Male Sprague-Dawley (SD) rats (180–200 g, 7 weeks old) were purchased from the Experimental Animal Center of Zhejiang Province (Zhejiang, China). The involvement of animals in the experiments was reviewed and approved by the Animal Policy and Welfare Committee of Wenzhou Medical University (Wenzhou, China). All animals were housed in groups of 4 rats per cage and maintained in controlled conditions (12 h light-dark cycle; 20–24°C; 45–55% humidity). Food and water were available ad libitum.

Sch B was obtained from Xi'an Plant Bio-Engineering Co., Ltd. (Xi'an, China). LY294002, a PI3K inhibitor, was obtained from Sigma-Aldrich; Merck Millipore (Darmstadt, Germany). Anti-phosphorylated (p)-Akt (Ser473; cat. no. 4060), anti-Akt (cat. no. 4685), anti-B-cell lymphoma 2 (Bcl-2; cat. no. 2876), anti-cleaved caspase-3 (cat. no. 9661), anti-GAPDH (cat. no. 5174) antibodies, and anti-rabbit horseradish-peroxidase (HRP)-conjugated immunoglobulin G (cat. no. 7074) secondary antibodies were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Anti-Bcl-2-like protein 4 (Bax; cat. no. sc-6236) antibody was purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA).

According to previous studies (9,10), preliminary experiments to assess three doses of Sch B (10, 30 and 60 mg/kg) showed that 60 mg/kg Sch B was the optimal dose based on the myocardial infarction size (81.3±7.1, 74.3±6.7 and 62.8±11.2%, respectively). In the subsequent experiments, the rats were randomly divided into five groups: i) Sham group; ii) I/R group; iii) Sch B+I/R group; iv) Sch B+LY+I/R group; and v) LY+I/R group. The Sch B-treated rats were administered with Sch B (60 mg/kg intragastrically, dissolved/suspended in olive oil, once a day) for 15 days, and the remaining rats received olive oil only. The LY-treated rats were administered with LY294002 [0.3 mg/kg dissolved in dimethyl sulfoxide (DMSO; 0.1%)] 30 min prior to reperfusion.

Following sacrifice of the rats with intraperitoneal pentobarbital (60 mg/kg), the rats were mechanically ventilated using a rodent respirator (DW-3000B; Huaibei Zhenghua Technology Co., Ltd., Anhui, China). A limb lead II electrocardiogram (ECG) was continuously monitored and recorded during the entire procedure. Following cardiac exposure, the left anterior descending (LAD) coronary artery was ligated with a 6–0 polypropylene suture below the inferior margin of the left auricle. At the top of the vessel, a vinyl tube was placed for reversible coronary occlusion. The success of ischemia was judged by the ST-segment elevation on the ECG. The myocardium underwent ischemia for 45 min, following which the slipknot was released for 24 h to mimic reperfusion.

Infarction size was evaluated using Evans Blue (EB)/triphenyl tetrazolium chloride (TTC) staining (14). In brief, at the end of reperfusion, 2 ml EB (2%; Sigma-Aldrich; Merck Millipore) was injected intravenously following ligation of the LAD. The heart was removed immediately and stored at −20°C for 20 min. The tissues were sectioned into five transverse slices perpendicular to the long axis (2 mm thick). The slices were incubated in a 1% TTC solution at 37°C for 20 min. The EB-stained area represented the area with normal blood supply. The infarct area (IA) was paler and the area stained red represented the viable tissue at risk. The IA and area at risk were analyzed using Image J software (version 1.44; National Institutes of Health, Bethesda, MD, USA).

Blood samples were collected from the rats at the end of reperfusion and were centrifuged at 3,000 × g for 10 min at 4°C. The activities of cTn-T and CK-MB in the plasma were estimated using commercially available assay kits (Shanghai XinFan Biotechnology Co., Ltd., Shanghai, China).

The hearts were collected at the end of reperfusion. Following excision of the superfluous tissue, the left ventricular tissue was fixed in 4% paraformaldehyde at room temperature for 24 h. Subsequently, the fixed tissues were embedded in paraffin and sectioned at 4 µm thicknesses. The myocardial sections were stained with hematoxylin and eosin at room temperature for 2 min and visualized under a light microscope (magnification, ×200; Nikon Corporation, Tokyo, Japan).

Following dewaxing and rehydration, the myocardial tissue sections (4 µm thick) were prepared and cell apoptosis was detected using the terminal deoxyribonucleotide transferase mediated dUTP nick end-labeling (TUNEL) assay (Ai Mingkang Bio-Reagent Co., Ltd., Chongqing, China). The stained sections were viewed using fluorescence microscopy (Nikon Corporation) and the TUNEL-positive cells were evaluated at ×400 magnification.

