The H9C2 cardiomyocyte line, a subclone of the original clonal cell line derived from embryonic rat heart tissue, was obtained from the American Type Cell Culture Collection. The cells were cultured in DMEM (Sigma-Aldrich; Merck KGaA) supplemented with (v/v) 10% FBS (Gibco; Thermo Fisher Scientific, Inc.), 100 U/ml penicillin and 100 U/ml streptomycin in a humidified incubator at 37°C with an atmosphere of 5% CO₂ and 95% air.

To create an in vitro model of MI/R injury, H9C2 cells were maintained in glucose-free Hanks' Balanced Salt Solution (Invitrogen; Thermo Fisher Scientific, Inc.) within an anaerobic chamber containing 95% N₂ and 5% CO₂ at 37°C for 6 h, which induced OGD. Subsequently, H9C2 cells from the OGD-treated groups were removed from the anoxic incubator and cultured under normal condition as aforementioned for a further 18 h to allow reoxygenation to occur. This process was called MI/R injury.

Small interfering (si)RNAs against HIF-1α (HIF-1α-siRNA) and non-specific control siRNA (NS-siRNA) were synthesized by Shanghai GenePharma Co., Ltd. The sense strand siRNA sequences are as follows: HIF-1α-siRNA, 5′-GCCGCUCAAUUUAUGAAUATT-3′ and NS-siRNA, 5′-UUCUCCGAACGUGUCACGUTT-3′. H9C2 cells were transfected with HIF-1α-siRNA (50 nM) or NS-siRNA (50 nM) using Lipofectamine^® RNAiMAX (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's instructions. Briefly, the cells were seeded in 6-well plates at a density of 5×10⁵ cells/per well. HIF-1α-siRNA, NS-siRNA or Lipofectamine^® RNAiMAX transfection reagent was diluted in Opti-MEM medium (Gibco; Thermo Fisher Scientific, Inc.). The diluted siRNA was added to the diluted Lipofectamine^® RNAiMAX reagent in a 1:1 ratio. Following incubation at room temperature for 5 min, the siRNA-lipid complexes were added to the cells and incubated for a further 6 h, after which the media was changed to complete media. The cells were harvested 48 h post-transfection and the transfection efficiency was determined using reverse transcription-quantitative (RT-q)PCR.

A previous study and preliminary experimental results indicated that troxerutin (10 µM) addition to cultured myocardial cells for 1 h before OGD exerts cardioprotective effects (31). LY294002 (20 µM; Merck KGaA), a specific inhibitor of the PI3K/AKT signaling pathway, was used to pretreat H9C2 cardiomyocytes for 2 h before troxerutin treatment (32).

In the present study, cells were divided into the following 6 groups: i) Control group, H9C2 cells were cultured under normal conditions in high-glucose DMEM; ii) OGD/R group, H9C2 cells were subjected to OGD for 6 h and reoxygenation for 18 h; iii) troxerutin + OGD/R group, H9C2 cells were incubated with troxerutin (10 µM) for 1 h followed by co-treatment with OGD/R; iv) LY294002 + troxerutin + OGD/R group, cells were pre-conditioned with LY2940021 (20 µM) for 2 h, troxerutin (10 µM) for 1 h and then subjected to OGD/R; v) siRNA+ troxerutin + OGD/R group, H9C2 cells were transfected with HIF-1α-siRNA (50 nM) or NS-siRNA (50 nM) for 24 h and then treated with troxerutin (10 µM) for 1 h followed by OGD/R; and vi) LY294002 + siRNA, H9C2 cells were incubated with LY2940021 (20 µM) for 24 h and treated with HIF-1α-siRNA (50 nM) or NS-siRNA (50 nM) for 24 h.

The cell viability of H9C2 cells treated as aforementioned was analyzed using an MTT assay. Briefly, the cells were seeded in 96-well plates at a density of 1×10⁴ cells/well. Following the aforementioned treatments, MTT solution (0.5 mg/ml) was added to the each well and incubated with the cells at 37°C for 4 h. Following this, the supernatants were removed and 150 µl DMSO was added to each well to dissolve the formazan crystals. The absorbance at 570 nm was measured using a microplate reader (Bio-Rad Laboratories, Inc.).

