-week-long adaptation period, the animals were randomized into five experimental groups of eight animals each as follows: sham-operated group + saline, MI/RI group + saline, MI/RI + sAT (60 mg/kg/day, orally), MI/RI + sAT (120 mg/kg/day, orally), MI/RI + sAT (240 mg/kg/day, orally). Pretreatment with sAT or saline was initiated 7 days prior to the operation and administered once a day for 7 days.

Experimental procedures for these animals were conducted according to the National Institutes of Health Guidelines for the Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee of the Fourth Military Medical University. Male Sprague-Dawley rats (250±30 g; 10±0.5 weeks) were anesthetized with 10% chloral hydrate (0.35 ml/100 g body weight) and ventilated using a positive-pressure respirator for small animals (HX-100E, Chengdu Technology & Market Co., Chengdu, China). Electrodes were placed subcutaneously and connected to an electrocardiograph (BL-420S; Taimeng Science Technology, Ltd., Chengdu, China). During surgery, body temperature was measured and maintained at 37°C by placing the rats on a heating pad. After left thoracotomy, the left anterior descending coronary artery was occluded with a 6-0 silk suture. After occlusion for 30 min, the suture was loosened, and the myocardium was reperfused for 3 h. Sham-operated rats underwent identical surgery, but the suture was not tightened around the coronary artery. ST-segment elevation in experimental animals was assessed as a measure of myocardial ischemia as previously described (20).

After 3 h of reperfusion, blood samples were collected from the abdominal aorta to measure serum LDH and CK-MB levels by using an enzyme-linked immunosorbent assay (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The assay was performed according to the manufacturer's instructions. Following anesthesia, the rats were sacrificed by exsanguination (blood was drawn from the abdominal aorta) and the hearts were harvested for double-staining.

Myocardial infarct size was measured using a double-staining technique with 3% Evans blue and 4% TTC adapted as previously described (21). After 6 h of reperfusion, the coronary artery was again occluded. To map the areas at risk for ischemia, 3% Evans blue solution was infused through the left jugular vein. The heart was quickly excised and frozen at −20°C, and the ventricles were then cross-sectioned into five sections. The sections were counterstained with 4% TTC (in phosphate buffer, pH 7.8) for 15 min at 37°C and photographed after overnight storage in 4% paraformaldehyde. The infarct size was presented as the left ventricular infarct area as a percentage of the ischemic area at risk.

To examine cardiac myocyte apoptosis, samples of tissue from the ischemic zones were fixed in 4% paraformaldehyde, embedded in paraffin, and cut into 5-mm transverse sections. The terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay was carried out using an apoptosis detection kit (Roche Applied Science, Mannheim, Germany) according to the manufacturer's instructions. 4′,6-Diamidino-2-phenylindole (DAPI) was used as a counterstain. The apoptotic cells were analyzed by laser-scanning confocal microscopy (SP5-FCS; Leica Microsystems, Wetzlar, Germany). The number of TUNEL-positive nuclei was calculated by ImageJ software (NIH, Bethesda, MD, USA). Quantitative analysis was carried out using the following formula: percentage of TUNEL-positive cells = TUNEL-positive cells/total cells.

Rat H9c2 cardiomyocytes were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA). The cells were cultured in DMEM with 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 mg/ml streptomycin and maintained in a humidified atmosphere of 5% CO₂ at 37°C. The medium was replaced every 2–3 days, and the cells were subcultured at 80–90% confluence by detaching with 0.05% trypsin-ethylenediaminetetraacetic acid and reseeding.

To mimic ischemic injury in vitro, ischemia and reperfusion were performed in rat H9c2 cells based on a previously described method (22). At 80% confluence, the cells were incubated in an 'ischemic buffer' for 2 h at 37°C in a hypoxic chamber (previously bubbled with 95% N₂ and 5% CO₂). The buffer contained 137 mM NaCl, 12 mM KCl, 0.49 mM MgCl₂, 0.9 mM CaCl₂·2H₂O, 4 mM HEPES, and 20 mM sodium lactate (pH 6.2). For the A/R studies, the medium was replaced by maintenance medium (DMEM with 2% FBS) during the re-oxygenation period.

