A H9c2 rat cardiomyocyte cell line was purchased from American Type Culture Collection (cat. no. CRL-1446) and cultured in Dulbecco’s modified Eagle’s medium (DMEM; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific, Inc.), 2 mM glutamine, 100 U/ml penicillin and 100 mg/ml streptomycin (all from Merck KGaA) at 37°C in a humidified incubator containing 5% CO₂.

The small interfering RNA (siRNA) duplexes corresponding to rat PGC-1α (siPGC-1α), Nrf2 (siNrf2) and the negative control siRNA (siNC) were purchased from Santa Cruz Biotechnology, Inc. siRNAs (100 nM) were transfected into H9c2 cells using Lipofectamine^® 2000 reagent (Thermo Fisher Scientific, Inc.), according to the manufacturer’s instructions. In brief, H9c2 cells (5×10⁵) were plated in a 6-well plate and cultured for 24 h. When the cells reached 80% confluence, Lipofectamine 2000 was added to the medium without serum and incubated for 5 min at room temperature. The diluted siRNA was gently mixed with the medium containing Lipofectamine 2000 and incubated for 20 min at room temperature prior to adding the mixture to each well containing cells and medium. The transfected cells were cultured at 37°C in a CO₂ incubator for 24 h prior to subsequent experiments. Untransfected cells were used as a blank control, and cells transfected with siNC were used as a negative control.

H9c2 cells were transfected with siPGC-1α, siNrf2 or siNC mimic 24 h prior to OGD stimulation. For the OGD/R experiments, when the transfected H9c2 cells reached 70–80% confluence, the culture medium was replaced by glucose-free DMEM (Thermo Fisher Scientific, Inc.) and the cells were cultured in an anaerobic chamber (95% N₂ and 5% CO₂) at 37°C for 4 h. The cells were then incubated with normal culture medium under normoxic conditions (95% air and 5% CO₂) at 37°C for the indicated duration (6, 12, 24 and 48 h) as reperfusion. Different concentrations of Mel (0.01, 0.1, 1 and 10 mM; Merck KGaA) or 1 μg/ml TNF-α antibody (cat. no. 11948; Cell Signaling Technology, Inc.) was added into the culture medium at the initiation of reperfusion. Untreated cells were used as the control.

Cell viability was measured by Cell Counting Kit-8 (CCK-8; Beyotime Institute of Biotechnology) according to the manufacturer’s instructions. In brief, H9c2 cells were seeded into 96-well plates at a density of 1×10⁴ cells/well. Following treatment, 10 μl CCK-8 solution was added to each well and incubated for 2 h at 37°C. The absorbance was measured at 450 nm using a microplate reader (Bio-Rad Laboratories, Inc.) to calculate the number of viable cells.

The levels of LDH and CK-MB in the supernatant of H9c2 cells were assessed using commercial kits (Nanjing Jiancheng Bioengineering Institute) according to the manufacturer’s instructions. Briefly, H9c2 cells were subjected to 4 h of OGD followed by reperfusion for 24 h. Different concentrations of Mel (0.01, 0.1, 1 and 10 mM) were added to the culture medium at the initiation of reperfusion and incubated for the indicated durations (6, 12, 24 and 48 h). The cells were harvested and centrifuged at 250 × g at room temperature for 2 min. A total of 100 μl working solution was added to each well and reacted for 30 min at room temperature. The reaction was stopped by adding 50 μl stop solution to each well. Absorbance was measured at 450 and 340 nm using a microplate reader (Bio-Rad Laboratories, Inc.).

Apoptosis was measured using an Annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI) detection kit (BD Biosciences). Briefly, following the indicated treatments, H9c2 cells were harvested, washed thrice with cold PBS and resuspended in binding buffer (BD Biosciences), followed by staining with 5 μl Annexin V-FITC in the dark for 10 min at 37°C. The cells were then incubated with 10 μl PI solution in the dark for 30 min. The apoptotic cells were determined by flow cytometry (BD FACScalibur; BD Biosciences) and analyzed using CellQuest software (BD Biosciences).