H9C2 cells were cultured in 96-well plates at a density of 1×10⁴ cells/well and subjected to the aforementioned treatments. Extracellular lactate dehydrogenase (LDH) activity was determined using a commercial LDH assay kit (Nanjing KeyGen Biotech Co., Ltd.), according to the manufacturer's instructions. The absorbance at 490 nm was measured using a microplate reader.

H9c2 cells were treated as aforementioned and the level of apoptosis was determined using an Annexin V-FITC/PI kit (Becton, Dickinson and Company), according to the manufacturer's instructions. After treatment, the cells were harvested, washed twice with PBS and stained with annexin V-FITC (5 µl) and propidium iodide (10 µl) solution for 15 min at 37°C in the dark. The level of apoptosis was determined using a flow cytometer (FACScan; BD Biosciences) and quantified using CELL Quest 3.0 software (BD Biosciences).

Total RNA was isolated from H9C2 cells using TRIzol Reagent (Thermo Fisher Scientific, Inc.), according to the manufacturer's instructions. First-strand complementary DNA was synthesized using 1 µg RNA using the PrimeScript RT Reagent kit with gDNA Eraser (Takara Bio, Inc.). The temperature protocol used for the RT reaction consisted of 37°C for 15 min and at 90°C for 5 sec. The mRNA levels of HIF-1α, interleukin (IL)-1β, IL-6 and tumor necrosis factor (TNF)-α were analyzed using the SYBR Premix Ex Taq kit (Takara Bio, Inc.) and an ABI PRISM 7500 PCR instrument (Applied Biosystems; Thermo Fisher Scientific, Inc.). qPCR thermocycling conditions were: 95°C for 90 sec for an initial denaturation, 45 denaturation cycles at 94°C for 6 sec, followed by 60°C for 30 sec for annealing and elongation, and final 72°C for 8 min for extension. β-actin was used as an internal control and the relative level of mRNA was analyzed using the 2^−ΔΔCq method (33). The primer sequences used are as follows: HIF-1α forward, 5′-GATGACGGCGACATGGTTTAC-3′ and reverse, 5′-CTCACTGGGCCATTTCTGTGT-3′; IL-1β forward, 5′-GCCTCGTGCTGTCGGACCCATAT-3′ and reverse, 5′-TCCTTTGAGGCCCAAGGCCACA-3′; IL-6 forward, 5′-ACAACCACGGCCTTCCCTACTT-3′ and reverse, 5′-CACGATTTCCCAGAGAACATGTG-3′; TNF-α forward, 5′-CCCTCCTGGCCAACGGCATG-3′ and reverse, 5′-TCGGGGCAGCCTTGTCCCTT-3′); and β-actin forward, 5′-CGTGCGTGACATCAAAGAGAAG-3′ and reverse, 5′-CCAAGAAGGAAGGCTGGAAAA-3′.

H9C2 cells were seeded at a density of 1×10⁵ cells/ml in 6-well plates and the production of intracellular ROS was determined using an ROS assay kit (Beyotime Institute of Biotechnology). Briefly, following the aforementioned treatments, H9C2 cells were washed twice with PBS and incubated with 2′,7′-dichlorodihydrofluorescein diacetate (10 µM) at 37°C for 20 min. The fluorescence intensity was observed under a fluorescence microscope (Olympus Corporation, magnification, ×200). For the quantitative assay, the fluorescence intensity was measured using a flow cytometer at an excitation wavelength of 485 nm and an emission wavelength of 525 nm.

The H9C2 cells from each group were homogenized with Scientz-IID Ultrasound Cell lysis Instrument (Changchun, Hangzhou, 15 sec/3 times pyrolysis and 15 sec for interval) on ice to determine the malondialdehyde (MDA) content, and the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px). The MDA content was determined using the thiobarbituric acid method, the activity of was measured using the xanthine oxidase method and the activity of GSH-Px was determined using the dithio-dinitrotoluidine method (all Nanjing Jiancheng Bioengineering Institute), according to the manufacturer's instructions.

