Propofol, 3-methyladenine (3-MA), acridine orange (AO), monodansylcadaverine (MDC) and the c-Jun N-terminal kinase (JNK) inhibitor SP600125 were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany) and were dissolved in dimethyl sulfoxide (DMSO), and further diluted in PBS. The final DMSO concentration was 0.1%, which did not affect cell function and the assay system. Fetal bovine serum (FBS) and Dulbecco's modified Eagle's medium (DMEM) were purchased from Gibco (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Primary antibodies used in the study included: Rabbit anti-microtubule-associated protein 1A/1B-light chain 3B (LC3B; cat. no. L7543; Sigma-Aldrich; Merck KGaA) and rabbit anti-p62 (cat. no. P0067; Sigma-Aldrich; Merck KGaA); rabbit anti-stress-activated protein kinase (SAPK)/JNK (cat. no. 9252; Cell Signaling Technologies, Danvers, MA, USA) and rabbit anti-phosphorylated (p)-SAPK/JNK (cat. no. 4668; Cell Signaling Technologies) and mouse anti-β-actin (cat. no. sc130301; Santa Cruz Biotechnology, Dallas TX, USA). Secondary antibodies included horseradish peroxidase-conjugated goat anti-mouse (cat. no. sc2031; Santa Cruz Biotechnology) and goat anti-rabbit immunoglobulin G (cat. no. sc2007; Santa Cruz Biotechnology).

The H9c2 embryonal rat heart-derived (cardiac muscle) cell line was obtained from the American Type Culture Collection (Manassas, VA, USA) and cultured in DMEM containing 4.5 g/l D-glucose, 1.5 g/l sodium bicarbonate and 110 mg/l sodium pyruvate, supplemented with 10% FBS, penicillin (100 U/ml) and streptomycin (100 Ag/ml), in a humidified incubator with 95% air and 5% CO₂ at 37°C.

Hypoxia was established in Modular Incubator Chambers (Billups-Rothenberg, Inc., Del Mar, CA, USA); the chambers were flushed with a gas mixture of 95% N₂ and 5% CO₂ at room temperature for 30 min at 10 l/min. Following flushing, the chambers were sealed and maintained at 37°C for 6 h. The concentration of O₂ in each chamber was monitored with an oxygen indicator (Mitsubishi Gas Chemical Company, Inc., Tokyo, Japan). Hypoxia was terminated by exposing the cells to fresh DMEM containing 10% FBS and incubating them in a normal incubator (95% air; 5% CO₂) for 4 h at 37°C to simulate reperfusion (reoxygenation). Propofol of concentrations between 12.5–100 µmol/l was added into the fresh medium at the commencement of reperfusion/reoxygenation. SP600125 and 3-MA treatments were administered at a final concentration of 10 µmol/l and 10 mmol/l, respectively, according to previously published methods (17,18), for 1 h prior to hypoxia exposure. The cells were trypsinized with 0.25% trypsin at 37°C, harvested at the centrifugation at 800 × g for 3 min at room temperature and washed twice with PBS at the end of reoxygenation.

H9c2 cells were separated into the following experimental groups: i) Control group of normally cultured H9c2 cells; ii) H/R group, which were subjected to H/R; iii) P-PostC group, which received propofol treatment upon H/R; iv) P-PostC + SP600125 group, which received SP600125 and P-PostC co-treatment upon H/R (group); v) P-PostC+3-MA group, which were subjected to 3-MA and P-PostC co-treatment under H/R; vi) P-PostC + 3-MA + SP600125 group, which received 3-MA, SP600125 and P-PostC co-treatment upon H/R; vii) vehicle-treated group, which were H9c2 cells normally cultured with 0.1% DMSO in the culture medium; and viii) propofol-treated group, which comprised vehicle-treated cells subjected to propofol administration without H/R.

To measure the extent of cell injury, the LDH activity was analyzed. Cells were plated in a 6-well plate at a density of 5×10⁵ cells/well. At the end of the reoxygenation, the supernatants were collected. The LDH assay kit (A020-1; Jiancheng Bioengineering Institute, Nanjing, China) was employed according to the manufacturer's protocols. Briefly, 0.1 ml culture medium was added into 3 ml LDH assay reaction mixture. After 3 min incubation at 37°C, the optical density was measured at a wavelength of 440 nm using a spectrophotometer. H9c2 cell viability in the different treatment groups was analyzed by MTT assay. Cells were plated at 1×10⁴ cells/well in 96-well microtiter plates. Each group was repeated in 10 wells. Following treatment, MTT (5 mg/ml in PBS) was added to each well and incubated for 3 h at 37°C. After careful removal of the medium, the formazan crystals were dissolved in 200 µl DMSO and absorbance (A) values were determined at 570 nm using a microplate reader (Model 550; Bio-Rad Laboratories, Inc., Hercules, CA, USA) within 30 min following the dissolution of the formazan crystals.

