H9c2 rat cardiomyocyte cell line was obtained from the American Type Culture Collection (Manassas, VA, USA.). Cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) with 10% (v/v) heat inactivated fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA), 100 U/ml penicillin and 100 µg/ml streptomycin (Sigma-Aldrich; Merck KGaA) at 37°C in a humidified incubator with 5% CO₂.

For treatment, H9c2 cells at a density of 1×10⁴ cells/well were pretreated with various concentrations of SA (0.1, 1 and 10 µM; Sigma-Aldrich; Merck KGaA) for 24 h. Then, the cultures were introduced into a humidified N₂ hypoxic chamber (2% O₂) at 37°C for 6 h and then reoxygenated 5 h at 37°C in 5% CO₂ (95% O₂). Normoxic control cells were incubated at 37°C in 5% CO₂.

Cell viability was detected using a Cell Counting kit-8 (CCK-8) assay. In brief, following treatment, the medium was removed and replaced with fresh DMEM (100 µl/well). Then, 10 µl CCK-8 solution (Dojindo Molecular Technologies, Inc., Kumamoto, Japan) was added to each well, and the microplates were incubated at 37°C for 2 h. The absorbance was measured at 490 nm using a microplate reader (Benchmark; Bio-Rad Laboratories, Inc., Hercules, CA, USA).

Cell cytotoxicity was measured by a lactate dehydrogenase (LDH) assay using the Cytotoxicity Detection kit (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocols. Briefly, H9c2 cells were seeded in 96-well plates at a density of 1×104 cells/well. Following treatment, the medium was collected to measure the LDH activity according to the manufacturer's protocol. The colorimetric compound was measured at 530 nm using a microplate reader (Benchmark; Bio-Rad Laboratories, Inc.).

H9c2 cells were seeded in 96-well plates at a density of 1×104 cells/well. Following treatment, the activity of SOD in media was analyzed using a SOD kit from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). The MDA level was measured with an MDA kit (Beyotime Institute of Biotechnology, Jiangsu, China). For colorimetric analysis, the absorbance at 532 nm was recorded using a microplate reader (Spectra Max 190; Molecular Devices, LLC, Sunnyvale, CA, USA).

The proteins were extracted from H9c2 cells using radioimmunoprecipitation lysis buffer (Beyotime Institute of Biotechnology) and the protein concentration was determined by a BCA protein assay kit (Invitrogen; Thermo Fisher Scientific, Inc.). A total of 30 µg protein was separated by 10% SDS-PAGE electrophoresis followed by electroblotting onto a nitrocellulose membrane (GE Healthcare Life Sciences, Little Chalfont, UK). Following blocking with 5% nonfat dry milk in PBS for 4 h, the membrane was incubated overnight at 4°C with primary rabbit anti-mouse antibodies (dilution, 1:1,000) targeting B-cell lymphoma 2 (Bcl-2; cat. no. sc-783), Bcl-2-like protein 4 (Bax; cat. no. sc-6236), cleaved caspase-3 (cat. no. sc-98785), phospho (p)-p38MAPK (cat. no. sc-101759), total p38MAPK (cat. no. sc-535), p-JNK (cat. no. sc-135642), total JNK (cat. no. sc-572) and GAPDH (cat. no. sc-25778; all from Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). Subsequently, the membrane was washed and incubated with goat anti-rabbit peroxidase-conjugated immunoglobulin G (cat. no. sc-516087; Santa Cruz Biotechnology, Inc.) diluted 1:3,000 in the blocking buffer for 1 h. Then the blot was washed in TBST buffer (TBS containing 0.1% Tween-20) and positive bands were visualized using enhanced chemiluminescence reagents (Bio-Rad Laboratories, Inc.). Densitometry was performed using Gel-Pro Analyzer software version 4.0 (Media Cybernetics, Inc., Rockville, MD, USA).

Data are presented as the mean ± standard deviation. Statistical analyses were performed using SPSS software version 13.0 (SPSS, Inc., Chicago, IL, USA). Statistical analysis was carried out using a one-way analysis of variance followed by a Bonferroni post hoc test for multiple comparisons. P<0.05 was considered to indicate a statistically significant difference.

The protective effects of SA on H9c2 cell cytotoxicity caused by H/R were investigated. The results of the CCK-8 assay demonstrated that, compared with the normoxia group, cell viability was significantly reduced following H/R treatment. Pretreatment with SA for 24 h before H9c2 cells suffered with 6/5 h H/R injury markedly inhibited H9c2 cell injury, compared with the H/R group (Fig. 1A).

It was further analyzed whether SA pretreatment influences the cell death of H9c2 cells. As demonstrated in Fig. 1B, H9c2 cells subjected to 6/5 h H/R injury caused an marked increase of LDH release. By contrast, pretreatment of cells with SA for 24 h downregulated LDH release compared with the H/R group.

Oxidative stress is widely reported to serve a critical role in mediating myocardial I/R injury and thus the effect of SA on oxidant stress in H9c2 cells in response to H/R injury was investigated. As demonstrated in Fig. 2A, H/R treatment significantly decreased SOD level in H9c2 cells compared with the normoxia group and this parameter was significantly increased by SA pretreatment. By contrast, pretreatment with SA significantly reduced MDA levels induced by H/R in H9c2 cells (Fig. 2B).

Oxidative stress is one of the major stimuli involved in the myocardial apoptosis observed during I/R. Thus, the effect of SA on H9c2 cell apoptosis in response to H/R injury was investigated. As demonstrated in Fig. 3, H9c2 cells exposed to 6/5 h H/R injury caused a marked increase of Bax expression. By contrast, pretreatment of cells with SA for 24 h markedly decreased the H/R-induced Bax expression. SA pretreatment significantly increased the expression of Bcl-2 in H9c2 cells and significantly decreased the level of cleaved caspase-3 following H/R.

Since p38MAPK and JNK are involved in myocardial injury caused by I/R, western blot analysis was performed to investigate whether SA inhibited the activation of p38MAPK and JNK in H9c2 cells. As demonstrated in Fig. 4, H/R treatment significantly induced the phosphorylation of p38MAPK and JNK, compared with the normoxia group. However, SA significantly alleviated H/R-induced phosphorylation of p38MAPK and JNK in H9c2 cells.