Sanggenon C is isolated from
Acute myocardial infarction (AMI) results from a disruption of coronary blood flow to the myocardial region that it supplies. AMI remains the leading cause of mortality and is associated with a heavy financial burden worldwide (
More recently, the role of autophagy in the pathogenesis of myocardial ischemic injury has been investigated. Autophagy is an evolutionarily conserved catabolic process that targets dysfunctional or damaged cytoplasmic constituents to the lysosome for degradation and recycling (
Sanggenon C, a flavanone Diel-Alder adduct compound, is isolated from the root bark of
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) (
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
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 (
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 (
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 (
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 (
Cellular energy levels are associated with the autophagy machinery by means of three major energy sensing pathways: AMPK, mTOR, and FOXO3a (
In the present study, it was demonstrated that Sanggenon C could increase cardiomyocyte autophagy in hypoxic conditions and prevent myocyte apoptosis. The increase in autophagy was associated with ROS clearance and the inhibition of inflammation, which would lead to cell death. The cardiac-protective effect of Sanggenon C was mediated by the modulation of AMPKα/mTOR/FOXO3a signaling.
In myocardial infarction and cardiomyocyte hypoxia, necrotic cardiomyocytes are capable of releasing a wide range of damage-associated molecular signals that activate innate immune pathways triggering an inflammatory response (
Autophagy involves many actions that are essential for cell survival. It preserves energy availability and removes damaged organelles through limited cellular catabolism. Removal of damaged mitochondria is particularly important since these organelles produce ROS and contribute to cell stress and damage. Autophagy reduces pro-inflammatory signals by eliminating intracellular organisms, degrading pro-inflammatory signaling platforms, and by controlling cytokine production and release. Studies have demonstrated that cells exposed to 2 h of simulated ischemia in the absence of oxygen exhibited a low level of autophagy (
To investigate the mechanism of induced autophagy following Sanggenon C treatment, the signal pathway associated with autophagy was detected. Autophagy is regulated by multiple signaling pathways, involving nutrients, stress, hormones, growth factors and intracellular energy information (
In conclusion, the current results supported the theory that Sanggenon C, administered as a pre-treatment prior to cardiomyocyte hypoxia, attenuates the inflammatory response and ROS production provoked during hypoxia. Notably, Sanggenon C was demonstrated to promote autophagy and render the cardiomyocyte resistant to hypoxic injury. The modulation of AMPKα/mTOR/FOXO3a signaling pathway restored autophagy as a reflex reaction.
This study was supported by the Applied and Technologic Research Program of Huai'an (grant no. HAS2014009) and the Research Fund for the Technology Development Project of Nanjing Medical University (grant no. 2013NJMU226).
Sanggenon C suppresses hypoxia-induced inflammation in cardiomyocytes. Reverse transcription-quantitative polymerase chain analysis of the mRNA levels of IL-1, IL-6, and TNFα induced by Sanggenon C (1, 10 and 100 µM) after hypoxia for 24 h (n=6). *P<0.05 vs. control group; #P<0.05 vs. hypoxia group. LD, low dose 1 µM Sanggenon C; MD, mid dose 10 µM Sanggenon C; HD, high dose 100 µM Sanggenon C; IL, interleukin; TNFα, tumor necrosis factor α.
Sanggenon C inhibits the oxidative stress induced by hypoxia in cardiomyocytes. (A) Sanggenon C (100 µM) inhibits the ROS production induced by hypoxia which was detected by florescence microscope, (n=3). (B) Effect of Sanggenon C (100 µM) on the production of NO and activity of SOD of hypoxia cardiomyocyte were determined by kit (n=6). *P<0.05 vs. normoxia-PBS; #P<0.05 vs. hypoxia-PBS. HD, high dose 100 µM Sanggenon C; ROS, reactive oxygen species; SOD, super oxide dismutase.
Sanggenon C activates autophagy in response to hypoxia in cardiomyocytes (A) Reverse transcription-quantitative polymerase chain reaction analysis of the mRNA levels of Atg5 and Beclin-1 in the indicated group (n=6). (B) Western blot analysis the effect of Sanggenon C (100 µM) on the transformation of LC3 I to LC3 II (n=6). Left, representative blots. Right, quantitative results. *P<0.05 vs. normoxia-PBS; #P<0.05 vs. hypoxia-PBS. HD, high dose 100 µM Sanggenon C; Atg5, autophagy related 5; LC3, microtubule-associated proteins 1A/1B light chain 3.
Sanggenon C attenuates hypoxia-induced apoptosis in cardiomyocytes. (A) Effect of Sanggenon C (100 µM) on the hypoxia-induced apoptosis among cardiomyocytes detected by TUNEL staining and (B) quantification (n=3). (C) The effect of Sanggenon C (100 µM) on the activation of apoptotic signaling pathways including Bax and Bcl-2 determined by western blot analysis and (D) densitometry semi-quantification (n=6). *P<0.05 vs. normoxia-PBS; #P<0.05 vs. hypoxia-PBS. HD, high dose 100 µM Sanggenon C; TUNEL, terminal deoxynucleotidyl-transferase-mediated dUTP nick-end labelling; Bax, Bcl-2 associated X apoptosis regulator; Bcl-2, Bcl-2 apoptosis regulator; c-caspase 3, cleaved caspase 3.
Effect of Sanggenon C on AMPKα/mTOR/FOXO3a signaling. (A) Western blot analysis the effect of Sanggenon C (100 µM) on the activation of AMPKα pathways including phosphorylated (p-) and total (T-) AMPKα, mTOR and FOXO3a with (B) densitometry analysis (n=6). (C) TUNEL staining of cardiomyocytes pretreated with Sanggenon C (100 µM) or AMPKa inhibitor, CpC (20 µM) and exposed to hypoxia for 24 h (n=3) and (D) quantification of TUNEL-positive cells. (E) Western blot analysis the effect of Sanggenon C (100 µM) and CpC on the transformation of LC3 I to LC3 II (n=6). *P<0.05 vs. normoxia-PBS; #P<0.05 vs. hypoxia-PBS. HD, high dose 100 µM Sanggenon C; p-, phospho; T-, total; AMPKα, AMP-activated protein kinase α; mTOR, mechanistic target of rapamycin; FOXO3a, forkhead box O3a; TUNEL, terminal deoxynucleotidyl-transferase-mediated dUTP nick-end labeling; LC3, microtubule-associated proteins 1A/1B light chain 3; CpC, compound C.