A Cell Counting kit-8 (CCK-8, cat no. ck04) was purchased from Dojindo Molecular Technologies, Inc., (Kumamoto, Japan). α-MG and CoCl₂ were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). Antibodies against apoptosis regulator Bcl-2 (rabbit anti-Bcl-2; cat no. 2870; 1:1,000), apoptosis regulator BAX (rabbit anti-Bax; cat no. 2772s; 1:1,000), rabbit anti-cleaved caspase-9 (cat no. 9507; 1:1,000), rabbit anti-cleaved caspase-3 (cat no. 9662; 1:1,000) and mouse anti-β-actin (cat. no. 3700) were purchased from Cell Signaling Technology, Inc., (Danvers, MA, USA). Annexin V/propidium iodide (PI) was purchased from BD Biosciences (Franklin Lakes, NJ, USA); and the ROS detection kit was purchased from Beyotime Institute of Biotechnology (Haimen, China).

H9C2 myocardial cells were purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). Briefly, the H9C2 cells were cultured in high-glucose Dulbecco's modified Eagle's medium (Thermo Fisher Scientific, Inc., Waltham, MA, USA) containing 10% fetal bovine serum (Invitrogen; Thermo Fisher Scientific, Inc.) in 6-well plates with 5% CO₂ and 95% air at 37°C. The medium was changed every 2–3 days.

To determine the appropriate treatment conditions, chemical hypoxia was achieved by adding different concentrations of CoCl₂ (50, 200, 400, 600 and 800 µM) to H9C2 cells for 24 h as the preliminary experiment (32). The proper concentration was determined by the CCK-8 assay. In order to investigate the effects of α-MG following CoCl₂-induced cell injury, CoCl₂-treated H9C2 cells were maintained in complete medium with 0.012, 0.06, 0.3, 0.6 or 1.2 mM α-MG. The control cells were incubated without α-MG or CoCl₂.

According to the results of the CCK-8 assay, H9C2 cells were randomly divided into the following groups: Group I (Control), cells treated without α-MG or CoCl₂; group II, cells treated with CoCl₂ alone; group III, cells pretreated with 0.06 mM α-MG for 24 h and with CoCl₂ over the next 24 h; and group IV, cells pretreated with 0.3 mM α-MG for 24 h and with CoCl₂ over the next 24 h.

CCK-8 was used to investigate the viability of H9C2 cells cultured in 96-well plates at a density of 5,000 cells/well. When the cells had grown to 90% confluence, 600 µM CoCl₂ was added into the plate treated for 24 h at 37°C, then cell injury was induced. Thereafter, 10 µl CCK-8 solution was added to each well and the cells were incubated for a further 2 h at 37°C. Absorbance was measured at 450 nm with a microplate reader. The mean optical density (OD) of four wells in each group was used to calculate cell viability as follows: Cell viability (%)=(OD_{treatment group}/OD_{control group}) ×100.

The dichlorofluorescein diacetate (DCFH-DA) method was used to detect the level of intracellular ROS (33). Intracellular ROS levels were determined by measuring the oxidative conversion of cell permeable 2′,7′-DCFH-DA to fluorescent dichlorofluorescein (DCF) in a BioTek Synergy 2 microplate reader (BioTek Instruments, Inc., Winooski, VT, USA). H9C2 cells were seeded (5×10⁵ cells/well), α-MG (0.06 or 0.3 mM) was added to the plates for 24 h and then the CoCl₂-induced cell injury protocol was performed. The cells were incubated as previously described. The cells were washed with D-Hank's (Thermo Fisher Scientific, Inc.) and incubated with DCFH-DA at 37°C for 20 min. The DCF fluorescence distribution of 2×10⁴ cells was detected by a microplate system at an excitation wavelength of 488 nm and an emission wavelength of 535 nm. The MDA concentration was measured by Lipid Peroxidation MDA Assay kit (Beyotime Institute of Biotechnology; cat no. S0131) and SOD activity was measured using the Superoxide Dismutase Assay kit (Beyotime Institute of Biotechnology; cat no. S0101), according to the manufacturer's protocols.

Apoptosis in H9C2 cells was quantified using Annexin V-FITC and PI double staining according to standard procedures using the aforementioned kit. Cell apoptosis was analyzed using CellQuest Pro software version 5.1 (BD Biosciences). Cellular fluorescence was measured by flow cytometer (FACS Calibur^™, BD Biosciences).

