Protective effect of α-mangostin against CoCl2-induced apoptosis by suppressing oxidative stress in H9C2 rat cardiomyoblasts

  • Authors:
    • Zhao Fang
    • Wanjun Luo
    • Yanli Luo
  • View Affiliations

  • Published online on: March 6, 2018     https://doi.org/10.3892/mmr.2018.8680
  • Pages: 6697-6704
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Abstract

Garcinia mangostana (a fruit) has been commonly used as a traditional drug in the treatment of various types of diseases. The aim of the present study was to evaluate the potential protective effect of α‑mangostin (α‑MG), a primary constituent extracted from the hull of the G. mangostana fruit (mangosteen), against CoCl2‑induced apoptotic damage in H9C2 rat cardiomyoblasts. α‑MG was demonstrated to significantly improve the viability of the CoCl2‑treated cells by up to 79.6%, attenuating CoCl2‑induced damage. Further studies revealed that α‑MG exerted a positive effect in terms of decreased reactive oxygen species generation, malondialdehyde concentration, cellular apoptosis, and increased superoxide dismutase activity. Furthermore, treatment with CoCl2 increased the cleavage of caspase‑9, caspase‑3 and apoptosis regulator BAX, and reduced apoptosis regulator Bcl‑2 in H9C2 cells, as measured by reverse transcription‑quantitative polymerase chain reaction and western blotting, which were significantly reversed by co‑treatment with α‑MG (0.06 and 0.3 mM). In conclusion, these results demonstrated that α‑MG protects H9C2 cells against CoCl2‑induced hypoxic injury, indicating that α‑MG is a potential therapeutic agent for cardiac hypoxic injury.

Introduction

Ischemic heart disease (IHD), caused by myocardial ischemic injuries, frequently presents as coronary heart disease and acute myocardial infarction (1,2). IHD was reported to cause 7.4 million mortalities in 2015, and is the leading cause of mortality globally according to the World Health Organization, representing a major socioeconomic burden. Therefore, finding novel therapies to reduce the incidence of IHD is urgently required.

Previous studies demonstrated that myocardial hypoxia-ischemia is an important event during the entire process of IHD (35), causing extensive cardiomyocyte death (6,7). Evidence suggests that oxidative stress is involved in myocardial ischemia and that antioxidant treatment may be beneficial in cardiac damage during ischemia (79). Markers of oxidative stress, including reactive oxygen species (ROS), malondialdehyde (MDA) and superoxide dismutase (SOD), are frequently used to monitor mitochondrial oxidative damage (10,11). Furthermore, it has been demonstrated that apoptosis serves an important role in ischemic-hypoxic myocardial injury (12,13); when the mitochondrial membrane potential is lost, the process of apoptosis is activated, leading to cell death (14).

Garcinia mangostana is a tropical tree commonly present in Southeast Asian countries, including Vietnam, the Philippines and Thailand. The pericarp of mangosteen, the fruit of G. mangostana, has been used as indigenous medicine for the treatment of skin infections, wounds and diarrhea for a number of years (15,16). Previously, α-mangostin (α-MG), a major constituent extracted from the hull of mangosteen (17,18), was demonstrated to possess a variety of pharmacological properties, including anti-inflammatory (19), antitumor (20), cardioprotective (2124), antidiabetic (25), antibacterial (26), antifungal (27), antioxidant (28) and antiobesity effects (29). α-MG has been demonstrated to arrest the cell cycle and induce apoptosis in various cancer cells via the mitochondrial pathway (20), and its prevention of cisplatin-induced apoptotic death in LLC-PK1 porcine kidney cells has been associated with the inhibition of ROS production (30). Previous in vivo studies have additionally demonstrated the cardioprotective effects of α-MG via the reduction of ROS generation (2224). However, the mechanism underlying the protective effect of α-MG on cardiomyocytes remains to be fully elucidated.

CoCl2, a chemical hypoxia-mimicking agent, is able to simulate the effect of ischemic-hypoxic myocardial injury (31). The H9C2 cell line is derived from rat embryonic cardiomyocytes, and may be used to investigate the electrophysiological and biochemical characteristics of myocardial tissue.