Following the aforementioned treatments, cells were lysed in ice-cold RIPA Buffer (Beyotime Institute of Biotechnology) containing 1% (v/v) Combined protease and phosphatase inhibitors (Thermo Fisher Scientific, Inc.). The cell lysates were centrifuged at 13,000 × g at 4°C for 10 min. The protein concentration in each sample was determined using the bicinchoninic acid method. Equal amounts of protein (30 µg) were separated on 10% SDS-PAGE and subsequently transferred to PVDF membranes. After blocking with 5% (w/v) non-fat dry milk in PBS with Tween (PBST) at room temperature for 2 h. The following primary antibodies were diluted 1:2,000 and incubated overnight at 4°C with the membranes (all Cell Signaling Technology, Inc.): PI3K (cat. no. 4249), phosphorylated AKT (p-AKT, cat. no. 9271), AKT (cat. no. 4691), HIF-1α (cat. no. 36169) and GAPDH (cat. no. 5174). After washing with TBST three times, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (Cell Signaling Technology, Inc., cat. no. 7074) at room temperature for 2 h. The membranes were washed and the protein bands were visualized using enhanced chemiluminescence (ECL; Pierce; Thermo Fisher Scientific, Inc.). Optical density values for each group were normalized to GAPDH and quantified using Quantity One 4.6.2 software (Bio-Rad, Laboratories, Inc.).

Following the aforementioned treatments, the levels of TNF-α (Thermo Fisher Scientific, Inc., cat. no. BMS622), IL-6 (Thermo Fisher Scientific, Inc., cat. no. BMS603-2) and IL-1β (E-EL-R0012c, Elabscience) in the supernatant were evaluated using ELISA, according to the manufacturer's instructions. The results of the ELISAs were measured using a plate reader at 450 nm.

Statistical analysis was performed using a one-way ANOVA followed by the least significant difference test using SPSS software, version 17.0 (SPSS, Inc.). All data are presented as the mean ± SD from three experiments. P<0.05 was considered to indicate a statistically significant difference.

To examine the protective effect of troxerutin on OGD/R injury, cell viability was assessed. The results revealed that the different concentrations of troxerutin treatment had no effect on cell viability (Fig. 1A), and Results from the MTT assay demonstrated that OGD/R treatment resulted in reduced cell viability, which was reversed by troxerutin (Fig. 1B). In addition, ODG/R-induced an increase in LDH activity that was also blocked by troxerutin (Fig. 1C). Subsequently, the effect of troxerutin on apoptosis in OGD/R-treated H9C2 cells was also measured. The results indicated that OGD/R treatment induced apoptosis (Fig. 1D) in H9C2 cells. However, these effects were attenuated by troxerutin treatment. These data demonstrated that troxerutin exerted cardioprotective effects against OGD/R injury in H9C2 cells.

To elucidate the potential underlying mechanism of troxerutin, the impact of troxerutin on the PI3K/AKT/HIF-1α signaling pathway was evaluated in OGD/R-injured H9C2 cells. OGD/R treatment significantly reduced the expressions of PI3K (Fig. 2A and B) and p-AKT (Fig. 2A and C), indicating that OGD/R inhibited the PI3K/AKT pathway. However, the expression of PI3K and p-AKT was reversed by troxerutin. In addition, troxerutin also significantly reversed the OGD/R-induced decrease in the expression of HIF-1α (Fig. 2A and D). Taken together, these data suggested that the PI3K/AKT/HIF-1α signaling pathway may play an important role in the cardioprotective effect of troxerutin against myocardial OGD/R damage.

Based on the aforementioned results, the effect of the PI3K/AKT pathway inhibitor LY294002 on the cardioprotective effects of troxerutin was investigated. LY294002 pretreatment significantly mitigated the troxerutin-induced upregulation of PI3K (Fig. 3A and B), p-AKT (Fig. 3A and C) and HIF-1α (Fig. 3A and D) in OGD/R-treated H9C2 cells, indicating that LY294002 results in the inhibition of the PI3K/AKT/HIF-1α signaling pathway. Meanwhile, compared with troxerutin + OGD/R group, pretreatment with LY294002 reversed troxerutin-induced increased cell viability (Fig. 3E) and decreased LDH activity (Fig. 3F) in OGD/R-treated H9C2 cells. These results suggested that troxerutin exerted its cardioprotective effects against OGD/R injury by enhancing PI3K/AKT-mediated HIF-1α signaling pathway.