Following treatment, myocytes were harvested with 0.25% trypsin and subjected to apoptosis analysis using an Annexin V-fluorescein isothiocyanate (FITC) detection kit (Beyotime Institute of Biotechnology, Haimen, China). Subsequently, the cell density was adjusted to 1×10⁶ cells/ml, 5 µl Annexin V-FITC and 10 µl propidium iodide (PI) (1 µg/ml) were then added to these cells and incubated for 10–20 min at room temperature (20–25°C) in the dark. The rate of apoptosis was then analyzed with a FACScalibur flow cytometer and calculated by CellQuest software (Becton Dickinson, Franklin Lakes, NJ, USA). Early apoptotic cells were positive for Annexin V-FITC and negative for PI.

H9c2 cells were seeded at a density of 1×10⁴ cells/well onto coverslips fixed in culture dishes and allowed to reach 70–80% confluence. MDC, a selective fluorescent marker preferentially accumulates in autophagic vacuoles, exhibits alterations in fluorescence that can be observed using a fluorescence microscope. Autophagy is characterized by increased formation of lysosomes and autophagolysosomes which can be stained with AO. Following H/R and P-PostC, cells were incubated with 0.05 mmol/l MDC or 1 mg/ml AO for 15 min, fixed with 3.7% paraformaldehyde in PBS for 30 min at 37°C, rinsed with PBS and dried. Cells were observed under an IX70 fluorescence microscope (Olympus Corporation, Tokyo, Japan) equipped with a filter system: MDC, excitation wavelength, 380 nm and emission filter, 525 nm; AO, excitation wavelength, 488 nm and emission filter, 525 nm. Five randomly selected fields were observed per slide. Data were obtained from ≥3 independent experiments.

H9c2 cells were plated at a density of 1×10⁶ cells/well in 6-well culture dishes. Following treatment, cells were washed with PBS and lysed on ice for 30 min in radioimmunoprecipitation assay buffer containing 50 mM Tris HCl (pH 7.5), 250 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM NaF, 1 mM PMSF, 1 mM DTT, 1 µg/ml leupeptin, 1 µg/ml aprotinin and 1 µg/ml pepstatin (Sigma-Aldrich, Merck KGaA). Cell lysates were then centrifuged at 13,000 × g for 10 min at 4°C, and protein concentrations were assayed using a Bicinchoninic Acid assay kit (Beyotime Institute of Biotechnology). Protein samples were separated by 12% SDS-PAGE and transferred to nitrocellulose membranes using an electro-blotting apparatus (Bio-Rad Laboratories, Inc.). The membrane was blocked with 5% nonfat milk in TBST solution, and incubated overnight with primary antibodies (anti-LC3B, 1:1,000; anti-p62, 1:1,000; anti-JNK, 1:1,000; anti-p-JNK, 1:1,000 and anti-β-actin, 1:500) in the blocking solution at 4°C. After three washes with TBST solution, the membrane was incubated at room temperature for 1 h, with horseradish peroxidase-conjugated secondary antibody diluted with TBST solution (1:3,000). The signals of detected proteins were visualized by an enhanced chemiluminescence reaction (ECL) system (Amersham, ECL kits). For semi-quantitative analysis, protein bands detected by ECL were scanned into Adobe Photoshop CS6 (Adobe Systems, Inc., San Jose, CA, USA) and analyzed using ImageJ software, version 1.6 (National Institutes of Health, Bethesda, MD, USA).

Data are reported as the mean ± standard deviation, and statistical significance was assessed using SPSS 18.0 software (SPSS, Inc., Chicago, IL, USA). Differences between groups were evaluated with analysis of variance (ANOVA) followed by a Bonferroni's post hoc test for multiple comparisons. Prior to ANOVA, Levene's test for equality of variances was performed; data were confirmed to be normally distributed. P<0.05 was considered to indicate a statistically significant difference.

To exclude possible interference by using animals, a heart-derived H9c2 cell line for in vitro observation was used in the present study. H9c2 cells have been extensively used in cardiological research, as they possess elements and properties of the signaling pathways of adult myocytes (19), thus offering a suitable in vitro experimental H/R model. To examine whether propofol treatment alone affected the viability of cultured cardiomyocytes, morphological observations and MTT cell viability assays were performed, following treatment with propofol for 4 and 12 h. Vehicle-treated cells (4 h) were considered to be 100% viable, the viability of vehicle-treated cells (12 h) was 150% compared with Vehicle-treated cells (4 h). No notable morphological alterations were observed in propofol-treated cells (Fig. 1A) and no significant differences in cell viability were detected (P>0.05; Fig. 1B) compared with the vehicle-treated group.

