1 INTRODUCTION

Ischemic heart diseases frequently cause a series of clinical symptoms, such as myocardial infarction, cardiac failure, and cardiac insufficiency. Hypoxic–ischemic is the early stage of acute myocardial infarction.1, 2 Recently, a large number of studies have demonstrated that abnormal apoptosis and necrosis of cardiomyocytes are invariably appeared under hypoxic–ischemic environment, which are closely related to cardiac dysfunction.3 Hypoxia is the leading cause of cardiomyocyte apoptosis, which is accompanied by the release of cytochrome C from mitochondria to cytosol and the activation of the caspase cascade.4, 5 Thus, inhibition of hypoxia-induced cardiomyocyte apoptosis is considered as an important treatment method for ischemic heart diseases. However, the mechanism for hypoxic myocardial injury is largely unclear and there is still a lack of effective medicines for myocardial protection and anti-apoptosis.

Autophagy (self-eating), a lysosomal degradation pathway, is a double-edged sword to regulate cell survival or death under different conditions.6 Autophagy frequently appears along with cell apoptosis.7 Recent studies extensively reported that autophagy and apoptosis not only can work synergistically but also can act antagonistically to each other.8 AMP-activated protein kinase (AMPK), a modulator of cell energy metabolism and survival, is involved in the promotion of a variety of cellular autophagy via decreasing ATP/AMP ratio and suppressing the activation of mTOR.6, 9 However, the role of AMPK and the mechanism of cell autophagy in the hypoxic-induced myocardial apoptosis are largely unknown.

Diosmetin (DIOS), a kind of natural flavonoid, has been reported to possess various pharmacological activities including anti-oxidation,10 anti-tumor,11-13 anti-inflammation,14 anti-bacteria, and anti-allergy.15, 16 A number of studies have reported that DIOS plays a role in anti-apoptosis to protect many kinds of non-cancerous cells from injury,17, 18 and induces the apoptosis of tumor cells.11, 12 To date, the function and mechanism of DIOS in the hypoxic-induced myocardial injury are unknown. The present study aims to investigate the role and internal mechanism of DIOS in two cardiomyocyte cell lines in a hypoxic environment and in a rat model of isoprenaline (ISO)-induced myocardial hypoxic injury. Our study could provide a potential drug and therapeutic target for hypoxia-induced myocardial diseases.

2 MATERIALS AND METHODS 2.1 Culture and groups

AC16 and HCM-a, two human cardiomyocyte cell lines were purchased from American Type Culture Collection (ATCC, Bethesda, MD) and grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS, Gibco, Carlsbad, CA), 50 U/mL penicillin, and 50 μg/mL streptomycin at 37°C in a humidified atmosphere with 5% CO2. Cells in logarithmic phase were inoculated in cell culture dishes until 80% confluence. To mimic hypoxic conditions in vitro, AC16 and HCM-a cells were exposed to oxygen–glucose deprivation (1% O2, 5% CO2, low glucose medium) for 24 h.19, 20

DIOS was obtained from Shanxi Huike Botanical Development Co., Ltd (Shanxi, China). It was dissolved in 0.1% DMSO (Sigma–Aldrich, St. Louis, MO) and with DMSO as a negative control.21 Cells were divided into three groups: Control, HYP, and DIOS+HYP. Cells in the Control group did not give any treatment. Cells in the HYP group were cultured under hypoxia environment for 24 h. Cells in the DIOS+HYP group were treated with 40 μM DIOS solution under hypoxia environment. Furthermore, 3-methyladenine (3-MA; 10 mM, Sigma–Aldrich, Santa Clara, CA), an inhibitor of autophagy, or Compound C (5 μM), an inhibitor of AMPK signaling, was added to cells treated with DIOS and HYP.22

2.2 Hypoxic–ischemic rat model inducting and grouping

Male Sprague–Dawley (SD) rats (7–10 weeks old, 259–284 g, n = 30) were purchased from the Laboratory Animal Center of Shanxi Medical University. Rats received standard feed and water ad libitum. Rats were housed in M-6-transparent plastic boxes at room temperature [(22.0 ± 0.5)°C] and kept in a humidity of 55 ± 10% with 12 h/12 h light/dark cycle.

