H9C2 rat cardiomyocytes were obtained from The Cell Bank of Type Culture Collection of Chinese Academy of Sciences (Shanghai, China). The H9C2 cells were treated as described in previous literature to establish an in vitro model of MI/R (20,21), and the protocols were further developed in the present study. Medium with no serum or glucose served as the ischemic buffer. The ischemic buffer was incubated in an atmosphere with a gas mixture of 95% N₂ and 5% CO₂ for 2 h. The cells were subsequently cultured in glucose-free Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (both from Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA), in an anoxic environment (95% N₂ and 5% CO₂) at 37°C for 2 h. The H9C2 cells were transferred to fresh medium (DMEM) at 37°C and the gas mixture of 95% N₂ and 5% CO₂ was replaced with air at a flow rate of velocity of 2 l/min (95% O₂ and 5% CO₂). Following incubation for 1 h, the MI/R cell model was harvested and cells were subsequently observed under an inverted microscope.

A total of five treatment groups were prepared in the present study: The control group (H9C2 cells with no treatment); the MI/R group (H9C2 cells treated with OGD/R injury); the 1 µM apigenin + MI/R group (H9C2 cells treated with OGD/R injury, and subsequently treated with 1 µM apigenin); the 6 µM apigenin + MI/R group (H9C2 cells treated with OGD/R injury, and subsequently treated with 6 µM apigenin); and the 25 µM apigenin + MI/R group (H9C2 cells treated with OGD/R injury, and subsequently treated with 25 µM apigenin). Inhibition of PI3K was performed via incubation with LY294002 (25 µM) for 2 h as previously described (22).

A Cell Counting Kit-8 (CCK-8; Beyotime Institute of Biotechnology, Haimen, China) was used to determine the cell viability of the H9C2 cells. H9C2 cells (6×10⁴ cells/ml) cultured in the logarithmic phase were seeded in 96-well plates, and subsequently maintained in a 5% CO₂ atmosphere at 37°C for 12 h. Apigenin at different concentrations (1, 3, 6, 12, 25 and 50 µM) was added to the cells. The H9C2 cells were maintained for 12, 24 and 48 h. Subsequently, 10 µl CCK-8 reagent was added to the wells of the 96-well plates and the H9C2 cells were maintained for a further 3 h. A microplate reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA) was used to record the absorbance at 450 nm. Cell viability was evaluated as the percentage of cell survival compared with the control.

H9C2 cells were seeded into 96-well plates (6×10³ cells/well) and treated according to the aforementioned protocol. Cells were subsequently collected and the content/activity of lactate dehydrogenase (LDH), malondialdehyde (MDA), catalase (CAT), Na⁺K⁺-ATPase and Ca²⁺-ATPase were determined using a LDH Assay Kit (cat. no. ab102526), MDA Assay Kit (cat. no. ab118970) (both from Abcam, Cambridge, UK), CAT Assay Kit (cat. no. BC0205), Na⁺K⁺-ATPase assay kit (cat. no. BC0065) and Ca²⁺-ATPase assay kit (cat. no. BC0960) (all from Beijing Solarbio Science & Technology Co., Ltd.), respectively, in accordance with the manufacturers' protocol.

Annexin V is a phospholipid binding protein, which has a high affinity for phosphatidylserine. Annexin V is a sensitive indicator for detecting early apoptosis of cells. Propidium iodide (PI) is a type of nucleic acid dye that is not able to penetrate an intact cell membrane. PI penetrates the cell membrane as cell membrane permeability increases in the late stage of apoptosis. Therefore, Annexin V and PI may be used to distinguish cells in different apoptotic periods. Flow cytometry (FCM) was conducted to assess the apoptosis of H9C2 cells. Following washing with PBS, cultured H9C2 cells were trypsinized with 0.25% trypsin (Beyotime Institute of Biotechnology, Haimen, China). The supernatant was collected and the H9C2 cells were prepared for assessment by suspension in an incubation buffer at a density of 1×10⁶ cells/ml. H9C2 cells were subsequently incubated with Annexin V-fluorescein isothiocyanate and PI (XiLong Scientific Co., Ltd., Shantou, China) in the dark at room temperature for 15 min. A flow cytometer (FACSCalibur; BD Biosciences, Franklin Lakes, CA, USA) with CellQuest software version 3.3 was used to measure cellular apoptosis.