Total RNA was extracted by TRIzol^™ (Invitrogen; Thermo Fisher Scientific, Inc.). A total of 100 ng total RNA was used for cDNA synthesis using the Stratagene AffinityScript qPCR cDNA Synthesis kit (Agilent Technologies, Inc., Santa Clara, CA, USA). The cDNA samples were diluted 10-fold with nuclease-free H₂O and 2 µl diluted template was combined with Brilliant III Ultra-Fast SYBR^® Green qPCR Master Mix (Applied Biosystems; Thermo Fisher Scientific, Inc.). β-actin was used as an internal reference control. The relative expression of target genes was determined by the 2^−∆∆Cq method (34). The qPCR cycling conditions comprised initial denaturation for 3 min at 95°C, followed by 45 cycles at 95°C (10 sec) and 58°C (45 sec); data were acquired at the end of the annealing/extension phase. Melt curve analysis was performed at the end of each run between 58 and 95°C. The gene primer sequences are exhibited in Table I. Data were analyzed using Microsoft Excel 2013 (Microsoft Corporation, Redmond, WA, USA).

Total proteins were extracted from cells using cell lysis buffer [50 mM Tris-HCl, (pH 8.0) 120 mM NaCl; 0.5% NP-40; 1 mM phenylmethylsulfonyl fluoride] and determined using bicinchoninic acid assay. A total of 40 µg protein extract was separated by 12% SDS-PAGE, followed by transfer to a polyvinylidene difluoride membrane (Bio-Rad Laboratories, Inc.), blocking with 5% non-fat milk in TBS-Tween buffer 7 (0.12 M Tris-base, 1.5 M NaCl, 0.1% Tween-20) for 1 h at room temperature, and incubation with the appropriate antibodies including rabbit anti-Bcl-2 (cat no. 2870; 1:1,000), rabbit anti-Bax (cat no. 2772s; 1:1,000), rabbit anti-cleaved caspase-9 (cat no. 9507; 1:1,000), rabbit anti-cleaved caspase-3 (cat no. 9662; 1:1,000) and mouse anti-actin (cat no. 3700) were purchased from Cell Signaling Technology, Inc., (Danvers, MA, USA), overnight at 4°C. Subsequently, the blots were incubated with horseradish peroxidase-conjugated anti-rabbit secondary antibody (1:10,000; cat no. 111-035-003; Jackson Immunoresearch Laboratories, Inc., West Grove, PA, USA) for 30 min at room temperature. The bound antibodies were detected using a chemiluminescence (ECL) system and autoradiography (cat. no. PI32209; Pierce; Thermo Fisher Scientific, Inc.). Quantity One analysis software version 4.6.9 (Bio-Rad Laboratories, Inc., Hercules, CA, USA) was used to quantify the relative band intensities from western blot images.

The activity of caspase-3 and caspase-9 was determined using a Caspase-3 Activity Assay kit and a Caspase-9 Activity Assay kit (Beyotime Institute of Biotechnology). This assay is based on the detection of the chromophore p-nitroaniline (pNA) by spectrophotometry following cleavage of pNA from the substrate by caspase-3/caspase-9. Assays were performed in 96-well plates (5×10⁴ cells/well), with each well containing 10 µl protein cell lysate, 80 µl reaction buffer and 10 µl substrate (Asp-Glu-Val-Asp-pNA for caspase-3 and Leu-Glu-His-Asp-pNA for caspase-9). The results were quantified spectrophotometrically using a BioTek Synergy 2 microplate reader (BioTek Instruments, Inc.) at a wavelength of 405 nm. Caspase activity was presented as a percentage relative to the control group.

The results are reported as the mean ± standard error of the mean for at least three analyses for each sample. Quantitative variables were compared using one-way analysis of variance to compare differences in two or more groups, and the Tukey test was performed for post-hoc subgroup analysis, as appropriate. Statistical analysis was performed using the SPSS 19.0 software package (IBM Corp., Armonk, NY, USA) and GraphPad Prism 5.0 (GraphPad Software, Inc., La Jolla, CA, USA). P<0.05 was considered to indicate a statistically significant difference.