We validated the effect of DIOS on hypoxic–ischemic-induced myocardial injury by ISO in vivo. Firstly, the rats were randomly divided into four groups: control (n = 6), Sham (n = 6), ISO (n = 9), and ISO + DIOS (n = 9). The animals in the control group received no treatment. The animals in the Sham group received 1% DMSO (subcutaneous injection [sc], 0.25 mL /100 g/day) as the negative control. Rats in the ISO group received 85 mg/kg/day ISO (sc) for 2 days and intraperitoneally injected (ip) with saline for 7 days. The animals in the ISO + DIOS group were treated with 85 mg/kg/day ISO (sc) for 2 days and 100 mg/kg/day DIOS (ip) for 7 days. On day seven, the changes of the mean arterial pressure (MAP), left ventricular systolic pressure (LVSP), and the heart rate (HR) were monitored. All the alive rats were anesthetized with pentobarbital sodium, after collecting the blood samples, all animals were sacrificed at day 7 and the myocardial tissues were obtained. Hematoxylin and Eosin (H&E) staining, Caspase-3 immunohistochemistry (IHC) and Western blot assays were used to assess the changes of myocardial apoptosis and autophagy as well as AMPKα activation. All animal experiments were approved by the Ethics Committee of The Inner Mongolia People’s Hospital (Approval NO. 20191210L) and complied with Regulations for the Administration of Affairs Concerning Experimental Animals.

2.3 Cell counting kit (CCK)-8 and MTT assays

5 × 103 cells were inoculated into 96-well plates and cultured for indicated times, and cell viability was tested by CCK-8 and MTT assays. For CCK-8 assay, 10 μL CCK-8 solution (Beyotime, Shanghai, China) was added to each well and incubated for 2 h at 37°C. The absorbance at 450 nm was detected using a microplate reader. For MTT assay, 10 μL MTT (5 mg/mL; Sigma–Aldrich) was added to each well and cultured at 37°C for 4 h. After discarding the supernatant, 100 μL DMSO was added to dissolve the formazan crystals. Absorbance value (OD) at 570 nm was measured using a microplate reader.

2.4 Cell apoptosis was detected by flow cytometry

The apoptosis of AC16 and HCM-a cells was analyzed by flow cytometry using an Annexin V Fluorescein Isothiocyanate (FITC) Apoptosis Detection Kit (BD Pharmingen, San Diego, CA) according to the manufacturer's instructions. In brief, each group of cells were collected and resuspended in 100 × binding buffer at a density of 1 × 106 cells per ml. Then, cells were incubated with Annexin V-FITC and propidium iodide (PI) for 15 min and analyzed with a BD FACSCalibur™ system (BD, Franklin Lakes, NJ). Apoptotic cells were those with Annexin V+PI+ and Annexin V+PI−.

2.5 Western blot

Cells and rat myocardial tissues in each group were lysed, and total protein was separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred onto nitrocellulose membranes (Millipore, Temecula, CA). The membranes were blocked by 3% nonfat milk for 1.5 h at room temperature, and then incubated with primary antibodies (Abcam, Cambridge, MA) including rabbit anti-cleaved-caspase3 (1:1000), Bcl-2 (1:900), Bax (1:800), AMPKα, p-AMPKα (1:1200), (1:1100), Cleaved caspase3 (1:800), and GAPDH (1:2000) at 4°C overnight. Then, the membranes were incubated with horseradish peroxidase-conjugated secondary antibody (donkey-anti-rabbit, 1:18,000, Santa Cruz Biotechnology, Santa Cruz, CA) for 1.5 h at room temperature. The protein bands were visualized using an enhanced chemiluminescence detection system. Densitometry values were normalized to levels of GAPDH. Quantitation analysis for western blot was performed using Image-Pro Plus 6.0 software (Media Cybernetics, Inc., Rockville, MD).

