A total of 60 adult male Sprague-Dawley rats (250–300 g; 1.5–2 months) were obtained from the Center of Experimental Animals in the Xi'an Jiaotong University (Xi'an, China). Animals were housed in a controlled room with a temperature of 22°C, 33% humidity, a 12 h light/dark cycle and free access to food and water. All animals used in the current study were cared for in accordance with the Guide for the Care and Use of Laboratory Animals published by the National Research Council (US) Committee (12) and all procedures were approved by The Committee of Experimental Animals of the Xi'an Jiaotong University.

PTB, the PI3K inhibitor LY294002 and DAPI were all purchased from Sigma-Aldrich; Merck KGaA (Darmstadt, Germany). Myocardial myeloperoxidase (MPO; cat. no. A044), creatine kinase-MB (CK-MB; cat. no. H197) and lactate dehydrogenase (LDH; cat. no. A020-1) assay kits were all purchased from Nanjing Jiancheng Bioengineering Research Institute (Nanjing, China). Interleukin-6 (IL-6; cat. no. R6000B) and tumor necrosis factor-α (TNF-α; cat. no. RTA00) ELISA kits were purchased from R&D Systems, Inc. (Minneapolis, MN, USA). IL-8 ELISA kit (cat. no. kt30449) was purchased from MSK Bio (Wuhan, China). Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) kits were purchased from Roche Applied Science (Mannheim, Germany). Antibodies against Akt (cat. no. 4685), p-Akt (Ser473; cat. no. 4058), Bcl-2 (cat. no. 3498), Bax (cat. no. 14796) and β-actin (cat. no. 4970) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). The horseradish peroxidase-conjugated immunoglobulin G secondary antibody (cat. no. A0208) was purchased from Beyotime Institute of Biotechnology (Haimen, China).

Sprague-Dawley rats were anesthetized intraperitoneally (i.p.) using sodium pentobarbital (Sigma-Aldrich; Merck KGaA; 40 mg/kg). Myocardial ischemia was induced by exteriorizing the heart with a left thoracic incision followed by a slipknot (5-0 silk) around the left anterior descending coronary artery (LAD). Following 30 min ischemia, the slipknot was released and the animal underwent 120 min reperfusion. The Sham group underwent the same procedure without LAD ligation.

Rats were randomly assigned to one of four groups (n=15): i) A sham group; ii) an MI/R group that received treatment vehicle with 0.9% sodium chloride i.p.; iii) an MI/R+PTB group that received PTB (10 mg/kg, i.p.) 10 min prior to reperfusion iv) and an MI/R+PTB+LY group that received PTB (10 mg/kg, i.p.) 10 min prior to reperfusion and LY (10 mg/kg, i.p.) every 2 days three times prior to surgery.

Tetrazolium chloride (TTC) staining was used to assess the myocardial infarct size. Following reperfusion, rats were anaesthetized and euthanized. Rat hearts were then immediately isolated, washed in PBS and sectioned into transverse slices 5 mm thick. Following incubation in 1% (0.01 g/ml) TTC at 37°C in PBS for 15 min, heart slices were photographed with a digital camera to distinguish between the red-stained viable tissues and the white-unstained infarcted tissues. Areas of infarct size were measured digitally using Image Pro Plus 6.0 software (Media Cybernetics Inc., Rockville, MD, USA).

Following reperfusion, blood (6 ml) was taken from the carotid artery of all rats and kept at room temperature for 30 min. Serum was collected by centrifugation at 3,000 × g at 4 °C for 15 min and kept at −70°C for preservation. The CK-MB and LDH assay kits were used to measure levels of CK-MB and LDH in the serum, following the manufacturer's instructions. Enzyme activity was expressed in U/l.

Following reperfusion, the myocardial tissue was kept at −70°C for preservation. An MPO assay kit was used to detect MPO levels in the myocardial tissue following the manufacturer's instructions. Levels of TNF-α, IL-6 and IL-8 were spectrophotometrically analyzed using ELISA kits following the manufacturer's instructions.

Myocardial apoptosis was determined using TUNEL staining. The tissue samples were fixed using 4% formaldehyde at 4°C for, 24 h and embedded in paraffin. The paraffin-embedded tissue was cut into sections 4–5 µm thick. Sections were incubated in 50 µl TUNEL mixture (solution A: Solution B=1:9) in a humidified chamber at 37°C for 1 h. Slides were incubated with DAPI for 5 min at room temperature in the dark to detect the nuclei. Sections were covered using cover slips with anti-fade mounting medium (cat. no. P0126; Beyotime Institute of Biotechnology, Haimen, China). Sections were observed using fluorescence microscopy in six random fields of view. The apoptotic index was calculated as the ratio of the number of TUNEL-positive neurons to the total number of nuclei.

