The primary antibodies included phosphorylated AMP-activated protein kinase α (p-AMPKα; cat. no. 2535), total (T)-AMPKα (cat. no. 2603P), p-mechanistic target of rapamycin (mTOR; cat. no. 2971), T-mTOR (cat. no. 2983), p-forkhead box O3a (FOXO3a; cat. no. 9465P), T-FOXO3a (cat. no. 2497P), Bcl-2 associated X apoptosis regulator (Bax; cat. no. 2722), Bcl-2 apoptosis regulator (Bcl-2; cat. no. 2870), and GAPDH (cat. no. 2118; all purchased from Cell Signaling Technology, Inc., Danvers, MA, USA). Sanggenon C (98% purity as determined by high-performance liquid chromatography analysis) was purchased from Shanghai Winherb Medical S&T Development Co., Ltd. (Shanghai, China). Fetal bovine serum was ordered from Gibco (Thermo Fisher Scientific, Inc., Waltham, MA, USA). The cell culture reagents were purchased from Gibco (Thermo Fisher Scientific, Inc.).

Rat cardiac H9c2 cells (Cell Bank of the Chinese Academy of Sciences, Shanghai, China) were cultured in Dulbecco's modified Eagle's medium (DMEM; cat. no. C11885; Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (FBS; cat. no. 10099-133; Gibco; Thermo Fisher Scientific, Inc.,), 100 U/ml penicillin/100 mg/ml streptomycin (cat. no. 15140; Gibco; Thermo Fisher Scientific, Inc.) and 5% CO₂ at 37°C. The media was changed every 1–2 days and subcultured to 70–80% confluency. Cells were plated at an appropriate density according to each experimental design. Cells were seeded at a density of 1×10⁶/well onto 6-well culture plates for mRNA extraction, 5×10³/well in 24-well plates for cell surface area examination and 10×10⁷/well onto 10-cm diameter culture plates for protein extraction. Following 24 h adherence, the culture medium was changed to serum-free DMEM 12 h prior to the experiment. Cells were pretreated with Sanggenon C (1, 10 and 100 µM) and/or Compound C (CpC; 20 µM) for 12 h in serum-free DMEM at 37°C and then cells were maintained at 37°C, under a hypoxic atmosphere of 95% N₂ and 5% CO₂ for 24 h.

Total RNA was extracted from frozen H9c2 cells using TRIzol (cat. no. 15596-026; Thermo Fisher Scientific, Inc.). RNA yield and purity were evaluated using a SmartSpec Plus Spectrophotometer (Bio-Rad Laboratories Inc., Hercules, CA, USA), comparing the absorbance (A) 260/A280 and A230/260 ratios. RT was performed on RNA (2 µg of each sample) to produce cDNA using oligo (dT) primers and the Transcriptor First Strand cDNA Synthesis kit (cat. no. 04896866001; Roche Diagnostics, Basel Switzerland). The PCR products were quantified using a LightCycler 480 SYBR-Green 1 Master mix (cat. no. 04707516001; Roche Diagnostics). Following an initial 5 min denaturation step at 95°C, a total of 42 primer-extension cycles were carried out. Each cycle consisted of a 10 sec denaturation step at 95°C, a 20 sec annealing step at 60°C, and a 20 sec incubation at 72°C for extension. Then a final extension step was performed at 72°C for 10 min. The double standard curve was used to quantify the PCR results. Calibrator normalized ratio = (concentration of sample target/concentrations of sample reference)/(concentration of calibrator target/concentration of calibrator reference) (15). The results were normalized against GAPDH gene expression. The sequences of the oligonucleotide primers (Sangon Biotech Co., Ltd., Shanghai, China) were as follows: TNFα forward, 5′-AGCATGATCCGAGATGTGGAA-3′ and reverse, 5′-TAGACAGAAGAGCGTGGTGGC-3′; interleukin-1 (IL-1) forward, 5′-GGGATGATGACGACCTGCTAG-3′ and reverse, 5′-ACCACTTGTTGGCTTATGTTCTG-3′; IL-6 forward, 5′-GTTGCCTTCTTGGGACTGATG-3′ and reverse, 5′-ATACTGGTCTGTTGTGGGTGGT-3′; Beclin-1 forward, 5′-AGCTTTTCTGGACTGTGTGC-3′ and reverse, 5′-TGAACTTGAGCGCCTTTGTC-3′; Atg5 forward, 5′-CAAGGATGCAGTTGAGGCTC-3′ and reverse, 5′-AGTTTCCGGTTGATGGTCCA-3′; p62 forward, 5′-AAGAGGCTCCATCACCAGAG-3′ and reverse, 5′-CCCCTTGACTCTGGCTGTAA-3′.

The level of intracellular ROS generation was assessed using the fluorescent dye 2′,7′-dichlorofluorescin diacetate (DCFH-DA). Following the indicated treatments, cells were washed twice with PBS and then incubated with serum-free DMEM and 1×10⁻⁵ mol/l DCFH-DA in a 37°C incubator for 30 min. Subsequently, cells were washed with PBS for three times to eliminate the residual DCFH-DA. A fluorescence microscope (BX51; Olympus Corporation, Tokyo, Japan) was also used to evaluate the DCFH florescence of cells on coverslips.

Antioxidative capacity was assessed by the release of NO and the activity of SOD according to the protocol of the NO detection kit, SOD detection kit, respectively (Beyotime Institute of Biotechnology, Haimen, China). For NO detection, the supernatant was collected and transferred to another 96-well plate. The NO released from H9c2 cells was measured at 540 nm using spectrophotometer according to the manufacturer's instructions (Beyotime Institute of Biotechnology). For SOD assessment, proteins were extracted and quantified before measuring the activity of SOD at 450 nm using the commercial kit (Beyotime Institute of Biotechnology).