Myocardial cells frequently suffer from ischemic and hypoxic death during the process of cardiac surgery; therefore, the use of α-MG to potentially decrease the damage in cardiac surgery may be beneficial. To investigate this, in the present study, α-MG was added prior to CoCl2 to H9C2 cells. CoCl2-treated H9C2 cells were used as a model to evaluate the effects of α-MG on cardiomyoblasts exhibiting chemical hypoxia-induced injury.

Materials and methods

Materials

A Cell Counting kit-8 (CCK-8, cat no. ck04) was purchased from Dojindo Molecular Technologies, Inc., (Kumamoto, Japan). α-MG and CoCl2 were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). Antibodies against apoptosis regulator Bcl-2 (rabbit anti-Bcl-2; cat no. 2870; 1:1,000), apoptosis regulator BAX (rabbit anti-Bax; cat no. 2772s; 1:1,000), rabbit anti-cleaved caspase-9 (cat no. 9507; 1:1,000), rabbit anti-cleaved caspase-3 (cat no. 9662; 1:1,000) and mouse anti-β-actin (cat. no. 3700) were purchased from Cell Signaling Technology, Inc., (Danvers, MA, USA). Annexin V/propidium iodide (PI) was purchased from BD Biosciences (Franklin Lakes, NJ, USA); and the ROS detection kit was purchased from Beyotime Institute of Biotechnology (Haimen, China).

Cell culture and treatment

H9C2 myocardial cells were purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). Briefly, the H9C2 cells were cultured in high-glucose Dulbecco's modified Eagle's medium (Thermo Fisher Scientific, Inc., Waltham, MA, USA) containing 10% fetal bovine serum (Invitrogen; Thermo Fisher Scientific, Inc.) in 6-well plates with 5% CO2 and 95% air at 37°C. The medium was changed every 2–3 days.

To determine the appropriate treatment conditions, chemical hypoxia was achieved by adding different concentrations of CoCl2 (50, 200, 400, 600 and 800 µM) to H9C2 cells for 24 h as the preliminary experiment (32). The proper concentration was determined by the CCK-8 assay. In order to investigate the effects of α-MG following CoCl2-induced cell injury, CoCl2-treated H9C2 cells were maintained in complete medium with 0.012, 0.06, 0.3, 0.6 or 1.2 mM α-MG. The control cells were incubated without α-MG or CoCl2.

Experimental design

According to the results of the CCK-8 assay, H9C2 cells were randomly divided into the following groups: Group I (Control), cells treated without α-MG or CoCl2; group II, cells treated with CoCl2 alone; group III, cells pretreated with 0.06 mM α-MG for 24 h and with CoCl2 over the next 24 h; and group IV, cells pretreated with 0.3 mM α-MG for 24 h and with CoCl2 over the next 24 h.

Cell viability assay

CCK-8 was used to investigate the viability of H9C2 cells cultured in 96-well plates at a density of 5,000 cells/well. When the cells had grown to 90% confluence, 600 µM CoCl2 was added into the plate treated for 24 h at 37°C, then cell injury was induced. Thereafter, 10 µl CCK-8 solution was added to each well and the cells were incubated for a further 2 h at 37°C. Absorbance was measured at 450 nm with a microplate reader. The mean optical density (OD) of four wells in each group was used to calculate cell viability as follows: Cell viability (%)=(ODtreatment group/ODcontrol group) ×100.

Determination of ROS, MDA and SOD levels

The dichlorofluorescein diacetate (DCFH-DA) method was used to detect the level of intracellular ROS (33). Intracellular ROS levels were determined by measuring the oxidative conversion of cell permeable 2′,7′-DCFH-DA to fluorescent dichlorofluorescein (DCF) in a BioTek Synergy 2 microplate reader (BioTek Instruments, Inc., Winooski, VT, USA). H9C2 cells were seeded (5×105 cells/well), α-MG (0.06 or 0.3 mM) was added to the plates for 24 h and then the CoCl2-induced cell injury protocol was performed. The cells were incubated as previously described. The cells were washed with D-Hank's (Thermo Fisher Scientific, Inc.) and incubated with DCFH-DA at 37°C for 20 min. The DCF fluorescence distribution of 2×104 cells was detected by a microplate system at an excitation wavelength of 488 nm and an emission wavelength of 535 nm. The MDA concentration was measured by Lipid Peroxidation MDA Assay kit (Beyotime Institute of Biotechnology; cat no. S0131) and SOD activity was measured using the Superoxide Dismutase Assay kit (Beyotime Institute of Biotechnology; cat no. S0101), according to the manufacturer's protocols.