2.6 Hochest33342 staining

The cells were seeded in 6-well plates (3 × 105 cells/well) and stained with Hoechst 33342 (5 μg/mL; Thermo Fisher Scientific, Carlsbad, CA) for 1 h at 37°C. Images of the cells were acquired with an inverted fluorescence microscope (Nikon TE2000; Nikon Corporation, Tokyo, Japan) after washing twice with PBS. The number of apoptotic cells was counted in five different fields of each group.

2.7 Cell immunofluorescence

The cells in each group were grew on cover slips and fixed with formaldehyde. The cell slips were blocked by 10% FBS for 2 h at room temperature. After removing the blocking buffer, sections were incubated by the primary antibodies rabbit anti-LC3 (1: 800, Abcam). After washing three times with 0.01 M phosphate-buffered saline, the sections were incubated with fluorescein-labeled secondary antibody for 2 h at room temperature. The sections were sealed up with anti-fluorescence quenching fluid. Tissues were examined under a Nikon fluorescence microscope.

2.8 H&E staining

H&E staining was performed on the myocardial tissues of rats in different groups. Samples were fixed in 2% paraformaldehyde at 4°C overnight, dehydrated by a graded series ethanol (50%, 70%, 80%, 90% ethanol, each step for 15 min; and 100% ethanol for 20 min), soaked twice in acetone (15 min each), and embedded in araldite. After 48 h of polymerization at 65°C, the embedded specimens were cut into ultrathin slices (70 nm) with an ultramicrotome (EM UC7, Leica, Wetzlar, Germany). The slices were stained with hematoxylin and eosin, and observed under a microscope (YYS-190E, Shanghai Optical Instrument, Shanghai, China).

2.9 Immunohistochemical (IHC) staining

IHC staining for caspase-3 was performed on the myocardial tissues in each group. The rat myocardial tissues were embedded in paraffin and sliced. The thickness of each tissue section was 5 μm. The primary antibody was rabbit anti-rat caspase-3 antibody (Abcam). The process of IHC was performed as described previously.21

2.10 Enzyme-linked immunosorbent assay (ELISA) assay

Ischemia modified albumin (IMA) is an important marker for myocardial ischemia.23 IMA changes in the serum of rats were tested by the IMA ELISA Kit (RenjieBio, Shanghai, China). Blood samples at day 7 were obtained from the carotid artery of rats and centrifuged for 20 min at 2000 rpm. Then, the supernatant was collected and analyzed according to the manufacturer's instructions.

2.11 Data statistics and analysis

All results are presented as the mean ± standard deviation (SD). Statistical analyses of the data were performed with SPSS 11.5 (SPSS Inc., Chicago, IL). Statistical significance was determined using one-factor ANOVA followed by Bonferroni post hoc tests. P values < 0.05 were considered statistically significant.

3 RESULTS 3.1 DIOS upregulated the viability of cardiomyocytes in a hypoxic environment

The cytotoxicity of DIOS was evaluated both in AC16 and HCM-a cells, and the results indicated that there was no cytotoxicity when DIOS concentration was no more than 40 μM (Figure 1A). Moreover, the effect of different concentrations of DIOS (≤40 μM) on HYP-treated AC16 and HCM-a cells was evaluated further with CCK-8 assay. As shown in Figure 1B, DIOS promoted cell viability in a dose-dependent manner in cells exposed to hypoxia. The viability of AC16 and HCM-a cells treated with 40 μM of DIOS in a hypoxic environment was determined by the CCK-8 assay at different time points (24, 48, 72, and 96 h). Compared with the control group, cell viability in the HYP group was suppressed (Figure 1C,D, P < 0.05), however, DIOS significantly facilitated the viability of these two cells. Whereas DIOS did not show any effect on the viability of cells in normal environment (Figure 1C,D, P < 0.05). Additionally, MTT assay was also used to examine the role of DIOS on the viability of AC16 and HCM-a cells, and the results were consistent with the CCK-8 assay (Figure 1E, P < 0.05). These results confirmed that DIOS played a protective effect on the hypoxia-injured cardiomyocytes.