Cell viability was determined colorimetrically using the MTT assay. Cells at the exponential phase were seeded at 1×10⁴ cells/well in 96-well plates. After different treatments, 20 µl of 5 mg/ml MTT solution was added to each well (0.5 mg/ml final concentration), and the plates were incubated for 4 h at 37°C. The supernatant was removed, the formazan crystals were dissolved in 150 µl DMSO, and the optical density at 490 nm was read on a microplate reader (Tecan, Mannedorf, Switzerland). The cell survival ratio was expressed as a percentage of the control.

The cell culture medium was collected after the A/R procedure. The concentrations of LDH and CK-MB were determined by a colorimetric method using commercial kits (Nanjing Jiancheng Bioengineering Institute) according to the manufacturer's protocol. The results were reported as U/l.

Briefly, H9c2 cardiomyocytes were washed with ice-cold PBS, fixed in 10% neutral buffered formalin for 10 min at room temperature, and washed again in ice-cold PBS. The cardiomyocytes were then exposed to Hoechst 33258 (2 µg/ml in PBS) and incubated for 20 min at room temperature. The cells were then washed three times in PBS and examined under a fluorescence microscope with an appropriate filter. Images were captured at a magnification of ×200 using an Olympus microscope (1X71; Olympus Corp., Tokyo, Japan).

Caspase-3 activity was determined in cytosolic protein extracts using a colorimetric activity assay kit (Beyotime Institute of Biotechnology, Haimen, China) according to the manufacturer's protocol. Specifically, after determining the protein concentration, the supernatant was incubated with the caspase-3 substrate (Ac-DEVD-pNA) on a 96-well-plate. The activity of caspase-3 was detected using a microplate reader (Tecan) at 405 nm.

H9c2 cells cultured in 6-well plates (3×10⁵/well) were washed twice with ice-cold PBS and lysed via incubation on ice for 30 min with lysis buffer (pH 7.5) containing 20 mM Tris-HCl, 120 mM NaCl, 1.0% Triton X-100, 10% glycerol, 2 mM ethylene-diaminetetraacetic acid, and protease inhibitor cocktail (Roche GmbH). Following centrifugation of the cell lysates at 4°C for 20 min at 10,000 × g, the supernatants (total cell extracts) were frozen and stored at −80°C. The protein concentration of each sample was determined using a bicinchoninic acid assay protein assay kit (Pierce Chemical, Rockford, IL, USA).

For the western blot analysis, equal amounts of cytosolic protein lysates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis on 10–15% gels. The gels were transferred onto polyvinylidene fluoride membranes (PVDF; Bio-Rad, Hercules, CA, USA), blocked for 30 min at 37°C with 5% non-fat dry milk, and incubated with the primary antibodies (1:1,000 dilution) overnight at 4°C. After three washes with Tris-buffered saline plus Tween-20, the membranes were incubated with secondary antibodies (1:200 dilution) in Tris-buffered saline plus Tween-20 for 30 min at 37°C and washed as described above. Subsequently, the blots were developed by enhanced chemiluminescence according to the manufacturer's instructions.

Data were presented as mean ± standard deviation. The statistical significance of differences between the means were determined using SPSS 19 software (SPSS, Inc., Chicago, IL, USA) for Windows using one-way analysis of variance followed by the Bonferroni post hoc test or Student's t-test, as appropriate. Differences with P-values of <0.05 were considered statistically significant.

Myocardial infarct size, plasma CK, and plasma LDH were the primary indicators used to assess myocardial injury after MI/RI. Representative areas at risk and infarct images are shown in Fig. 1A and B. No myocardial infarction was observed in hearts from the sham-operated group. However, significant infarction was observed in rats in the MI/RI group compared to rats in the sham group [50.8±1.5% (infarct area as a percentage of area at risk), P<0.01]. Treatment with 120 and 240 mg/kg sAT significantly decreased infarct size compared to the MI/RI-group (39.6±3.3%, P<0.05 and 22.0±3.0%, P<0.01, respectively). There was no significant difference in the area at risk between any of the groups.

Injury to the cardiomyocytes was determined by measuring the levels of LDH and CK-MB in the serum at the end of reperfusion (Fig. 1C and D). MI/RI significantly increased LDH and CK-MB levels compared to those in the sham group (1185.5±83.7%, P<0.01 and 3523.4±326.9%, P<0.01, respectively), while 120 and 240 mg/kg sAT markedly reduced the levels of LDH and CK-MB after MI/RI (P<0.01).