Heart tissues were lysed with RIPA lysis buffer (cat. no. sc-24948, Santa Cruz Biotechnology, Inc., Dallas, TX, USA). Following sonication at 4°C for 25 sec at a frequency of 20 kHz, lysates were centrifuged at 14,000 × g at 4°C for 30 min and protein concentration was detected using the BCA determination method. Proteins (30 µg/lane) were then separated by SDS-PAGE (8–15%) and transferred to a polyvinylidene difluoride membrane (EMD Millipore, Billerica, MA, USA). Following blocking with 5% skimmed milk in Tris-buffered saline at room temperature for 1 h, the membrane was incubated with primary antibodies against p-Akt (1:1,000), Akt (1:1,000), Bcl-2 (1:1,000), Bax (1:1,000) and β-actin (1:1,000) overnight at 4°C. The membrane was then washed with PBS and incubated with horseradish peroxidase-conjugated immunoglobulin G secondary antibody (1:10,000) for 1 h at 37°C. Blots were developed using a SuperSignal chemiluminescence detection kit (Thermo Fisher Scientific, Inc., Waltham, MA, USA) and immunoblotting was visualized and analyzed using the Quantity One System 4.62 (Bio-Rad, Inc., Hercules, CA, USA).

Data are presented as the mean ± standard error of the mean. SPSS 18.0 (SPSS, Inc., Chicago, IL, USA) was used to perform statistical analysis. Differences among groups were evaluated by one-way analysis of variance followed by a Dunnett's t-test for multiple comparisons. P<0.05 was determined to indicate a statistically significant difference.

A myocardial infarct was induced all groups apart from the Sham group (Fig. 1). Treatment with PTB significantly decreased the myocardial infarct size compared with the M/IR group (P<0.05; Fig. 1). However, the protective effect of PTB was reversed by LY treatment, as the myocardial infarct size was significantly increased in the MI/R+PTB+LY group compared with the MI/R+PTB group (P<0.05; Fig. 1).

The release of CK-MB and LDH is an indicator of myocardial injury; therefore, serum levels of CK-MB and LDH were measured. There was a significant increase in the release of serum CK-MB and LDH in the MI/R group compared with the Sham group (both P<0.05; Fig. 2). However, there was a significant decrease in the release of CK-MB and LDH in the MI/R+PTB group compared with the MI/R group (both P<0.05; Fig. 2). The release of CK-MB and LDH was significantly increased in the MI/R+PTB+LY group compared with the MI/R+PTB group (P<0.05; Fig. 2) indicating that PI3K was involved in the protective effect of PTB against MI/R injury.

Increased MPO activity in the myocardium indicates neutrophil infiltration. MPO activity in the MI/R group was significantly increased compared with the Sham group (P<0.05; Fig. 3A). However, PTB significantly decreased myocardial MPO activity in the MI/R+PTB group compared with the MI/R group and LY treatment reversed the effect of PTB (both P<0.05; Fig. 3A). Levels of TNF-α, IL-6, and IL-8 in the plasma were all significantly increased following MI/R injury compared with the Sham group (all P<0.05; Fig. 3B-D). Treatment with PTB significantly decreased cytokine levels in the serum (all P<0.05; Fig. 3B-D) and these effects were reversed following LY treatment (all P<0.05; Fig. 3B-D).

TUNEL staining was performed to evaluate apoptosis in the rat hearts (Fig. 4). The apoptotic index of the MI/R+PTB group was significantly decreased compared with the MI/R group (P<0.05; Fig. 4). However, treatment with LY again significantly reversed the protective effects of PTB (P<0.05; Fig. 4), indicating that PI3K activation is involved in the mechanism of action of PTB against MI/R-induced myocardial apoptosis.

MI/R injury in the MI/R group significantly increased the phosphorylation of Akt compared with the Sham group (P<0.05; Fig. 5A and B). The phosphorylation of Akt was further increased following treatment with PTB (P<0.05, Fig. 5A and B). However, treatment with LY reversed the increase in Akt phosphorylation that was induced by PTB (P<0.05; Fig. 5A and B). In addition, MI/R injury significantly decreased Bcl-2 expression in the MI/R group compared with the Sham group (P<0.05; Fig. 5C). Treatment with PTB significantly increased the expression of Bcl-2 (P<0.05), which was subsequently decreased by co-administration with LY (both P<0.05; Fig. 5C). By contrast, MI/R injury induced a significant increase in Bax expression compared with the Sham group (P<0.05; Fig. 5D). PTB administration significantly decreased the expression of Bax in the MI/R group compared with the MI/R+PTB group (P<0.05), which was significantly reversed by co-administration with LY (both P<0.05; Fig. 5D).