PBS was added to the cultured H9C2 cells until the cell density reached 1×10⁶/ml. FL1-H [Multisciences (Lianke) Biotech Co., Ltd., Hangzhou, China] was added to the H9C2 cells. H9C2 cells were incubated in the dark at room temperature for 10 min. The supernatant was collected and 100 µl PBS was added to the H9C2 cells. ROS have the ability to transform dichloro-dihydro-fluorescein diacetate (DCFH-DA) into 2′,7′-dichlorofluorescein (DCF). The fluorescence of DCF indicates the ROS level. The DCFH-DA dye (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) was used to detect ROS generation, according to previous literature (23,24). The fluorescence dye Rhodamine-123 (Rho-123) is able to penetrate the cell membrane. As described in previous studies (25,26), Rho-123 (Sigma Aldrich; Merck KGaA) was used to detect the MMP in the present study. FCM was performed with an excitation wavelength of 488 nm to assess the MMP and ROS levels in H9C2 cells. Cells (1×10⁴) were collected from each sample, and the data were analyzed using BD CellQuest^™ Pro software (version 3.3; BD Biosciences).

Cultured cells were lysed on ice in a radioimmunoprecipitation assay lysis buffer (Thermo Scientific Inc.; cat. no. 89900). The cells were fragmented following treatment with an ultrasonic cell disruptor. Following centrifugation at 5,000 × g at 4°C, fractionation occurred and the supernatant was collected. The protein expression level was measured using a protein assay reagent (Bio-Rad Laboratories, Inc.) and was performed following the manufacturer's protocol. From each sample, an equal quantity of protein (50 µg) was separated using 5% SDS-PAGE. The proteins were obtained and transferred to nitrocellulose membranes (EMD Millipore, Billerica, MA, USA) for 1.5 h. Nonspecific proteins were blocked by immersing the membranes into 5% low fat dried milk at room temperature for 2 h following washing with PBS. The membranes were incubated with primary antibodies: Anti-poly ADP-ribose polymerase (PARP; 1:5,000; cat. no. ab32138); anti-cleaved-caspase-3 (1:500; cat. no. ab49822); tumor necrosis factor receptor superfamily member 6 (Fas; 1:2,000; cat. no. ab133619); tumor necrosis factor ligand superfamily member 6 (Fasl; 1:8,000; cat. no. ab186671); anti-phospho (p)-PI3K (1:800; cat. no. ab182651); anti-PI3K (1:1,000; cat. no. ab189403); anti-p-Akt (1:500; ab38449); anti-Akt (1:6,000; cat. no. ab81283); anti-apoptosis regulator Bcl-2 (Bcl-2; 1:1,000; cat. no. ab59348); anti-p-serine/threonine-protein kinase mTOR (mTOR; phospho S2448; 1:1,000; cat. no. ab109268); anti-mTOR (1:1,000; cat. no. ab32028); and anti-GAPDH (1:2,500; cat. no. ab9485) (all from Abcam) overnight at 4°C. Horseradish peroxidase-conjugated secondary antibodies (1:5,000; Abcam; cat. no. ab205718; goat anti-rabbit) were added and incubated at room temperature for 1 h. Enhanced chemiluminescent reagents (EMD Millipore) with an enhanced chemiluminescence system (GE Healthcare Life Sciences, Little Chalfont, UK) were performed to assess the results. Densitometric analysis was performed using Quantity One software (version 4.6.9; Bio-Rad Laboratories, Inc.).