Consistent with these results, the MI/RI group exhibited a significant increase in TUNEL-positive cells compared with the sham-group (85.5±3.7%, P<0.01) (Fig. 2). Pretreatment with 120 or 240 mg/kg sAT substantially reduced the number of TUNEL-positive cells (62.9±4.8%, P<0.01 and 36.6±5.7%, P<0.01, respectively). Taken together, these data suggested that sAT decreased post-MI/RI myocardial apoptosis in vivo.

The toxicity of sAT was examined in cultured H9c2 cells incubated with sAT (1–100 µg/ml) for 24 h. As shown in Fig. 3A, sAT did not alter cell viability at concentrations reaching 100 µg/ml. The time course of the loss of cell viability following A/R injury was also assessed using MTT assays (Fig. 3B). Based on these data, 4 h was selected as the re-oxygenation time to measure the markers of cell injury. Although the A/R treatment significantly reduced cell survival, pretreatment with sAT markedly increased survival in a concentration-dependent manner. The 50 µg/ml concentration was selected for subsequent investigation (Fig. 3C). LDH and CK-MB activities were measured in the supernatants of culture media. An increase in LDH and CK-MB levels was observed in the A/R group compared to the corresponding levels in the control group (197.2±3.4%, P<0.01 and 143.5±9.7%, P<0.01, respectively; Fig. 3D and E). The levels of LDH and CK-MB were both decreased by sAT as compared to the corresponding levels in the A/R group (P<0.01).

Subsequent to A/R injury, cardiomyocytes exhibited typical characteristics of apoptosis, including shrinkage of the nuclei, chromatin condensation, and the appearance of apoptotic bodies. However, the number of cells with nuclear condensation and fragmentation was significantly decreased in cardiomyocytes incubated with sAT for 24 h following A/R injury (Fig. 4A). Quantitative analysis results are shown in Fig. 4B. Similar data were obtained from observation of the effects of sAT on caspase-3 activity (Fig. 4C) and protein expression of cleaved caspase-3 (Fig. 4D) and cytochrome c (Cyto-c; Fig. 4E). The expression of Bax, a pro-apoptotic protein, was markedly increased after A/R injury, but was decreased in cells treated with sAT compared to A/R alone (Fig. 4F). At the same time, the expression of Bcl-2, an anti-apoptotic protein, decreased in the A/R group and increased in the sAT-treated groups. The Bcl-2/Bax ratio was also decreased in H9c2 cells following A/R injury compared to normoxic control cells (0.54±0.12%, P<0.01), and this decrease was reversed by treatment with sAT (2.27±0.31%, P<0.01 and 2.78±0.17%, P<0.01, respectively).

To investigate the possible mechanisms involved in the cardioprotective effects of sAT, the expression levels of proteins associated with the AMPK pathway were determined by western blot analysis. As shown in Fig. 5A, the phosphorylation of AMPK was investigated in H9c2 cardiomyocytes exposed to A/R with different re-oxygenation times (10, 20, 40, 60 and 120 min). Since the highest level of p-AMPK (relative to total AMPK) was reached at 40 min, this time point was selected for subsequent mechanistic studies. Enhancement of AMPK and ACC phosphorylation was observed in the cells incubated with sAT after A/R injury compared with the control group (Fig. 5B and C).

To investigate whether the AMPK pathway was required for the protective effect of sAT in cardiomyocytes subjected to A/R injury, cells were treated with the AMPK inhibitor, compound C (0.5 mM) for 1 h prior to incubation with sAT. Enhancement of AMPK phosphorylation (Fig. 6A) and ACC phosphorylation (Fig. 6B) by sAT was eradicated by treatment with compound C. In addition, the expression of Bax and Bcl-2 and the activity of caspase-3 were measured following the incubation of cells under A/R with or without compound C in the presence of sAT. As shown in Fig. 6C and D, compared with the sAT-treated group, treatment with sAT plus compound C markedly inhibited the increase in the Bcl-2/Bax ratio (1.38±0.31%, P<0.05) and the reduction in caspase-3 activity (229.0±10.6%, P<0.05).