Morphological assessment of apoptotic cells by Annexin V-fluorescein isothiocyanate (FITC) and PI double staining

Apoptosis in H9C2 cells was quantified using Annexin V-FITC and PI double staining according to standard procedures using the aforementioned kit. Cell apoptosis was analyzed using CellQuest Pro software version 5.1 (BD Biosciences). Cellular fluorescence was measured by flow cytometer (FACS Calibur, BD Biosciences).

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR)

Total RNA was extracted by TRIzol (Invitrogen; Thermo Fisher Scientific, Inc.). A total of 100 ng total RNA was used for cDNA synthesis using the Stratagene AffinityScript qPCR cDNA Synthesis kit (Agilent Technologies, Inc., Santa Clara, CA, USA). The cDNA samples were diluted 10-fold with nuclease-free H2O and 2 µl diluted template was combined with Brilliant III Ultra-Fast SYBR® Green qPCR Master Mix (Applied Biosystems; Thermo Fisher Scientific, Inc.). β-actin was used as an internal reference control. The relative expression of target genes was determined by the 2−∆∆Cq method (34). The qPCR cycling conditions comprised initial denaturation for 3 min at 95°C, followed by 45 cycles at 95°C (10 sec) and 58°C (45 sec); data were acquired at the end of the annealing/extension phase. Melt curve analysis was performed at the end of each run between 58 and 95°C. The gene primer sequences are exhibited in Table I. Data were analyzed using Microsoft Excel 2013 (Microsoft Corporation, Redmond, WA, USA).

Table I.

Primer sequences of Bcl-2, Bax, caspase-3, caspase-9 and β-actin.

Table I.

Primer sequences of Bcl-2, Bax, caspase-3, caspase-9 and β-actin.

GenePrimer sequences
Bcl-2FP: 5′-GGATGACTGAGTACCTGAA-3′
RP: 5′-GCCATATAGTTCCACAAAGG-3′
BaxFP: 5′-GATGAACTGGACAACAACAT-3′
RP: 5′-CACGGAAGAAGACCTCTC-3′
Caspase-3FP: 5′-ATTATGGAATTGATGGATAGTGTT-3′
RP: 5′-GTAGTCGCCTCTGAAGAA-3′
Caspase-9FP: 5′-ACTGCCTCATCATCAACA-3′
RP: 5′-GTTCTTCACCTCCACCAT-3′
β-actinFP: 5′-CGTAAAGACCTCTATGCCAACA-3′
RP: 5′-AGCCACCAATCCACACAGAG-3′

[i] FP, forward primer; RP, reverse primer; Bax, apoptosis regulator BAX; Bcl-2, apoptosis regulator Bcl-2.

Western blot analysis

Total proteins were extracted from cells using cell lysis buffer [50 mM Tris-HCl, (pH 8.0) 120 mM NaCl; 0.5% NP-40; 1 mM phenylmethylsulfonyl fluoride] and determined using bicinchoninic acid assay. A total of 40 µg protein extract was separated by 12% SDS-PAGE, followed by transfer to a polyvinylidene difluoride membrane (Bio-Rad Laboratories, Inc.), blocking with 5% non-fat milk in TBS-Tween buffer 7 (0.12 M Tris-base, 1.5 M NaCl, 0.1% Tween-20) for 1 h at room temperature, and incubation with the appropriate antibodies including rabbit anti-Bcl-2 (cat no. 2870; 1:1,000), rabbit anti-Bax (cat no. 2772s; 1:1,000), rabbit anti-cleaved caspase-9 (cat no. 9507; 1:1,000), rabbit anti-cleaved caspase-3 (cat no. 9662; 1:1,000) and mouse anti-actin (cat no. 3700) were purchased from Cell Signaling Technology, Inc., (Danvers, MA, USA), overnight at 4°C. Subsequently, the blots were incubated with horseradish peroxidase-conjugated anti-rabbit secondary antibody (1:10,000; cat no. 111-035-003; Jackson Immunoresearch Laboratories, Inc., West Grove, PA, USA) for 30 min at room temperature. The bound antibodies were detected using a chemiluminescence (ECL) system and autoradiography (cat. no. PI32209; Pierce; Thermo Fisher Scientific, Inc.). Quantity One analysis software version 4.6.9 (Bio-Rad Laboratories, Inc., Hercules, CA, USA) was used to quantify the relative band intensities from western blot images.