Diosmetin (DIOS) increased the viability of hypoxic cardiomyocytes. (A) The cell viability was detected by CCK-8 assay in AC16 and HCM-a cells at 72 h after addition of different concentrations of DIOS. (B) CCK-8 assay was used to detect the viability of hypoxic cardiomyocytes at 72 h after addition of different concentrations of DIOS. Cell viability was detected by CCK-8 assay at 24, 48, 72, and 96 h (C, D) and with MTT assay at 48 h (E) after addition of 40 μM DIOS. HYP: hypoxia. *, p < 0.05 versus control; #, p < 0.05 versus HYP. Data are expressed as means ± SD. Each experiment was independently repeated at least three times

3.2 Diosmetin inhibited apoptosis of hypoxic cardiomyocytes

A number of studies have reported that DIOS plays an anti-apoptotic role to protect many kinds of non-cancerous cells from injury.17, 18 In the present study, the apoptosis rates of AC16 and HCM-a cells were upregulated in the HYP group compared with those in the control group (Figure 2A, P < 0.05). Intriguingly, DIOS significantly attenuated cell apoptosis induced by hypoxia (Figure 2A, P < 0.05). The levels of cleaved-caspase 3 and the ratio of Bax to Bcl-2 in the two cells were all downregulated in DIOS group compared to those in the HYP group (Figure 2B–D, P < 0.05). Furthermore, to examine change in the nucleus morphology, AC16 and HCM-a cells were stained with hochest33342. The blue fluorescence of apoptotic cells is enhanced than the normal cells, and exhibits nuclear fragmentation and condensation.24 Compared with the control cells, the fluorescence intensity was much brighter in AC16 and HCM-a cells treated with HYP. And AC16 and HCM-a cells in the DIOS group displayed relatively weak fluorescence intensity compared with that in the HYP group (Figure 2E,F, P < 0.05). These results indicated that DIOS could attenuate hypoxia-injured cardiomyocyte apoptosis.

Diosmetin (DIOS) inhibited the apoptosis of hypoxic cardiomyocytes. (A) The apoptosis of the cardiomyocytes was detected by flow cytometry. (B) The expression levels of cleaved-caspase3 were tested by Western blot. (C) The ratio of Bax/Bcl-2 was measured by Western blot. (D) The images of Western blot. (E) and (F) The apoptosis-like nuclear of the cardiomyocytes were analyzed using hochest33342 staining. *, p < 0.05 versus control; #, p < 0.05 versus HYP. HYP: hypoxia. Data are expressed as means ± SD. Each experiment was independently repeated at least three times

3.3 Diosmetin induced autophagy of hypoxic cardiomyocytes

The present results showed that compared with the control group, LC3 puncta of AC16 and HCM-a cells were reduced in the HYP group (Figure 3A,B, P < 0.05). DIOS increased LC3 puncta compared with that in the HYP group (Figure 3A,B, P < 0.05). Western blot results showed that the protein expression levels of Beclin1 and the LC3-II/LC3-I ratio were upregulated but the expression level of p62 was downregulated in the DIOS group when compared with that in the HYP group (Figure 3C–F, P < 0.05). These results demonstrated that DIOS could induce the autophagy of hypoxic cardiomyocytes.