Cellular damage was assessed by measuring the activity levels of the cytosolic enzyme LDH released into the medium, induced by cell membrane leakage (20). Following H/R exposure, cells exhibited a significant increase in LDH activity in the culture medium, 1.9-fold of the level in the control group (Fig. 2A). This increase was significantly reduced by P-PostC treatments at various concentrations (P<0.01 vs. H/R; Fig. 2A). The cardioprotection of P-PostC against H/R injury was further examined by determining the rate of cardiomyocyte apoptosis. The percentage of early apoptotic cells in the untreated control and H/R groups were 4.72±1.3 and 12.45±1.6%, respectively (P<0.05). The proportion of Annexin V-FITC-stained cells in the 12.5–50 µmol/l P-PostC-treated groups decreased significantly and in a dose-dependent manner, compared with the H/R group (Fig. 2B) There was a marked reduction in the number of early apoptotic cells, ranging between 4.7±0.8 and 8.6±1.3%, with different concentrations (12.5–50 µmol/l) of propofol compared with H/R. This indicated that 12.5–50 µmol/l P-PostC conferred dose-dependent protection against H/R injury to cardiomyocytes. However, the percentage of early apoptotic cells in the 100 µmol/l P-PostC group was 8.5±1.1%, compared with in the 50 µmol/l P-PostC-treated group (4.7±0.8%, P<0.05); the optimal concentration of propofol for P-PostC was demonstrated to be 50 µmol/l. P-PostC (12.5–50 µmol/l) protected the H/R cardiomyocytes from apoptosis, whereas propofol concentrations >100 µmol/l exhibited less positive effects on cell apoptosis. These data were consistent with a previous study and may be due to the known inhibitory effect of calcium channels at high concentrations (21).

The fluorescent dye MDC is an autofluorescent marker that accumulates in autophagic vacuoles, and is used to detect autophagic vesicle formation (22). Autophagy was induced by incubating H9c2 cells for 6 h in hypoxic conditions followed by 4 h of reoxygenation. The level of autophagic vacuole formation was evaluated by staining the cells with MDC. The results demonstrated that autophagosome formation was enhanced under H/R conditions (Fig. 3A), and P-PostC (50 µmol/l) further induced autophagy compared with untreated control cells. Fluorescence microscopic analysis identified MDC-positive structures in the H/R and P-PostC groups, particularly in the P-PostC group, whereas MDC-labeled structures were almost undetectable in normal cells. In addition, the acidic vesicular organelles in cells were also stained with AO. Fluorescence microscopic analysis demonstrated that the H/R and P-PostC treated cells exhibited abundant cytoplasmic acidic vesicular organelle formation, compared with the Control group (Fig. 3B).

The induction of autophagy was confirmed by detecting increased protein expression of LC3-II and the enhanced ratio of LC3-II/LC3-I expression. LC3-II expression was enhanced in H9c2 cells in the H/R group (Fig. 3C), and preconditioning with different concentrations of propofol (12.5–100 µmol/l) significantly increased the expression of LC3-II in a dose-dependent manner, compared with expression in the H/R group (Fig. 3C). The levels of p62 expression in H9c2 cells were reduced following H/R exposure and decreased in response to P-PostC. Accompanied by increased LC3 lipidation, the steady-state levels of p62 were reduced following H/R exposure and P-PostC, which confirmed the autophagy within H9c2 cells (Fig. 3D).

To study the role of the SAPK/JNK signaling pathway in P-PostC-induced autophagy, the activation of JNK in the H/R and H/R + P-PostC groups was detected by western blotting (Fig. 4). H/R exposure slightly increased JNK phosphorylation compared with in control; Fig. 4, and P-PostC co-treatment further enhanced the phosphorylation of JNK (P<0.05 vs. H/R; Fig. 4).

As presented in Fig. 5, H/R induced the apoptosis of cardiomyocytes compared with in the control group, while P-PostC (50 µmol/l) markedly reduced the percentage of apoptosis. To investigate the association between the apoptosis and autophagy induced in the myocardiocytes, cells of the P-PostC+3-MA group were pre-treated with the autophagy inhibitor 3-MA (10 mmol/1) for 1 h prior to hypoxia, and then treated with H/R and P-PostC (50 µmol/l). The results revealed that pre-treatment with 3-MA markedly increased the percentage of apoptotic cells compared with the P-PostC (50 µmol/l) group (P<0.01 vs. P-PostC 50 µmol/l; Fig. 5). As autophagy was inhibited in the 3-MA-treated cardiomyocytes, the cardioprotection of P-PostC (50 µmol/l) was also eliminated.

To further investigate the role of JNK in P-PostC-mediated autophagy, cells were pretreated with 10 µmol/l SP600125, a JNK activity-specific inhibitor, for 1 h, followed by exposure to H/R or P-PostC (50 µmol/l) treatment. SP600125 significantly eliminated the phosphorylation of JNK induced by P-PostC (50 µmol/l) on H/R cells (Fig. 6A). In addition, SP600125 could reverse the autophagy-induced protective effects of P-PostC (50 µmol/l). Compared with P-PostC treatment, pretreatment with SP600125 inhibited the increase of LC3-II induced by P-PostC (50 µmol/l; Fig. 6B) and had similar effects as the 3-MA pretreatment (Fig. 6B). These results indicated that the JNK-specific inhibitor could block autophagy induced by P-PostC in H9c2 cardiac cells.