H9c2 rat cardiomyocyte cells were purchased from The Cell Bank of Type Culture Collection of the Chinese Academy of Sciences and cultured in high-glucose DMEM (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.), 100 U/ml penicillin and 100 U/ml streptomycin at 37°C in a humidified atmosphere containing 5% CO₂. The cells were passaged every 4 days and the culture medium was replaced every 2 days.

H9c2 cells (5×10³ cells per well) were seeded in 96-well plates and incubated at 37°C in a 5% CO₂ humidified incubator overnight. Subsequently, the cells were exposed to different H₂O₂ concentrations (200, 400, 600 and 800 µM) and harvested at different time points (4, 8 and 12 h). The appropriate concentration and exposure time of H₂O₂ used for the establishment of the oxidative stress model in H9c2 cells was determined by the Cell Counting Κit-8 (CCK-8) assay.

GB was purchased from Sigma-Aldrich; Merck KGaA. The stock solution was prepared by dissolving GB in DMSO at 100 mM. The working solution of GB was obtained by diluting the stock solution in DMEM to the desired concentrations. To avoid the DMSO-induced cytotoxicity, the final concentration of DMSO was retained to <1%. H9c2 cells were pretreated with GB for 20 h prior to co-incubation with H₂O₂. The total time of GB-pretreatment group is the same as single GB-treated group. Prior to cell pretreatment with GB at 0.01, 0.1, 1, 10 and 100 µM for 1 h, the cells were treated with PI3K inhibitor LY294002 (Selleck Chemicals) at concentrations of 5, 10, 20 and 40 µM.

Cell viability was determined by the CCK-8 assay (Dojindo Molecular Technologies, Inc.), according to the manufacturer's protocol. H9c2 cells (5×10³ cells per well) were seeded in 96-well plates overnight and were pretreated with different concentrations of LY294002, GB and H₂O₂. To measure cell viability, 10 µl CCK-8 assay solution was added into each well, containing 90 µl medium, and the cells were then incubated in the dark at 37°C for additional 2 h. Absorbance was measured at 450 nm in a microplate reader (BioTek Instruments, Inc.).

Typical morphological features of apoptotic cells were observed by Hoechst 33342 staining (Nanjing KeyGen Biotech Co., Ltd.). Briefly, the cells were washed twice with PBS following H₂O₂ incubation with or without GB pretreatment and stained with Hoechst 33342 for 15 min in a 5% CO₂ incubator at 37°C in the dark. Finally, the cells were washed once with PBS and the images were captured by fluorescence microscopy (magnification, ×200; Zeiss Axiovert A1). The apoptotic cells with nuclear chromatin condensation and fragmentation were stained bright blue. A total of three fluorescence images per well were captured randomly, and the percentage of apoptotic cells was calculated using the following equation: (The number of apoptotic cells/the total number of cells) ×100.

The induction of apoptosis was examined using the Annexin V-FITC/PI double staining assay (BD Biosciences; Becton-Dickinson and Company). Briefly, H9c2 cells were gently washed with ice-cold PBS, digested with 0.25% Trypsin without ethylenediaminetetraacetic acid (Gibco; Thermo Fisher Scientific, Inc.) and resuspended in complete culture medium. The cells were then centrifuged at 120 × g for 5 min at 4°C and the cell pellet was resuspended in ice-cold PBS and centrifuged at the same conditions. Finally, the cells were resuspended in 500 µl 1X binding buffer supplemented with 5 µl Annexin V-FITC and 5 µl PI. The cells were incubated at room temperature for 15 min in the dark and were analyzed by flow cytometry (BD FACSCalibur™; BD Biosciences; Becton-Dickinson and Company). Data was analyzed using FlowJo version 7.6.1 software (FlowJo LLC).