Diosmetin (DIOS) induced the autophagy of hypoxic cardiomyocytes. (A) The number of LC3 puncta per cell was calculated. (B) The localization of the LC3-puncta was observed by cell immunofluorescence. (C) The expression levels of Beclin1 were tested by Western blot. (D) The LC3-II/I ratio was measured by Western blot. (E) The expression levels of p62 were detected by Western blot. (F) The images of Western blot. *, p < 0.05 versus control; #, p < 0.05 versus HYP. HYP: hypoxia. Data are expressed as means ± SD. Each experiment was independently repeated at least three times

3.4 Diosmetin alleviated hypoxia-induced cardiomyocyte apoptosis via inducing autophagy

To confirm the role of DIOS on autophagy, 10 mM 3-MA, an inhibitor of autophagy, was co-treated with DIOS in hypoxia-induced AC16 cells. The results indicated that the autophagy markers Beclin1 and LC3-II/I were all downregulated in the DIOS +3-MA + HYP group compared to those in the DIOS+HYP group. (Figure 4A,B,F, P < 0.05), while the apoptosis rates of AC16 cells were upregulated in the DIOS+3-MA + HYP group (Figure 4C, P < 0.05). Compared with the DIOS+HYP group, the expression level of cleaved-caspase 3, Bax/Bcl-2 ratio, and the fluorescence intensity were all upregulated in the DIOS+3-MA + HYP group (Figure 4D–H, P < 0.05). These findings suggested that 3-MA partly eliminated the inhibition of DIOS on hypoxia-induced cardiomyocyte apoptosis.

The role of autophagy in diosmetin (DIOS)-regulated apoptosis of AC16 cells under hypoxia was studied. (A) The expression levels of Beclin1 were tested by Western blot. (B) The ratio of LC3-II/I was measured by Western blot. (C) The apoptosis of cardiomyocytes was examined by flow cytometry. (D) The expression levels of cleaved-caspase3 were determined by Western blot. (E) The ratio of Bax/Bcl-2 was measured by Western blot. (F) The images of Western blot. (G) and (H) The apoptosis-like nuclear of the cardiomyocytes were observed using hochest33342 staining. HYP: hypoxia. DIOS: diosmetin. #, p < 0.05 versus HYP; *, p < 0.05 versus DIOS+HYP. Data are expressed as means ± SD. Each experiment was independently repeated at least three times

3.5 Diosmetin alleviated hypoxia-induced cardiomyocyte apoptosis via inducing AMPK activation-induced autophagy

Autophagy has been reported to be mediated by the cellular energy sensor AMPK.25 Western blot analysis indicated that the level of p-AMPKα/AMPKα were upregulated in the DIOS group compared with the HYP group (Figure 5A,B, P < 0.05). Thus, Compound C, an inhibitor of AMPK, was used to confirm the effects of AMPK activation on the DIOS-treated cardiomyocytes. In comparison to the DIOS+HYP group, Compound C significantly weakened the effects of DIOS on cell autophagy and the levels of Beclin1 and LC3-II/I in AC16 cells (Figure 5C–E,H, P < 0.05). Furthermore, Compound C also attenuated the inhibitory role of DIOS on cleaved-caspase3 levels, Bax/Bcl-2 ratio, fluorescence intensity and the apoptosis rate in the cells (Figure 5F–K, P < 0.05). These results indicated that DIOS inhibited hypoxia-induced cardiomyocyte apoptosis via accelerating AMPK activation-induced autophagy.

Diosmetin (DIOS) alleviated hypoxia-induced myocardial apoptosis via inducing AMPK activation-induced autophagy. (A) The ratio of p-AMPKα to AMPKα was measured by Western blot. (B) The western blot images of p-AMPKα and AMPKα. (C) The number of LC3 puncta per cell was calculated. (D) The expression levels of Beclin1 were tested by Western blot. (E) The ratio of LC3-II/I was measured by Western blot. (F) The expression levels of cleaved-caspase3 were assayed by Western blot. (G) The ratio of Bax to Bcl-2 was measured by Western blot. (H) The western blot images of Beclin1, LC3-II/I, cleaved-caspase3, Bax and Bcl-2. (I) The apoptosis of cardiomyocytes was examined by flow cytometry. (J) and (K) The apoptosis-like nuclear of the cardiomyocytes were observed by hochest33342 staining. *, p < 0.05 versus control; #, p < 0.05 versus HYP; &, p < 0.05 versus DIOS+HYP. HYP: hypoxia. Compound C: An inhibitor of AMPK signaling. Data are expressed as means ± SD. Each experiment was independently repeated at least three times