Following dewaxing and rehydraton, the myocardial tissue sections (4 µm thick) were blocked with 5% bovine serum albumin (BSA; Sigma-Aldrich; Merck Millipore) and incubated with anti-Bax antibody (1:100) or anti-Bcl-2 antibody (1:100) overnight at 4°C. Subsequently the sections were incubated with the HRP-conjugated secondary antibody (1:200) at room temperature for 30 min and visualized under a light microscope (magnification, ×200; Nikon Corporation).

Proteins were extracted from the myocardial tissues using lysis buffer containing protease (Beyotime Institute of Biotechnology, Haimen, China) and phosphatase inhibitors (Cell Signaling Technology, Inc., Danvers, MA, USA) under homogenization, and the concentrations of protein were measured using a Bicinchoninic Acid Protein Assay kit (Beyotime Institute of Biotechnology). Following separation by 12% SDS-PAGE, a total of 20 µg proteins were transferred onto a polyvinylidene fluoride membrane, blocked with 5% BSA at room temperature for 1 h, and incubated with specific primary antibodies (1:1,000) overnight at 4°C. Subsequently, immunoreactive bands were incubated with a HRP-conjugated secondary antibody (1:1,000) at room temperature for 1 h and detected using enhanced chemiluminescence (EMD Millipore, Billerica, MA, USA).

Data are expressed as the mean ± standard deviation. GraphPad Prism software (version 5.0; GraphPad Software, Inc., La Jolla, CA, USA) was used for statistical analysis. One-way analysis of variance and Student-Newman-Keuls test were used. P<0.05 was considered to indicate a statistically significant difference.

Representative images of the TTC and EB staining for the rats in each group are shown in Fig. 1. Compared with the control rats, Sch B administration led to a significant reduction in myocardial infarction size (62.8±11.2 vs. 84.5±4.1%; P<0.05). However, the protective effect of the medication was eliminated by LY294002 treatment (I/R+Sch B+LY group, 83.8±2.9%), compared with the I/R+ Sch B group (62.8±11.2%; P<0.05; Fig. 1A).

Compared with the sham-operation rats, the levels of myocardial infarction-associated markers in plasma samples were distinctly increased in the control rats. Consistently, Sch B treatment reduced the levels of cTn-T, compared with that in the control rats (468.3±92.0 vs. 761.8±114.0 ng/l, respectively; P<0.05; Fig. 1A) and reduced the level of CK-MB, compared with that in the control rats (136.8±16.3 vs. 162.1±12.9 U/l, respectively; P<0.05 Fig. 1B). However, the inhibitory effect of Sch B on the level of cTn-T was eliminated by LY294002 administration, compared with that in the Sch B group (468.3±92.0 vs. 725.6±122.2 ng/l, respectively; P<0.05), with the same observed for CK-MB (136.8±16.3 vs. 155.7±17.2 U/l, respectively; P<0.05; Fig. 1).

Compared with the hearts of the sham group, disorganization of cell structures was observed in the myocardium of untreated rats. Cardiomyocyte necrosis and inflammatory cell infiltration were decreased in the Sch B treatment group, compared with the untreated rats. However, treatment with LY294002 eliminated the protective effects of Sch B in myocardial injury (Fig. 1D).

The results of the TUNEL assay revealed that the number of TUNEL-positive cells was higher in the untreated rats (74.50±5.97%), compared with that in the sham group (5.75±2.50%). Treatment with Sch B reduced the number of TUNEL-positive nuclei stained (41.25±7.27%; P<0.05). However, this anti-apoptotic effect of Sch B was attenuated by LY294002 administration (41.25±7.27 vs. 70.50±6.76%, respectively; P<0.05), as shown in Fig. 2A and B.

Compared with the untreated rats, the concentrations of Bax were significantly decreased and the concentrations of Bcl-2 were increased in the Sch B treatment groups, however, these effects of Sch B were attenuated by LY294002 treatment (Fig. 3A-F).

Treatment with Sch B significantly increased the concentrations of p-Akt, compared with that in the untreated rats (0.20±0.02 vs. 0.12±0.02; P<0.05; Fig. 4A and B). However, the administration of LY294002, an inhibitor of PI3K, attenuated the Sch B-induced upregulation of myocardial p-Akt in the I/R + Sch B + LY group (0.11±0.03). The concentrations of cleaved caspase-3 in the myocardium of the Sch B+I/R group were also lower, compared with those in the I/R and Sch B+LY+I/R groups (Fig. 4C and D).