H9c2 cells were washed with ice-cold PBS, pelleted by cell scraper and lysed in 100 µl lysis buffer containing 1 µl phosphatase inhibitor, 0.1 µl protease inhibitor and 0.5 µl PMSF. The extracted proteins were quantified with the bicinchoninic acid Protein assay kit (Thermo Fisher Scientific, Inc.). A total of 20 µg protein extracts per lane were separated by 6–15% SDS-PAGE. The proteins were transferred onto polyvinylidene difluoride membranes or nitrocellulose filter membrane. The membranes were blocked with 5% BSA (Gibco; Thermo Fisher Scientific, Inc.) at room temperature for 2 h and incubated overnight at 4°C with primary antibodies against rabbit anti-Bax (1:1,000; cat. no. 2772), rabbit anti-Bcl-2 (1:1,000; cat. no. 2870), rabbit anti-cleaved caspase-3 (1:1,000; cat. no. 9664), rabbit anti-caspase-3 (1:1,000; cat. no. 9662), rabbit anti-Akt (1:1,000; cat. no. 9272), rabbit anti-phosphorylated (p)-Akt (p-Akt; 1:1,000; cat. no. 4060), rabbit anti-mTOR (1:1,000; cat. no. 2983), rabbit anti-p-mTOR (p-mTOR; 1:1,000; cat. no. 5536), and rabbit anti-GAPDH (1:1,000; cat. no. 5174; all from Cell Signaling Technology, Inc.). The membranes were subsequently washed for 2 h with TBST (10 mM Tris, pH 7.5, 150 mM NaCl, and 0.05% Tween-20), and incubated with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (1:500; cat. no. sc-2004; Santa Cruz Biotechnology, Inc.) for 1 h at room temperature. The antigen-antibody complexes on the membranes were detected using the SuperSignal™ West Femto Maximum Sensitivity substrate (Thermo Fisher Scientific, Inc.) and quantified on the ChemiDoc™ XRS Imaging system (Bio-Rad Laboratories, Inc.).

All experiments were repeated three times. The data are presented as the mean ± SD. Statistical differences among groups were analyzed using a one-way ANOVA, followed by a Tukey's post-hoc test. All statistical analyses were performed using the GraphPad Prism 5 software (GraphPad Software, Inc.). P<0.05 or P<0.001 were considered to indicate a statistically significant difference.

H₂O₂ reduced cell viability in a dose- and time- dependent manner (Fig. 1A). For instance, treatment of H9c2 cells with 600 µM H₂O₂ for 4 h reduced cell viability to 63.11% compared with that noted in untreated cells. The H9c2 cell state in this condition was not too poor to conduct subsequent experiments. Therefore, 600 µΜ H₂O₂ was selected as the optimal treatment concentration for subsequent experiments. The effect of GB alone on cell viability was also investigated. Cell viability was not significantly affected by GB treatment for 24 h even at concentrations up to 100 µM (Fig. 1B). Subsequently, the effects of H9c2 cell-GB pretreatment in the protection of the cells against H₂O₂-induced cytotoxicity were examined. GB pretreatment at 10 µM concentration for 20 h prior to co-incubation with 600 µM H₂O₂ for 4 h revealed the highest inhibitory effect on the induction of cell cytotoxicity caused by treatment of 600 µM H₂O₂ for 4 h (Fig. 1C). Therefore, 10 µM GB was used in subsequent experiments.

Chromatin condensation and fragmentation are the typical features of apoptotic cells so Hoechst 33342 staining was used to evaluate induction of cell apoptosis caused by H₂O₂. Pretreatment of the cells with 10 µM GB for 20 h significantly decreased the percentage of apoptotic cells, compared with that noted following treatment of the cells with 600 µM H₂O₂ for 4 h (Fig. 2A and B). Furthermore, the Annexin V-FITC/PI assay was further used to evaluate the induction of cell apoptosis. Treatment of the cells with 600 µM H₂O₂ for 4 h led to a significant increase in cell apoptosis compared with that noted in the control group, whereas this effect was inhibited by GB pretreatment (Fig. 2C and D). The expression levels of the apoptotic proteins Bcl-2, Bax, caspase-3 and cleaved caspase-3, were determined by western blot analysis. Both Bax and cleaved caspase-3 protein levels were significantly elevated in the H₂O₂-treated group, whereas Bcl-2 levels were downregulated (Fig. 2E-H). In addition, GB pretreatment significantly decreased the expression levels of Bax and cleaved caspase-3 and increased the expression levels of Bcl-2 compared with those of the H₂O₂-treated group. Both the Bax/Bcl-2 and the cleaved caspase-3/caspase-3 ratios were significantly decreased in the GB-pretreatment group compared with those of the H₂O₂-treated group.

The expression levels of p-Akt and p-mTOR were downregulated in the H₂O₂-treated group, whereas GB pretreatment was able to reverse the effects of H₂O₂ (Fig. 3A and B).

To determine whether the activation of Akt and mTOR was associated with the protective effects of GB, the LY294002 inhibitor was used to investigate the expression levels of p-Akt and to assess cell viability. Pretreatment of the cells with LY294002 and GB downregulated the expression levels of p-Akt in a dose-dependent manner, compared with those noted in the single GB-pretreatment group. In addition, LY294002 and GB pretreatment reduced cell viability compared with that of the single GB-pretreatment group. These results indicated that GB elicited its protective effects via activation of the PI3K/Akt/mTOR signaling pathway (Fig. 4A and B).