3.6 DIOS inhibited the apoptosis and promoted the autophagy of myocardia cells in ISO-induced hypoxic rats

In order to verify the role of DIOS in vivo, the hypoxic–ischemic myocardial injury rat models were built. After 7 days treatment, all the mice were survival. H&E staining confirmed that ISO could injure the myocardium (Figure 6A). The levels of IMA were significantly upregulated by ISO (Figure 6B, P < 0.05). Additionally, we found that caspase-3 levels were upregulated (Figure 6C,D, P < 0.05), the Bax/Bcl-2 ratio was significantly increased (Figure 6E,I, P < 0.05), but the expression level of Beclin1 and LC3-II/LC3-I ratio were downregulated (Figure 6F,G,I, P < 0.05) in the myocardium of ISO-treated rats compared with those in the control group. However, the myocardial injury was repaired (Figure 6A), and the IMA levels were decreased in the ISO + DIOS group (Figure 6B, P < 0.05). Furthermore, the levels of apoptosis markers (caspase-3 and Bax/Bcl-2 ratio) were decreased and the levels of autophagy markers (Beclin1 and LC3-II/LC3-I ratio) were increased (Figure 6C–G,I, P < 0.05) in rats in the ISO + DIOS group when compared with those in the ISO group. Additionally, the p-AMPKα/AMPKα ratio was upregulated in the DIOS-injected ISO rats compared with those in the ISO group (Figure 6H,I, P < 0.05). All the findings demonstrated that DIOS inhibited apoptosis, accelerated autophagy, and activated AMPKα pathway in ISO-induced hypoxic rats in vivo.

Diosmetin (DIOS) alleviated isoprenaline (ISO)-induced hypoxic–ischemic injury in rats. (A) The effects of ISO and/or DIOS on the histology and morphology of myocardial tissues of rats were examined by H&E staining. (B) The serum levels of IMA were tested by ELISA, (C) and (D) IHC was used to detect the apoptosis levels in myocardial tissues by determining caspase-3 level. (E-H) The levels of Bax/Bcl-2, Beclin1, LC3-II/I, and p-AMPKα/AMPKα in myocardial tissues were detected by Western blot. (I) the images of Western blot. *, p < 0.05 versus control; #, p < 0.05 versus ISO. Data are expressed as means ± SD. Each experiment was independently repeated at least three times

4 DISCUSSION

In the present study, DIOS played an anti-apoptosis role in the hypoxic cardiomyocytes in vitro and ISO-induced hypoxic rats in vivo for the first time. More importantly, AMPK activation-mediated cell autophagy was confirmed to be a key mechanism of DIOS-alleviated cardiomyocyte apoptosis under hypoxic environment.

Although DIOS frequently induced the apoptosis of tumor cells,11, 12 it has been widely reported to inhibit the abnormal apoptosis of non-cancerous cells. Studies have demonstrated that DIOS could inhibit H2O2-induced apoptosis and intracellular ROS production of hepatocytes,26 and suppress endotoxin-induced apoptosis, inflammation, and oxidation of hepatocytes.18 DIOS relieved the renal tubule apoptosis induced by ischemia/reperfusion.17 In vivo, the apoptosis of retinal cells in SD rats was attenuated by DIOS.27 Consistent with these studies, our results confirmed that 40 μM DIOS inhibited the apoptosis of hypoxic cardiomyocytes. Furthermore, the expression levels of apoptosis markers such as cleaved-caspase3 and Bax/Bcl-2 ratio were also downregulated by DIOS treatment in hypoxic cardiomyocytes. Besides, according to the results of hochest33342 staining, the hypoxic-induced apoptosis was significantly decreased after DIOS treatment. Thus, these findings confirmed that DIOS played an inhibitory role in the apoptosis of hypoxia-induced cardiomyocytes. The dose of DIOS used in this study referred many articles.. Studies have revealed that 20–40 μM DIOS increased the viability of hepatocytes in the presence of H2O2,26 however, 20 μg/mL (66.7 μM) DIOS inhibited the proliferation of human hepatocellular carcinoma HepG2 cells.28 Satué et al.29 found that DIOS at greater than or equal to 100 μM revealed a toxic effect on osteoblast cells. In the present study, we found that 40 μM DIOS increased the viability of hypoxic cardiomyocytes. Taken together, these results indicated that DIOS accelerated the viability and attenuated the apoptosis of cardiomyocytes affected by hypoxia.

