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Diosmetin has shown great potential in the control of several diseases. The aim of the present study was to evaluate the role of diosmetin as a candidate agent for the treatment of myocardial infarction which was mainly caused by hypoxia. The model of hypoxia-injured myocardial cells was established using the H9c2 cell line. Cell viability was determined using Cell Counting Kit-8, cell apoptosis was determined by Annexin V-FITC Apoptosis Detection Kit and cleaved caspase-3 level was assessed by western blot analysis. Autophagy was monitored using a commercial kit, and a well-established reporter system was used to confirm the role of diosmetin in autophagy. The activity of adenosine 5′-monophosphate-activated protein kinase (AMPK) signaling was detected by western blot analysis. Cell viability assay indicated that diosmetin alleviated hypoxia-induced cell death of H9c2 cells in a dose-dependent manner. Data of the apoptosis assay revealed that diosmetin reduced the proportion of apoptotic cells in the hypoxia-injured H9c2 cells. It was also found that the occurrence of autophagy was promoted when hypoxia-injured cells were treated with diosmetin alone, and results of the western blot analysis revealed that AMPK signaling was activated by diosmetin. Administration of diosmetin together with an inhibitor of autophagy (3-methyladenine, 3-MA) or AMPK (Compound C) was able to decrease the diosmetin-induced autophagy as well as the cytoprotective effects in the hypoxia-injured cells. Our study concluded that diosmetin exhibits a cytoprotective effect on hypoxia-injured myocardial cells by inducing autophagy and alleviating apoptosis. AMPK was demonstrated to regulate the observed effects caused by diosmetin. This investigation confirmed diosmetin as a promising drug candidate for myocardial infarction treatment. The present findings regarding the inherent molecular mechanisms involved in the protective effects of diosmetin promote the clinical application of diosmetin.
Myocardial infarction is defined worldwide as one of the most common cardiovascular diseases with an estimated number of cases of more than 600,000 in the US (
Diosmetin (3′,5,7-trihydroxy-4′-methoxy flavone) is the aglycone of the flavonoid glycoside diosmin, which is abundant in
Autophagy is characterized as a survival mechanism that is responsible for removal of misfolded or wrongly assembled proteins, clearance of damaged organelles and elimination of intracellular pathogens (
The present study was designed with an aim to explore the effects of diosmetin on hypoxia-injured myocardial cells. Subsequently, the potential involvement of autophagy in the diosmetin-mediated effects was focused on. Then the activity of AMPK signaling in diosmetin-treated cells was assessed to elucidate the intrinsic molecular mechanisms.
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 (
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) (
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
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 (
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 (
Myocardial infarction is globally recognized as one of the most common cardiovascular diseases consistently threatening human health (
The results of the present study revealed that diosmetin efficiently protected and increased the cell viability of H9c2 cells by inhibiting apoptosis indicated by decreased cleaved caspase-3. It was further found that diosmetin promoted cell autophagy, although hypoxia induced slight autophagy. Autophagy is characterized as a conserved process involving the degradation of cytoplasmic components (
ULK1, a protein kinase, is required for autophagy and has been reported as a substrate of adenosine monophosphate-activated protein kinase (AMPK) (
The
In conclusion, the present study revealed that diosmetin may protect myocardial cells from hypoxia-mediated cell death by inducing autophagy. Further investigation demonstrated that AMPK is involved in the regulation of diosmetin-induced autophagy. This study suggests diosmetin as a drug candidate for myocardial infarction treatment, and the results regarding AMPK contribute to the clinical application of diosmetin.
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No funding was received.
The datasets used and/analyzed during the current study are available from the corresponding author on reasonable request.
NZ was involved in the conception and design of the study. QS, YS and DH were responsible for the data acquisition, analysis and interpretation. QS wrote the manuscript.
Not applicable.
Not applicable.
The authors declare that they have no competing interests.
Effects of diosmetin on the H9c2 cell line. (A) Cells were treated with diosmetin at different concentrations (0, 5, 10 and 15 µg/ml) prior to being cultured in a hypoxic condition. Cells constantly cultured in a normoxic condition were considered as the non-treated group (NT). Cell viability was determined using CCK-8 assay. (B and C) Cell apoptosis was detected using a Annexin V-FITC Apoptosis Detection Kit. Double-staining cells were defined as apoptotic cells. (D and E) Cleaved caspase-3, an apoptosis-related protein, was assessed using western blot analysis. Three independent experiments were conducted for all assays. *P<0.05, **P<0.01, ****P<0.0001.
Diosmetin functions as an autophagy inducer. (A and B) The occurrence of autophagy was detected using a CYTO-ID® Autophagy detection kit in hypoxia-injured cells previously treated with diosmetin. Median fluorescence intensity (MFI) of channel FL1 (green fluorescence) was assessed using flow cytometry, and the higher fluorescence intensity represented stronger autophagy. (C) Images were captured using a fluorescence microscope; blue fluorescence represents Hoechst 33342 staining of cell nuclei and green fluorescence indicates CYTO-ID green staining of autophagic vesicles. (D) An independent Autophagy LC3 HiBiT Reporter Assay System based on 293T cells was performed to verify the role of diosmetin in autophagy; a lower luminescence signal represents stronger autophagy. At least six independent experiments were conducted for above assays. ***P<0.001, ****P<0.0001. To note, cells constantly cultured in a normoxic condition were considered as the non-treated group (NT).
Induction of autophagy is essential for the cytoprotective effects of diosmetin on hypoxia-injured cells. (A) Cells were treated with diosmetin alone or together with 3-MA prior to being cultured in a hypoxic condition. The cells constantly cultured in a normoxic condition were considered as the non-treated group (NT). Cell viability was determined using the CCK-8 assay. (B and C) Cell apoptosis was detected using the Annexin V-FITC Apoptosis Detection Kit. The double-staining cells were defined as apoptotic cells. (D and E) Cleaved caspase-3 was assessed by western blot analysis. Three independent experiments were conducted for all assays. *P<0.05, **P<0.01 ***P<0.001, ****P<0.0001.
AMPK is involved in the regulatory effects of diosmetin on autophagy and apoptosis. (A and B) Expression of AMPK and ULK1 as well as their phosphorylation levels (p-AMPK and p-ULK1) were detected by western blot analysis in diosmetin-treated and hypoxia-exposed cells. (C and D) Cells were treated with diosmetin alone or together with compound C prior to being cultured in a hypoxic condition. Autophagy was monitored using a CYTO-ID® Autophagy detection kit by flow cytometry and fluorescence microscopy. Blue fluorescence represents Hoechst 33342 staining of cell nuclei and green fluorescence indicates CYTO-ID green staining of autophagic vesicles. (E and F) Cell apoptosis was detected using the Annexin V-FITC Apoptosis Detection Kit. Three independent experiments were conducted for all assays. **P<0.01, ***P<0.001, ****P<0.0001. To note, cells constantly cultured in a normoxic condition were considered as the non-treated group (NT). AMPK, adenosine 5′-monophosphate-activated protein kinase; ULK1, Unc-51 like autophagy activating kinase 1.