The cardiomyocyte cell line H9c2 derived from the rat was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). Cells were maintained in DMEM (HyClone Laboratories Inc./GE Healthcare) as recommended. All medium used were supplemented with 10% fetal bovine serum (FBS; HyClone Laboratories Inc./GE Healthcare) and cells were routinely cultured in a humidified incubator at 37°C under 5% CO₂.

H9c2 cells at 70–80% confluence were maintained in serum-free medium with low glucose for 12 h for cell starvation. Subsequently, 5, 10 or 15 µg/ml diosmetin (Selleck Chemicals) dissolved in DMSO, 5 mM 3-methyladenine (3-MA; Selleck Chemicals) dissolved in PBS or 10 µM compound C (Selleck Chemicals) dissolved in DMSO were added to the cell cultures immediately after serum starvation. The cells were then cultured in normoxic conditions (74% N₂, 5% CO₂ and 21% O₂) for 1 h prior to being cultured in hypoxic conditions (94% N₂, 5% CO₂ and 1% O₂) for 48 h, a duration which was frequently used in several previous studies (20,21). As the non-treated group (NT), cells were maintained in normoxic conditions for the duration of the experiment.

For determination of cell viability, H9c2 cells exposed to hypoxia or the non-treated group were plated into a flat bottom 96-well plate at 4×10³ cells per well in triplicate with 100 µl medium. Ten microliters of Cell Counting Kit-8 reagent (CCK-8; Dojindo) was added into each well. After incubation for 2 h, optical density (OD) value at the wavelength of 450 nm was measured using a microplate reader (Bio-Rad Laboratories, Inc.).

Cell apoptosis was detected using the Annexin V-FITC Apoptosis Detection Kit (Calbiochem/EMD/Merck KGaA) according to the manufacturer's instructions.

Cell lysates were prepared using RIPA lysis buffer (Cell Signaling Technology) supplemented with protease inhibitor and phosphatase inhibitors for protein extraction. Concentration of total protein was determined by the Pierce Coomassie (Bradford) Protein Assay Kit (Thermo Fisher Scientific, Inc.). Then protein samples (20 µg) were resolved on 8% SDS-PAGE and transferred to polyvinylidene difluoride membranes. The rinsed membranes were incubated with primary antibodies targeting cleaved caspase-3 (cat. no. 9654), phospho-AMPKα (cat. no. 2531), AMPK (cat. no. 5832), phospho-ULK1 (cat. no. ab229537), ULK1 (cat. no. 8054) or β-actin (cat. no. 4970) at 4°C overnight. After washing, the membrane was incubated with a secondary antibody conjugated with horseradish peroxidase (cat. no. 7074). The signals were developed using Super Signal West Dura Extended Duration chemiluminescence substrate (Thermo Fisher Scientific, Inc.) and measured by ChemiDoc™ XRS+ System (Bio-Rad Laboratories, Inc.). All primary antibodies and secondary antibodies were purchased from Cell Signaling Technology except for phospho-ULK1 antibody which was purchased from Abcam. All antibodies were used in a 1:1,000 dilution as recommended by the supplier.

Cell autophagy was detected using a CYTO-ID^® Autophagy detection kit (Enzo Life Sciences) following the manufacturer's instructions. Briefly, H9c2 cells were harvested and washed with PBS. Cells were then stained with CYTO-ID green fluorescence regents for 30 min at 37°C. After being washed with assay buffer provided in the CYTO-ID^® Autophagy detection kit, cells were fixed with 4% paraformaldehyde in PBS for 20 min. Cells labeled with fluorescence were analyzed by flow cytometry and photographed using a Leica DM6000B fluorescence microscope (Leica Microsystems, Inc.). Higher fluorescence intensity indicated a higher level of autophagy.

An Autophagy LC3 HiBiT Reporter Assay System (Promega) provides a quick, efficient and common method with which to assess the effects of compounds on autophagy. The effects of diosmetin on autophagy were confirmed using this assay. Briefly, 293T cells provided by this system were routinely cultured with DMEM supplemented with 10% FBS and 500 µg/ml G418 (Selleck Chemicals). For the assays, 293T cells were plated into an opaque, white tissue-culture 96-well plate at 2×10⁴ cells per well in triplicate. After incubation for 24 h, the cells were treated with diosmetin at different concentrations (5, 10 and 15 µg/ml); cells treated with an equal amount of solvent were as control. After incubation at 37°C for another 48 h, the luminescence signal was detected with an EnVision 2105 Multimode Plate Reader (PerkinElmer) following the manufacturer's instructions. The luminescence signal of treated cells was normalized to the control. Three independent experiments were performed.

Statistical analysis was performed using SPSS 19.0 (SPSS, Inc.). Data are presented as a mean ± standard deviation of three parallel experiments. One-way ANOVA analysis was used for comparison of more than two groups, followed by Newman-Keuls post hoc test. A P-value <0.05 was considered to indicate a statistically significant difference.