Caspase-3 and caspase-9 activity assay

The activity of caspase-3 and caspase-9 was determined using a Caspase-3 Activity Assay kit and a Caspase-9 Activity Assay kit (Beyotime Institute of Biotechnology). This assay is based on the detection of the chromophore p-nitroaniline (pNA) by spectrophotometry following cleavage of pNA from the substrate by caspase-3/caspase-9. Assays were performed in 96-well plates (5×104 cells/well), with each well containing 10 µl protein cell lysate, 80 µl reaction buffer and 10 µl substrate (Asp-Glu-Val-Asp-pNA for caspase-3 and Leu-Glu-His-Asp-pNA for caspase-9). The results were quantified spectrophotometrically using a BioTek Synergy 2 microplate reader (BioTek Instruments, Inc.) at a wavelength of 405 nm. Caspase activity was presented as a percentage relative to the control group.

Statistical analysis

The results are reported as the mean ± standard error of the mean for at least three analyses for each sample. Quantitative variables were compared using one-way analysis of variance to compare differences in two or more groups, and the Tukey test was performed for post-hoc subgroup analysis, as appropriate. Statistical analysis was performed using the SPSS 19.0 software package (IBM Corp., Armonk, NY, USA) and GraphPad Prism 5.0 (GraphPad Software, Inc., La Jolla, CA, USA). P<0.05 was considered to indicate a statistically significant difference.

Results

Effect of α-MG on CoCl2-induced cell viability

As exhibited in Fig. 1, H9C2 cells treated with CoCl2 (≥600 µM) for 24 h exhibited significantly reduced cell viability (P<0.001). The choice of 600 µM for the concentration of CoCl2 applied to all experiments was based on these results from the CCK-8 assay. However, in H9C2 cells pretreated with various concentrations of α-MG, CoCl2-induced hypoxic injury was significantly reduced (P<0.05; Fig. 2). Cells pretreated with α-MG at concentrations between 0 and 12 mM presented as an ‘inverted U shape’ in the present study: At concentrations of 0.012–0.06 mM, cell viability increased gradually (P<0.05), reaching a maximum at 0.06 mM; at concentrations of 0.06–1.2 mM the curve exhibited a steep decline, indicating a decrease in cell viability with increasing concentrations, although cell viability remained higher compared with the group treated with CoCl2 alone (P<0.05). Among the α-MG pretreatment groups, all exhibited significant differences compared with the 0.06 mM group (P<0.01), apart from the 0.3 mM group (P>0.05).

Effects of α-MG on CoCl2-induced alterations in ROS generation, MDA concentration and SOD activity

The involvement of ROS in the CoCl2-induced apoptosis of H9C2 cells was evaluated by measuring the level of ROS production. Following treatment of H9C2 cells with 600 µM CoCl2 for 24 h, the intracellular ROS level increased significantly compared with that in the control group (P<0.001). However, treatment with α-MG decreased the intracellular ROS levels significantly (P<0.001), further demonstrating its antioxidant effects (Fig. 3A). As exhibited in Fig. 3B and C, H9C2 cells treated with CoCl2 exhibited significantly increased MDA concentrations and reduced SOD concentration (P<0.01 and P<0.001, respectively). The oxidative abnormalities were ameliorated by α-MG at a concentration of 0.06 mM, as demonstrated by the significant reduction in MDA concentration and the increase in SOD activity (P<0.05).