Cells were cultured on cover slips in a 24-well plate, fixed in 4% paraformaldehyde for 5 min at room temperature and then permeabilized in 0.1% Triton X-100 for 5 min in room temperature following treatment. TUNEL staining according to the protocol of ApopTag^® Plus Fluorescein in situ Apoptosis Detection kit (EMD Millipore, Billerica, MA, USA) for 1 h at 37°C. Nuclei were labeled with DAPI and DNA fragmentation was quantified under fluorescence microscope (magnification, ×200; BX51TRF; Olympus Corporation). The percentages of TUNEL-positive cells relative to DAPI-positive cells were calculated by an investigator in a blinded manner.

Cultured cardiac H9c2 cells were lysed in radioimmunoprecipitation (RIPA) lysis buffer [720 µl RIPA, 20 µl phenylmethylsulfonyl fluoride (1 mM), 100 µl cOmplete^™ protease inhibitor cocktail (cat. no. 04693124001; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany)], 100 µl phosSTOP (cat. no. 04906837001; Roche Diagnostics), 50 µl NaF (1 mM), 10 µl Na₃VO₄/ml and the protein concentration was measured by the bincinchoninic assay method. A total of 30 µg cell lysate was used for protein separation using SDS-PAGE on a 10% gel. The proteins were then transferred to polyvinylidene difluoride (PVDF) membranes (EMD Millipore). Specific protein expression levels were normalized to the GAPDH protein levels of the total cell lysate and cytosolic proteins on the same PVDF membranes, which were blocked with 5% non-fat milk at room temperature for 2 h. The following primary antibodies were used: p-AMPKα, T-AMPKα, p-mTOR, T-mTOR, p-FOXO3a, T-FOXO3a, Bax, Bcl-2, and GAPDH. The primary antibodies were diluted at 1:1,000. Antibody incubation was performed overnight with gentle shaking at 4°C. Quantification of the western blots was performed using an Odyssey infrared imaging system (LI-COR, Lincoln, NE, USA). The secondary antibodies, goat anti-rabbit IRdye^® 800 CW (cat. no. 926-32211; LI-COR) IgG and goat anti-mouse IRdye 800 CW (cat. no. 926-32210; LI-COR), were used at a 1:10,000 dilution at 37°C in Odyssey blocking for 1 h. The blots were scanned using an infrared LI-COR scanner, allowing for simultaneous detection of two targets (phosphorylated and total protein) within the same experiment.

Data is expressed as the mean ± standard error of the mean. Differences among groups were determined by a two-way analysis of variance followed by Tukey's post hoc test. Statistical analyses were conducted using SPSS software (version 19.0; IBM Corp., Armonk, NY, USA). P<0.05 was considered to indicate a statistically significant difference.

The effects of Sanggenon C on the induction of TNFα, IL-1β and IL-6 in cardiomyocytes exposed to hypoxia were measured by RT-qPCR. The expression levels of pro-inflammatory cytokines TNFα, IL-1β and IL-6 were significantly increased in the hypoxia group. Sanggenon C treatment significantly attenuated this increase in a concentration-dependent manner (Fig. 1).

Cells incubated with DCFH-DA were used to determine the ROS production. A marked increase of ROS was observed in H9c2 cells exposed to hypoxia, while 100 µM Sanggenon C suppressed hypoxia-induced ROS generation. The effect of Sanggenon C on the release of antioxidants was also measured. Exposure to hypoxia for 24 h significantly decreased the release of NO and SOD activity in H9c2 cells and 100 µM Sanggenon C significantly attenuated this decrease (Fig. 2A and B).

The effect of Sanggenon C on cardiomyocyte autophagy in response to hypoxia was explored. Previous studies have demonstrated that Beclin-1, p62, Atg5 and LC3 are autophagy-associated proteins and enhanced autophagy is accompanied by an increased ratio of LC3 II/LC3 I and Atg5 and Beclin-1 expression (16). The results demonstrated that autophagy significantly decreased in hypoxia-exposed cardiomyocytes, however increased in 100 µM Sanggenon C pre-treated cardiomyocytes (Fig. 3A and B).

To demonstrate the effect of Sanggenon C on cardiomyocyte apoptosis in response to hypoxia, TUNEL staining and western blotting were performed to detect cell apoptosis. Results revealed that the rate of apoptotic cells increased significantly after 24 h of hypoxia. Sanggenon C significantly reduced hypoxia-induced apoptosis (Fig. 4A and B). As expected, changes in expression of apoptosis-associated proteins were also consistent with the results above (Fig. 4C and D).

Cellular energy levels are associated with the autophagy machinery by means of three major energy sensing pathways: AMPK, mTOR, and FOXO3a (16). In the current study, activated AMPKα was decreased, and phosphorylated mTOR and FOXO3a were increased in cardiomyocytes under hypoxia. Sanggenon C pre-treatment significantly increased the activation of AMPKα, and reduced the phosphorylation of mTOR and FOXO3a (Fig. 5A and B). To confirm the protective effects of Sanggenon C on hypoxia-induced cardiomyocyte injury were mediated by AMPKα signaling, an AMPKα inhibitor, CpC (20 µM) was used. The anti-apoptotic and pro-autophagy effects of Sanggenon C in response to hypoxia were abolished by CpC (Fig. 5C-E). The data indicated that enhanced autophagy and reduced inflammation, oxidative stress, apoptosis in Sanggenon C-pretreated cardiomyocytes exposed to hypoxia may be mediated through the regulation of the AMPKα/mTOR/FOXO3a signaling pathway.