5,5′, 6,6′-Tetracholoro-1,1′3,3′-tetraethybenzimidazole-carbocyanide iodine (JC-1; Beyotime Institute of Biotechnology) was used to measure the MMP of H9c2 cells according to the manufacturer’s instructions. Briefly, following the indicated treatments, H9c2 cells were harvested and resuspended in 1 ml culture medium (DMEM + FBS) with JC-1 fluorescent dye. Following incubation at 37°C for 20 min, the cells were centrifuged at 600 × g at 4°C for 3 min, and the supernatant was removed. The cells were resuspended in 1X JC-1 buffer for observation under a fluorescent microscope at ×100 magnification (Carl Zeiss AG). In each sample, five random fields were selected for observation and analysis.

The activity of caspase-3 and caspase-9 in H9c2 cells subjected to different treatments was measured by Caspase Activity kits (Beyotime Institute of Biotechnology) using the substrate peptides acetyl-Asp-Glu-Val-Asp p-nitroanilide (Ac-DEVD-pNA) and acetyl-Leu-Glu-His-Asp p-nitroanilide (Ac-LEHD-pNA), respectively. In brief, following treatment, the cells were lysed using RIPA lysis buffer (Beyotime Institute of Biotechnology), and the supernatants were mixed with buffer (Beyotime Institute of Biotechnology) containing the substrate peptides and incubated at 37°C for 2 h. The release of pNA was quantified by determining the absorbance at 405 nm using a microplate reader (Bio-Rad Laboratories, Inc.). The caspase activity was expressed as a relative percentage of the control value.

Intracellular ROS levels were monitored using 2′,7′-dichlorofluorescein diacetate (DCFH-DA; Thermo Fisher Scientific, Inc.) according to the manufacturer’s instructions. In brief, following the indicated treatments, H9c2 cells were loaded with 10 μM DCFH-DA at 37°C for 30 min and washed with PBS thrice. Absorbance was measured at 485 and 525 nm using a fluorescent microplate reader (Carl Zeiss AG) to determine the intensity of DCF fluorescence.

The MDA content and SOD, CAT and GSH-Px activity levels were measured using commercial kits (Nanjing Jiancheng Bioengineering Institute) according to the manufacturers’ instructions. In brief, following the indicated treatments, H9c2 cells were lysed using RIPA lysis buffer (Beyotime Institute of Biotechnology) and the supernatants were collected to determine MDA content and SOD, CAT and GSH-Px activity levels. The absorbance was measured at 530 (MDA), 450 (SOD), 405 (CAT) and 412 (GSH-Px) nm using a microplate reader (Bio-Rad Laboratories, Inc.).

Following treatment, H9c2 cells were washed twice with PBS, lysed in RIPA buffer (Beyotime Institute of Biotechnology) and centrifuged at 1,000 × g at 4°C for 3 min. The supernatants were collected and quantified for protein concentration using a BCA kit (Beyotime Institute of Biotechnology). Equal amounts (30 μg/lane) of protein from each sample were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes (EMD Millipore). The membranes were blocked with 5% non-fat milk in TBST buffer (50 mM Tris, pH 7.4, 250 mM NaCl, 0.1% Tween-20) for 1 h at room temperature and probed with antibodies against PGC-1α (1:1,000; cat. no. ab54481), Nrf2 (1:2,000; cat. no. ab137550), HO-1 (1:1,000; cat. no. ab189491) and NQO1 (1:1,000; cat. no. ab97385; all from Abcam), caspase-3 (1:1,000; cat. no. 14220), cleaved (cl)-caspase-3 (1:1,000; cat. no. 9664), cytochrome c (1:1,000; cat. no. 11940), Bax (1:1,000; cat. no. 5023), Bcl-2 (1:1,000; cat. no. 3498), TNF-α (1:1,000; cat. no. 6945) and β-actin (1:1,000; cat. no. 4967; all from Cell Signaling Technology, Inc.) overnight at 4°C. Following washing with TBST thrice, the membranes were incubated with horseradish peroxidase-conjugated anti-rabbit IgG (1:2,000; cat. no. 7074; Cell Signaling Technology, Inc.) for 1 h at room temperature and visualized by enhanced chemiluminescence reaction reagents (EMD Millipore). Protein band densities were quantified using Quantity One software (Bio-Rad Laboratories, Inc.). The results were normalized to the internal control β-actin and calculated as fold-changes compared with the control group in each experiment.