Effects of α-MG on CoCl2-induced cell apoptosis

To further investigate the functional effect of α-MG in H9C2 cells treated with CoCl2, cellular apoptosis in H9C2 cells was assessed using the Annexin V-FITC/PI method with flow cytometry. The results demonstrated that CoCl2 increased the percentage of apoptotic H9C2 cells to ~60%, which was attenuated by treatment with 0.06 mM α-MG (reduced to ~5.8%) and 0.3 mM α-MG (P<0.001; reduced to ~15.34%), indicating the cytoprotective effect of α-MG against chemical hypoxia-induced apoptosis (Fig. 4).

Effect of α-MG on cell apoptosis-regulating genes

The results of the RT-qPCR (Fig. 5) revealed that treatment with CoCl2 significantly decreased the expression level of Bcl-2 to 64.9% and increased the expression levels of Bax, caspase-9 and caspase-3 to 194, 138 and 193% relative to the control group, respectively (P<0.001 vs. control group). However, pretreatment with α-MG at a concentration of 0.06 mM increased the expression level of Bcl-2 to 81.4% and attenuated the expression levels of Bax, caspase-9 and caspase-3 to 110, 120 and 133% relative to the control group, respectively (P<0.001 vs. CoCl2-induced injury group), while pretreatment with α-MG at a concentration of 0.3 mM increased the expression level of Bcl-2 to 70.8% and attenuated the expression levels of Bax, caspase-9 and caspase-3 to 142, 126 and 182% relative to the control group, respectively (P<0.05 vs. CoCl2-induced injury group).

Effect of α-MG on cellular apoptosis-regulating protein expression

As depicted in Fig. 6, protein expression was analyzed by western blotting. Treatment with CoCl2 significantly downregulated the expression level of Bcl-2 to 49.6% and upregulated the levels of Bax, caspase-9 and caspase-3 to 285, 1,111 and 307% relative to the control group, respectively (P<0.001). Pretreatment with α-MG at a concentration of 0.06 mM upregulated the expression level of Bcl-2 to 87.6% and downregulated the expression levels of Bax, caspase-9 and caspase-3 to 138, 474 and 157%, respectively (P<0.01), while pretreatment with α-MG at a concentration of 0.3 mM downregulated the expression levels of Bax, caspase-9 and caspase-3 to 165, 650 and 183%, respectively (P<0.01), while there was not significant difference with Bcl-2.

Effect of α-MG on the activation of caspase-3 and caspase-9

The activation of caspase-3 and caspase-9 has important influences on apoptosis. When compared with the control group (Fig. 7), treatment with CoCl2 significantly increased the activity of caspase-3 and caspase-9 to 270 and 906%, respectively (P<0.001). When pretreated with α-MG (0.06 and 0.3 mM), the activities of caspase-3 (140 and 167%, respectively) and caspase-9 (447 and 573%, respectively) were significantly attenuated (P<0.001 vs. CoCl2-induced injury group).

Discussion

The findings of the present study revealed that α-MG, a promising cardioprotective natural extract, was able to suppress oxidative stress and inhibit apoptosis in H9C2 cells by attenuating cellular oxidative damage. To the best of the authors' knowledge, this is the first report describing the anti-apoptotic effect of α-MG in H9C2 cells.

Mangosteen fruit has been used for centuries to alleviate a number of pathological conditions in humans. In 1855, α-MG was identified among the major xanthones isolated from the pericarp of the mangosteen fruit (35). This compound is yellowish in color and may additionally be obtained from other parts of the plant. The structure of this xanthone was interpreted by Dragendorff [reviewed in (36)]. The molecular formula, type and position of the substituent groups of α-MG were subsequently determined by Stout et al (37). Preclinical studies with purified α-MG, the major constituent of the pericarp of mangosteen, have demonstrated its beneficial effects in various diseases. However, the cardioprotective effects of α-MG have not been extensively investigated. In a study conducted by Devi Sampath and Vijayaraghavan (22), rats were administered oral α-MG at a dose of 200 mg/kg prior to the induction of myocardial infarction by isoproterenol (ISO). On comparing the experimental groups, the myocardial injury markers and oxidation products were increased significantly in the blood and myocardia of rats not treated with oral α-MG, whereas they were significantly reduced in rats with myocardial injury that received oral α-MG, demonstrating that α-MG exerts a protective effect against lipid peroxidation and enhances the antioxidant tissue defense system during ISO-induced myocardial infarction in rats. A further study on the activity of rat myocardium mitochondrial enzymes revealed significantly increased enzyme activity, indicating mitochondrial damage, following treatment with ISO in rats that did not receive oral α-MG, whereas oral α-MG was able to reverse this result, suggesting that α-MG may reduce the occurrence of ISO-induced myocardial infarction, mitochondrial dysfunction and associated oxidative stress (23). Buelna-Chontal et al (24) reported that α-MG exerts a protective effect in the post-ischemic heart, which is associated with the prevention of oxidative stress secondary to reperfusion injury. To date, studies have focused on animal research, whereas a study in vitro has not been reported.