TRIzol^® reagent (Beyotime Institute of Biotechnology) was used to extract the total RNA from the cultured H9C2 cells. RNA was reverse transcribed to cDNA using an RT kit (Beyotime Institute of Biotechnology), according to the manufacturers' protocol. The temperature protocol used for RT was as follows: 25°C for 10 min, 42°C for 50 min and 70°C for 5 min. RT-qPCR analysis was performed using SYBR-Green PCR Master Mix (Thermo Fisher Scientific, Inc.) on an ABI 7500 Thermocycler (Applied Biosystems; Thermo Fisher Scientific, Inc.). PCR cycling conditions were as follows: Pretreatment at 95°C for 10 min; 96°C for 15 sec; 65°C for 45 sec (45 cycles); 96°C for 15 sec; 65°C for 1 min; 95°C for 15 sec; and a final extension at 75°C for 10 min, maintained at 4°C. The results were quantified using the 2^−ΔΔCq method (27). The primers were purchased from Invitrogen (Thermo Fisher Scientific, Inc.): PARP forward, 5′-CTGTTAATGCTAATCGTGAT-3′ and reverse, 5′-AACTGACTCCTACATATTAG-3′ (product, 223 bp); Fas forward, 5′-AGTACAGCCGGGAAGACAAT-3′ and reverse, 5′-TTTCTGGGCCATGCTTCTCT-3′ (product, 269 bp); Fasl forward, 5′-CAGAAAGCATGATCCGCGAC-3′ and reverse, 5′-GGTCTGGGCCATAGAACTGA-3′ (product, 215 bp); GAPDH forward, 5′-TCTGAACTCCAACGATGCCT-3′ and reverse, 5′-TCTTGTCCTTAAGCCTGGGG-3′ (product, 225 bp). GAPDH was used as the control to normalize the input RNA level.

The results are presented as the mean ± standard deviation. The data was evaluated using one-way analysis of variance followed by Tukey's multiple comparisons. Statistical analyses were performed using GraphPad Prism software (version 6.0; GraphPad Software, Inc., La Jolla, CA, USA). P<0.05 was considered to indicate a statistically significant difference. All experiments were performed in triplicate.

The cultured cardiomyocytes were observed with an inverted microscope (Fig. 1A). The shape of the H9C2 cells was round prior to adhesion. However, after 4–6 h, the majority of the cardiomyocytes began to adhere to the wall of culture bottle, exhibiting a round or spindle shape. Following culture for 24 h, the cells were arrayed in long fusiform, with a few arranged in triangular or irregular polygonal form. Additionally, cell nuclei were small and the cytoplasm was dark. After 48 h, cells dispersed gradually at the bottom of culture bottle, forming a cell monolayer. The cardiomyocytes were subsequently harvested for further experiments.

To evaluate the cytotoxic activity of apigenin on normal H9C2 cells, a CCK-8 assay was performed to determine the cell viability of H9C2 cells. The present results demonstrate that apigenin at concentrations of 1, 3, 6, 12 and 25 µM did not induce significant cytoxicity in H9C2 cells. The cell viability of H9C2 cells treated with apigenin was >80% with the exception that the cell viability of H9C2 cells was significantly lower compared with the control when the concentration of apigenin was 50 µM (Fig. 1B; P<0.05). Thus, we selected 1, 6 and 25 µM to treat the H9C2 cells for 12 h for the subsequent experiments.

Numerous oxidative stress markers in H9C2 cells, including LDH, MDA, CAT and ATPase activity, were assessed in the present study. The data indicated that the LDH content was significantly increased by MI/R injury (Fig. 2A; P<0.05); however, reduced upon treatment with apigenin. MDA content in H9C2 cells additionally exhibited similar trends following treatment with MI/R injury and apigenin (Fig. 2B; P<0.05) at different concentrations. The CAT activity in H9C2 cell treated with MI/R injury was significantly decreased compared with the control group (Fig. 2C; P<0.01). However, apigenin markedly enhanced the activity of CAT in MI/R-induced H9C2 cells (Fig. 2C). It was additionally observed that apigenin significantly increased the activities of Na⁺K⁺-ATPase and Ca²⁺-ATPase in H9C2 cells induced by MI/R injury (Fig. 2D; P<0.05).