Next, an siRNA against HIF-1α was used to further investigate the underlying mechanism of troxerutin. H9C2 cells were transfected with HIF-1α-siRNA or NS-siRNA and the transfection efficiency was determined using RT-qPCR. The results showed that compared with NS-siRNA transfection group, HIF-1α-siRNA transfection significantly decreased the level of HIF-1α mRNA in the control, OGD/R group and troxerutin + OGD/R groups (Fig. 4A). HIF-1α-siRNA further reduced cell viability and reduced the troxerutin-induced increase in cell viability in OGD/R-treated H9C2 cells (Fig. 4B). HIF-1α-siRNA also exacerbated the OGD/R-mediated increase in LDH activity, and, notably, HIF-1α-siRNA did not eliminate the inhibition of troxerutin on LDH activity in troxerutin + OGD/R-treated H9C2 cells (Fig. 4C). Additionally, HIF-1α-siRNA aggravated apoptosis in the OGD/R-treated group (compared with the NS-siRNA + OGD/R group) and reduced the troxerutin-induced decrease in apoptosis rate in the troxerutin + OGD/R-treated group (compared with the troxerutin + OGD/R group) (Fig. 4D and E). Notably, the effects of troxerutin on apoptosis were not abolished by HIF-1α-siRNA, meaning other pathways may contribute to the cardioprotection of troxerutin against apoptosis. These results indicated that troxerutin protected against OGD/R-induced cytotoxicity partly depending on the PI3K/AKT/HIF-1α signaling pathway, while the effects of troxerutin attenuated apoptosis independent of the PI3K/AKT/HIF-1α signaling pathway.

To further explore the effect of troxerutin on oxidative stress and the role of the PI3K/AKT/HIF-1α signaling pathway in this process, oxidative stress markers were assessed in H9C2 cells. Compared with the control group, OGD/R treatment increased ROS generation (Fig. 5A and B) and the MDA content (Fig. 5C) in H9C2 cells, while compared with OGD/R group, troxerutin pretreatment decreased ROS production (Fig. 5A and B) and the MDA content (Fig. 5C) in OGD/R-treated H9C2 cells, highlighting the anti-oxidative properties of troxerutin under OGD/R condition in H9C2 cells. However, compared with NS-siRNA group, the inhibition of troxerutin on oxidative stress was partly reversed by HIF-1α-siRNA in TX + OGD/R group. In addition, troxerutin alleviated the OGD/R-induced decreases in antioxidant stress indicators, including SOD (Fig. 5D) and GSH-Px activities (Fig. 5E), while these effects were reduced by HIF-1α-siRNA. Compared with the NS-siRNA transfection group, HIF-1α-siRNA induced oxidative stress in the OGD/R treatment group as shown by the increased generation of ROS and MDA content, and the reduced activities of SOD and GSH-Px. Notably, the effect of troxerutin on oxidative stress was not totally abolished by the knockdown of HIF-1α, the effect was only reduced, meaning that other molecule or pathways may be involved in the protection of troxerutin against oxidative stress. These results suggested that troxerutin attenuated OGD/R-induced oxidative stress, partly by enhancing PI3K/AKT/HIF-1α signaling pathway.

The effect of troxerutin on inflammation in OGD/R-treated H9C2 cells was investigated. Troxerutin treatment mitigated OGD/R-induced increases in inflammatory markers including IL-1β (Fig. 6A), IL-6 (Fig. 6B) and TNF-α (Fig. 6C) mRNA expression in H9C2 cells, while these effects were reduced by HIF-1α-siRNA. In addition, OGD/R-induced an increase in the levels of IL-1β (Fig. 6D), IL-6 (Fig. 6E), and TNF-α (Fig. 6F) in the culture supernatant, which were also reversed by troxerutin treatment. However, these effects of troxerutin on the inflammatory markers reduced by HIF-1α-siRNA. These results indicated that troxerutin attenuated OGD/R-induced inflammation by promoting the PI3K/AKT/HIF-1α signaling pathway.