Total RNA was extracted from H9c2 cells following indicated treatments using TRIzol^® reagent (Thermo Fisher Scientific Inc.). Complementary DNA was synthesized by RT reaction with PrimeScript RT Master Mix (Takara Biotechnology Co., Ltd.). The reverse transcription protocol used was 37°C for 15 min and 85°C for 30 sec. qPCR assay was performed using SYBR Premix Ex Taq kit (Takara Biotechnology Co., Ltd.) according to the manufacturer’s instructions. The amplification conditions were as follows: 95°C for 10 min, followed by 40 cycles of 95°C for 10 sec and 60°C for 60 sec. Reactions were conducted on the ABI Prism 7500 Real-Time PCR System (Thermo Fisher Scientific, Inc.). The mRNA levels were calculated using the 2^−ΔΔCq method (26). GAPDH was used for normalization. The primers used were as follows: TNF-α forward, 5′-CCTCTTCTCATTCCTGCTCG-3′ and reverse, 5′-GGTATGAAATGGCAAATCGG-3′; interleukin (IL)-6 forward, 5′-CTGCGCAGCTTTAAGGAGTTC-3′ and reverse, 5′-TCTGAGGTGCCCATGCTACA-3′; IL-1β forward, 5′-CAACCAACAAGTGATATTCTCCATG-3′and reverse, 5′-GATCCACACTCTCCAGCTGCA-3′; IL-8 forward, 5′-GGCAGCCTTCCTGATTTCTG-3′ and reverse, 5′-CTTGGCAAAACTGCACCTTCA-3′; monocyte chemotactic protein-1 (MCP-1) forward, 5′-CTCTCGCCTCCAGCATGAA-3′ and reverse, 5′-GGGAATGAAGGTGGCTGCTA-3′; GAPDH forward, 5′-CCATCACCATCTTCCAGGAG-3′ and reverse, 5′-CCTGCTTCACCACCTTCTTG-3′.

Following the indicated treatments, the culture media were collected and centrifuged at 600 × g at room temperature for 5 min. The levels of TNF-α, IL-6, IL-1β, IL-8 and MCP-1 in the supernatants were determined using commercially available ELISA kits (R&D systems, Inc.) according to the manufacturer’s instructions and expressed as pg/ml.

The data are presented as the mean ± SD from three independent experiments. The results were compared by one-way analysis of variance, followed by least significant difference post-hoc test using SPSS 16.0 software (SPSS, Inc.). P<0.05 was considered to indicate a statistically significant difference.

First, the potential effects of Mel on the viability of OGD/R-treated H9c2 cells were explored. CCK-8 assay results demonstrated that OGD/R significantly reduced the viability of H9c2 cells compared with that of the control group. Mel (0.01, 0.1, 1 and 10 mM) significantly attenuated the decrease in OGD/R-stimulated H9c2 cell viability (Fig. 1A). Next, the activity levels of LDH and CK-MB, two indicators of cardiomyocyte injury, were measured using commercial kits. A notable OGD/R-induced increase in the activity levels of LDH and CK-MB was observed in H9c2 cells, whereas Mel (0.01, 0.1, 1 and 10 mM) significantly prevented injury (Fig. 1B and C). In addition, Mel markedly enhanced cell viability (Fig. 1D) and decreased LDH and CK-MB activity (Fig. 1E and F) in OGD/R-exposed H9c2 cells. These results indicated that Mel protected H9c2 cells from OGD/R-induced damage. Mel (1 mM) treatment for 24 h was chosen for subsequent experiments due to the observed effects against OGD/R-induced H9c2 cell injury.

OGD/R can result in cardiomyocyte apoptosis (27). Flow cytometry results demonstrated that OGD/R increased the percentage of apoptotic cells compared with that in the control group, whereas Mel reduced the OGD/R-induced apoptosis of H9c2 cells (Fig. 2A and B). The MMP in H9c2 cells subjected to OGD/R was decreased; however, this effect was markedly attenuated in the presence of Mel (Fig. 2C). To assess the involvement of caspase activation in the inhibition of OGD/R-induced apoptosis by Mel, the activity levels of caspase-3 and caspase-9 in the mitochondria-mediated apoptosis pathway were examined. Caspase-3 and caspase-9 activity levels were enhanced by OGD/R exposure compared with the control group (Fig. 2D and E). However, Mel significantly reduced the OGD/R-induced caspase-3 and caspase-9 activity. Consistent with the change in caspase-3 activity, the protein expression levels of cl-caspase-3 and cytochrome c were increased in OGD/R-insulted H9c2 cells. Mel reversed the upregulation of cl-caspase-3 and cytochrome c expression induced by OGD/R (Fig. 2F). Western blot analysis also revealed significant upregulation of Bax and downregulation of Bcl-2 following OGD/R insult in H9c2 cells, which was reversed by Mel treatment (Fig. 2F). These results suggested that Mel inhibited OGD/R-induced H9c2 cell apoptosis.