The ROS content was measured in H9C2 cells from each treatment group. According to the FCM data, MI/R injury significantly increased the ROS production in H9C2 cells (Fig. 3; P<0.001). However, following treatment with apigenin at different concentration levels, a dose-dependent decrease in the ROS content in MI/R-induced H9C2 cells was observed, which was significant at 6 and 25 µM compared with MI/R injury (Fig. 3; P<0.05).

Furthermore, in the present study, MMP level in H9C2 cells, which were treated with MI/R and apigenin at different concentrations, was measured. Based on the FCM data, it was observed that MI/R significantly reduced the MMP level in H9C2 cells compared with the control (Fig. 4; P<0.05). Following treatment with apigenin at different concentrations, the MMP level in MI/R-induced H9C2 cells significantly recovered (Fig. 4; P<0.05).

As the aforementioned data suggested that apigenin may decrease the oxidative stress in MI/R-induced H9C2 cells, the apoptosis capacity of MI/R-induced H9C2 cells that treated with apigenin was evaluated. The FCM data demonstrated that the percentage of apoptotic H9C2 cells in the MI/R group was 16.27%, which was significantly increased compared with the control (Fig. 5; 1.77%; P<0.001). Furthermore, following treatment with apigenin at different concentrations of 1, 6 and 25 µM, the apoptosis rate of MI/R-induced H9C2 cells decreased from 16.27 to 13.18, 10.43 and 9.74%, respectively (Fig. 5; P<0.01). These data indicated that apigenin suppressed the apoptosis capacity of MI/R-induced H9C2 cells in a dose-dependent manner.

It was demonstrated that apigenin inhibited apoptosis of H9C2 cells induced by MI/R; therefore, apoptosis-associated protein expression in H9C2 cells was measured. According to the RT-qPCR data, PARP, Fas and Fasl expression in H9C2 cells from the MI/R group was significantly higher than the control group (Fig. 6A; P<0.001). The PARP, Fas, and Fasl expression in MI/R-induced H9C2 cells was markedly decreased upon treatment with apigenin in a dose-dependent manner (Fig. 6A). Furthermore, western blot analysis additionally demonstrated that the expression levels of PARP, cleaved caspase-3, Fas and Fasl in H9C2 cells were significantly upregulated upon MI/R injury compared with the control (Fig. 6B; P<0.0001); whereas, the expression of these molecules was markedly decreased with the addition of apigenin at different concentrations (Fig. 6B).

In addition, the mechanisms of apigenin in protecting H9C2 cells against MI/R induced injury were examined. The p-PI3K, PI3K, p-Akt, and Akt level in H9C2 cells was measured. Among the H9C2 cells from MI/R groups, the level of p-PI3K was significantly decreased compared with the control (Fig. 7A; P<0.001). However, significant increases were observed in the p-PI3K level in MI/R-induced H9C2 cells treated with apigenin at different concentrations (Fig. 7A; P<0.01). Furthermore, there was no significant difference in the expression of PI3K in H9C2 cells between each treatment group (Fig. 7A; P>0.05). Additionally, the level of p-Akt in H9C2 cells was significantly downregulated by MI/R injury (Fig. 5B; P<0.001). The decreased level of p-Akt in MI/R-induced cardiomyocytes was significantly recovered upon treatment with apigenin (Fig. 5B; P<0.001). Additionally, there was no significant difference in the expression level of Akt in H9C2 cells among the treatment groups (Fig. 7B; P>0.05).

To further confirm the role of PI3K/Akt in the present study model, LY294002 was used to block the activity of PI3K. It was revealed that the apoptosis rate was significantly increased in the MI/R + apigenin + LY294002 group compared with the MI/R + apigenin group (Fig. 8A; P<0.05). Furthermore, the expression of Bcl-2 and p-mTOR decreased upon treatment with LY294002 compared with the apigenin + MI/R group (Fig. 8B; P<0.01).