Cell autophagy could be induced by DIOS. Recent studies have shown that DIOS triggered the autophagy of hepatocellular carcinoma cells by via decreasing the phosphorylation of Akt and Erk1/2 and then stimulating AMPK phosphorylation and decreasing the expression of mTOR.28 It has been reported that the autophagy of liver cells could also be induced by DIOS.30 Similarly, in the present study, the levels of Beclin1 and LC3-II/LC3-I were upregulated but p62was downregulated in the DIOS-treated cells under hypoxia. Additionally, DIOS induced the phosphorylation of AMPKαwhich indicated the activation of AMPK signaling. It was confirmed that DIOS could trigger the autophagy in hypoxia-induced cardiomyocytes.

It has been extensively reported that autophagy and apoptosis work antagonistically to each other.31-33 Foglio et al.34 found that exogenous high mobility group box-1 protein (HMGB1) induced the autophagy of cardiomyocytes but reduced cardiomyocyte apoptosis in the border zone of infarcted mice, and the upregulation of autophagy by HMGB1 treatment was related to the activation of AMPK. In retinal Müller cells, high glucose-induced apoptosis was inhibited by activating the AMPK/mTOR signaling pathway-induced autophagy.32 Yao et al.31 demonstrated that exacerbation of cardiac cell apoptosis could inhibit autophagy under diabetic condition. Similarly, the present study revealed that the activation of autophagy and AMPK signaling by DIOS remarkably inhibited the apoptosis of the hypoxia-induced cardiomyocytes. ISO, a myocardial necrosis inducer,35 has been reported to induce apoptosis of cardiomyocytes, but this effect was attenuated by activating kinase 1-mediated autophagy.36 In the present study, the results also suggested that ISO induced myocardial apoptosis but suppressed autophagy in vivo; however, DIOS remarkably reversed the effects of ISO on myocardial apoptosis and autophagy. Furthermore, this study confirmed that DIOS inhibited apoptosis of hypoxia-induced cardiomyocytes by accelerating autophagy via activating the AMPK signaling. AMPK inhibitor, Compound C, was used to block AMPK activation. As was expected, Compound C inhibited the effects of DIOS on the autophagy and apoptosis of cardiomyocytes. Moreover, 3-MA, an autophagy inhibitor, reversed the influences of DIOS on cardiomyocyte apoptosis. All these results in the present study indicated that DIOS inhibited the apoptosis of hypoxia-induced cardiomyocytes by inducing autophagy via activating AMPK.

Although this study found that DIOS exhibited potent activities on inhibiting apoptosis and inducing autophagy of hypoxia-induced cardiomyocytes, while, there are many limitations in present study, further studies need to be done to develop DIOS as a drug. For example, on the one hand, more mechanism studies need to be done to clarify the mechanism of DIOS protecting cardiomyocytes against hypoxic environment, just as the effect of DIOS on mitochondrial study.37 On the other hand, more studies on the safety and metabolism evaluation in more animals even in humans need to be done in the future.

5 CONCLUSIONS

The present study investigated the role and the internal mechanism of DIOS in cardiomyocytes under hypoxic environment. We found that DIOS inhibited the apoptosis and induced the autophagy of hypoxia-induced cardiomyocytes, and DIOS alleviated hypoxia-induced myocardial apoptosis via inducing autophagy through AMPK activation. Our study provides novel and potential therapeutic targets for hypoxia-induced myocardial injury.

CONFLICT OF INTEREST

The authors declare no conflict of interest.