To ascertain the effects of diosmetin on hypoxia-induced injury in cardiomyoblasts, H9c2 cells were treated with gradient dilutions of diosmetin (5, 10, 15 µg/ml) before exposure to hypoxia. Results of the CCK-8 assay indicated that cell viability was significantly lower in the cells exposed to hypoxia when compared to the cells not exposed to hypoxia (NT) (Fig. 1A). It was also observed that cell viability was obviously higher in the cells treated with 10 and 15 µg/ml diosmetin than that in cells exposed only to hypoxia among all hypoxia-treated groups (Fig. 1A). These data demonstrated that exposure to hypoxia decreased cell survival, while diosmetin protected H9c2 cells from hypoxia-induced decrease in cell survival in a dose-dependent manner. Furthermore, cell apoptosis was detected by PI/Annexin V staining assay. The results revealed that exposure to hypoxia significantly induced H9c2 apoptosis, resulting in considerable double-labeled population (46.1%, Fig. 1B). Notably, the double-labeled apoptotic population was 35.1, 16.0 and 5.8 in the cells pretreated with 5, 10 and 15 µg/ml diosmetin, respectively (Fig. 1B and C). Moreover, western blot analysis showed that hypoxia upregulated expression of cleaved caspase-3, while the upregulated cleaved caspase-3 was effectively suppressed when cells were treated with diosmetin at increasing concentrations (Fig. 1D and E). Collectively, diosmetin may protect H9c2 cells from hypoxia-induced cell death via alleviating hypoxia-induced cell apoptosis in a dose-dependent manner.

The effects of diosmetin on cell autophagy were also investigated in the present study. CYTO-ID^® Autophagy detection kit was used to detect cell autophagy, providing a rapid, specific and quantitative approach for monitoring autophagy. As shown in Fig. 2A-C, exposure to hypoxia induced slight cell autophagy which was subsequently strengthened by additional treatment of diosmetin in a dose-dependent manner. An Autophagy LC3 HiBiT Reporter Assay System was used to investigate whether diosmetin functions as an autophagy inducer or inhibitor. Currently, the direct effects of diosmetin on cell autophagy were estimated using this reporter assay system. The results indicated that luminescence intensity was comparably lower in cells which were treated with 10 and 15 µg/ml diosmetin (Fig. 2D), confirming the role of diosmetin as an autophagy inducer in a dose-dependent manner.

3-Methyladenine (3-MA) is a well-studied autophagy inhibitor. 3-MA was used as a cell autophagy inhibitor to confirm whether diosmetin-induced autophagy is essential in maintaining its protective effect on cell survival. Results of the CCK-8 assay illustrated that diosmetin treatment significantly enhanced the cell viability of the hypoxia-injured cells, which was previously evidenced. Subsequently, it was demonstrated that an additional treatment of 3-MA observably decreased cell viability in the hypoxia-injured cells treated with diosmetin (Fig. 3A). It can be concluded that the protective effects of diosmetin on hypoxia-injured cells were suppressed by autophagy inhibition; namely, autophagy is an indispensable mechanism that mediates the protection of diosmetin in regards to cell proliferation. apoptosis analysis also revealed that 3-MA partially reversed apoptosis inhibition induced by diosmetin (Fig. 3B and C). Cleaved caspase-3 was also upregulated in cells treated with diosmetin and 3-MA as compared to cells only treated with diosmetin (Fig. 3D and E). The above findings imply that autophagy is the main mechanism responsible for diosmetin-induced proliferation protection.

Activation of Unc-51 like autophagy activating kinase 1 (ULK1), a kinase that is importantly involved in cell autophagy, can be regulated by adenosine 5′-monophosphate-activated protein kinase (AMPK) in a direct or even an indirect manner (19). Therefore, we aimed to ascertain whether the AMPK signaling pathway is involved in diosmetin-induced autophagy and apoptosis inhibition. Phosphorylated (p)-AMPK, AMPK, p-ULK1 and ULK1 were detected by western blot analysis. The results indicated that the levels of p-AMPK and p-ULK1 in hypoxia-injured cells were significantly increased by diosmetin (Fig. 4A and B). Compound C, an effective and widely used AMPK inhibitor, was used together with diosmetin in cells before hypoxia treatment. Cell autophagy was detected using CYTO-ID^® Autophagy detection kit in hypoxia-injured cells treated with diosmetin alone or combined with compound C. It was found that additional administration of compound C indeed significantly attenuated autophagy as compared to treatment with diosmetin alone (Fig. 4C and D). PI/Annexin V staining assay also showed that the apoptotic population in the hypoxia-injured cells treated with diosmetin was increased from 10.6 to 31.1% when compound C was additionally applied (Fig. 4E and F). Based on the observed results, it was proposed that diosmetin-induced autophagy and apoptosis inhibition were partially mediated by activation of AMPK.