As exhibited in Fig. 1, H9C2 cells treated with CoCl₂ (≥600 µM) for 24 h exhibited significantly reduced cell viability (P<0.001). The choice of 600 µM for the concentration of CoCl₂ applied to all experiments was based on these results from the CCK-8 assay. However, in H9C2 cells pretreated with various concentrations of α-MG, CoCl₂-induced hypoxic injury was significantly reduced (P<0.05; Fig. 2). Cells pretreated with α-MG at concentrations between 0 and 12 mM presented as an ‘inverted U shape’ in the present study: At concentrations of 0.012–0.06 mM, cell viability increased gradually (P<0.05), reaching a maximum at 0.06 mM; at concentrations of 0.06–1.2 mM the curve exhibited a steep decline, indicating a decrease in cell viability with increasing concentrations, although cell viability remained higher compared with the group treated with CoCl₂ alone (P<0.05). Among the α-MG pretreatment groups, all exhibited significant differences compared with the 0.06 mM group (P<0.01), apart from the 0.3 mM group (P>0.05).

The involvement of ROS in the CoCl₂-induced apoptosis of H9C2 cells was evaluated by measuring the level of ROS production. Following treatment of H9C2 cells with 600 µM CoCl₂ for 24 h, the intracellular ROS level increased significantly compared with that in the control group (P<0.001). However, treatment with α-MG decreased the intracellular ROS levels significantly (P<0.001), further demonstrating its antioxidant effects (Fig. 3A). As exhibited in Fig. 3B and C, H9C2 cells treated with CoCl₂ exhibited significantly increased MDA concentrations and reduced SOD concentration (P<0.01 and P<0.001, respectively). The oxidative abnormalities were ameliorated by α-MG at a concentration of 0.06 mM, as demonstrated by the significant reduction in MDA concentration and the increase in SOD activity (P<0.05).

To further investigate the functional effect of α-MG in H9C2 cells treated with CoCl₂, cellular apoptosis in H9C2 cells was assessed using the Annexin V-FITC/PI method with flow cytometry. The results demonstrated that CoCl₂ increased the percentage of apoptotic H9C2 cells to ~60%, which was attenuated by treatment with 0.06 mM α-MG (reduced to ~5.8%) and 0.3 mM α-MG (P<0.001; reduced to ~15.34%), indicating the cytoprotective effect of α-MG against chemical hypoxia-induced apoptosis (Fig. 4).

The results of the RT-qPCR (Fig. 5) revealed that treatment with CoCl₂ significantly decreased the expression level of Bcl-2 to 64.9% and increased the expression levels of Bax, caspase-9 and caspase-3 to 194, 138 and 193% relative to the control group, respectively (P<0.001 vs. control group). However, pretreatment with α-MG at a concentration of 0.06 mM increased the expression level of Bcl-2 to 81.4% and attenuated the expression levels of Bax, caspase-9 and caspase-3 to 110, 120 and 133% relative to the control group, respectively (P<0.001 vs. CoCl₂-induced injury group), while pretreatment with α-MG at a concentration of 0.3 mM increased the expression level of Bcl-2 to 70.8% and attenuated the expression levels of Bax, caspase-9 and caspase-3 to 142, 126 and 182% relative to the control group, respectively (P<0.05 vs. CoCl₂-induced injury group).

As depicted in Fig. 6, protein expression was analyzed by western blotting. Treatment with CoCl₂ significantly downregulated the expression level of Bcl-2 to 49.6% and upregulated the levels of Bax, caspase-9 and caspase-3 to 285, 1,111 and 307% relative to the control group, respectively (P<0.001). Pretreatment with α-MG at a concentration of 0.06 mM upregulated the expression level of Bcl-2 to 87.6% and downregulated the expression levels of Bax, caspase-9 and caspase-3 to 138, 474 and 157%, respectively (P<0.01), while pretreatment with α-MG at a concentration of 0.3 mM downregulated the expression levels of Bax, caspase-9 and caspase-3 to 165, 650 and 183%, respectively (P<0.01), while there was not significant difference with Bcl-2.

The activation of caspase-3 and caspase-9 has important influences on apoptosis. When compared with the control group (Fig. 7), treatment with CoCl₂ significantly increased the activity of caspase-3 and caspase-9 to 270 and 906%, respectively (P<0.001). When pretreated with α-MG (0.06 and 0.3 mM), the activities of caspase-3 (140 and 167%, respectively) and caspase-9 (447 and 573%, respectively) were significantly attenuated (P<0.001 vs. CoCl₂-induced injury group).