OGD/R induces oxidative stress (28). As demonstrated in Fig. 3A, OGD/R promoted intracellular ROS production in H9c2 cells compared with that in the control group. Of note, Mel significantly inhibited OGD/R-induced ROS generation. In addition, the content of MDA, which is an indicator of lipid peroxidation, was significantly reduced by Mel in OGD/R-insulted H9c2 cells (Fig. 3B). Compared with the OGD/R group, Mel also significantly increased the activity of endogenous antioxidant enzymes, including SOD (Fig. 3C), CAT (Fig. 3D) and GSH-Px (Fig. 3E) in H9c2 cells. These results demonstrated that Mel exerted an antioxidant effect in OGD/R-insulted H9c2 cells.

PGC-1α knockdown reduced the expression of Nrf2, HO-1 and NQO-1, whereas Nrf2 knockdown suppressed HO-1 and NQO-1 expression in OGD/R-stimulated H9c2 cells (Fig. 4A). OGD/R led to an upregulation of PGC-1α, Nrf2, HO-1 and NOQ1 expression in H9c2 cells. Mel further enhanced the levels of PGC-1α, Nrf2, HO-1and NOQ1 in OGD/R-insulted H9c2 cells (Fig. 4B). In addition, the Mel-induced decrease in ROS generation in OGD/R-exposed H9c2 cells was attenuated by PGC-1α or Nrf2 silencing (Fig. 4C). PGC-1α or Nrf2 knockdown counteracted the Mel-induced increase in the viability of OGD/R-exposed H9c2 cells (Fig. 4D). PGC-1α or Nrf2 knockdown also reversed the Mel-mediated reduction in apoptosis in OGD/R-insulted H9c2 cells (Fig. 4E). These results demonstrated that the antioxidant effects of Mel were dependent on the activation of PGC-1α/Nrf2 signaling in OGD/R-insulted H9c2 cells.

To examine whether Mel serves an inhibitory role in OGD/R-induced inflammation, the expression and release of several pro-inflammatory cytokines in H9c2 cells was measured by RT-qPCR and ELISA. The RT-qPCR results revealed that the expression of TNF-α (Fig. 5A), IL-6 (Fig. 5B), IL-1β (Fig. 5C), IL-8 (Fig. 5D) and MCP-1 (Fig. 5E) were considerably higher in the OGD/R-exposed H9c2 cells compared with those in the control cells, and that Mel partially reversed the OGD/R-induced increase in the TNF-α, IL-6, IL-1β, IL-8 and MCP-1 levels. Consistently, the production of TNF-α (Fig. 5F), IL-6 (Fig. 5G), IL-1β (Fig. 5H), IL-8 (Fig. 5I) and MCP-1 (Fig. 5J) was significantly suppressed by Mel in OGD/R-treated H9c2 cells. These results demonstrated that Mel reduced the levels of pro-inflammatory cytokines in OGD/R-exposed H9c2 cells.

PGC-1α can decrease TNF-α production in myotubes via TNF-α stimulation (29). TNF-α is implicated in OGD/R-induced H9c2 cell apoptosis (27). As demonstrated in Fig. 6A, siPGC-1α transfection led to an upregulation of TNF-α expression in OGD/R-exposed H9c2 cells. OGD/R induced a significant increase in PGC-1α and TNF-α expression in H9c2 cells, whereas Mel further increased PGC-1α expression but reduced the level of TNF-α (Fig. 6B). In OGD/R-insulted H9c2 cells, the Mel-mediated decrease in the production of TNF-α was reversed by PGC-1α knockdown. However, the addition of the TNF-α antibody reduced TNF-α production (Fig. 6C). In addition, PGC-1α silencing resulted in a decrease in the Mel-induced enhancement in the viability of OGD/R-exposed H9c2 cells, which was rescued by the addition of the TNF-α antibody (Fig. 6D). Incubation with the TNF-α antibody reversed the PGC-1α depletion-induced increase in OGD/R-stimulated H9c2 cell apoptosis (Fig. 6E). These results revealed that Mel exerted an anti-apoptotic effect in OGD/R-treated H9c2 cells via the PGC-1α/TNF-α signaling pathway.