Hypoxia may induce ROS generation and lipid peroxidation (38). ROS, a series of cellular molecules generated during oxygen metabolism, are generally considered to be important mediators of oxidative stress injury (39). The MDA concentration frequently reflects the degree of lipid peroxidation, indirectly reflecting the degree of cellular oxidative injury (40). SOD decrease cellular free oxygen radicals (41). The present study demonstrated that the ROS level and MDA concentration were increased in H9C2 cells treated with CoCl2, while the activity of SOD was suppressed. In H9C2 cells treated with α-MG, the results were reversed, demonstrating the antioxidant effects of α-MG, which coincide with the observations in vivo.

It was previously reported that apoptosis is rare in the healthy myocardium, with a percentage of 0.001–0.002% (42); however, when the cells become damaged, they may undergo apoptosis. Oxidative stress usually causes DNA damage, and instability of the membrane, cellular lipids and proteins, leading to cellular dysfunction and apoptosis (43). In the present study, myocardial cell viability decreased following treatment with CoCl2, and the apoptosis rate increased. α-MG-treated groups (at concentrations of 0.06 and 0.3 mM) exhibited decreased oxidative stress and apoptosis rates, indicating the protective effect of α-MG against CoCl2-induced apoptosis.

The first limitation of the present study is associated with the use of CoCl2, which is commonly used for constructing hypoxic models in different cell lines (44,45). CoCl2-treated cells were used as a model of chemical hypoxia-induced injury; however, there are several other effects of hypoxia that are not achieved by treatment with CoCl2. For example, CoCl2 may stabilize the expression of hypoxia-inducible factor 1α (HIF-1α) (46,47). Second, the mechanisms underlying the antioxidant and anti-apoptotic properties of α-MG are not fully elucidated, and continued research is required in the future.

In conclusion, in the present study, a myocardial cell model of hypoxia was successfully constructed and α-MG was demonstrated to be effective in reducing apoptosis and oxidative stress induced by CoCl2.

Acknowledgements

Not applicable.

Funding

The present study was supported by the Celebrity Award of Xiangya Hospital.

Availability of data and materials

All data generated or analyzed during the present study are included in this published article.

Authors' contributions

FZ contributed to acquisition, analysis and interpretation of data, writing the main manuscript text, LYL and LWJ designed the study and contributed by revising the manuscript.

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Glossary

Abbreviations

Abbreviations:

α-MG

α-mangostin

ROS

reactive oxygen species

MDA

malondialdehyde

SOD

superoxide dismutase

IHD

ischemic heart disease

CCK-8

cell-counting kit 8

FACS

fluorescence activated cell sorting

OD

optical density

PI

propidium iodide

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May-2018
Volume 17 Issue 5

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Online ISSN:1791-3004

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Copy and paste a formatted citation
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Spandidos Publications style
Fang Z, Luo W and Luo Y: Protective effect of α-mangostin against CoCl2-induced apoptosis by suppressing oxidative stress in H9C2 rat cardiomyoblasts. Mol Med Rep 17: 6697-6704, 2018
APA
Fang, Z., Luo, W., & Luo, Y. (2018). Protective effect of α-mangostin against CoCl2-induced apoptosis by suppressing oxidative stress in H9C2 rat cardiomyoblasts. Molecular Medicine Reports, 17, 6697-6704. https://doi.org/10.3892/mmr.2018.8680
MLA
Fang, Z., Luo, W., Luo, Y."Protective effect of α-mangostin against CoCl2-induced apoptosis by suppressing oxidative stress in H9C2 rat cardiomyoblasts". Molecular Medicine Reports 17.5 (2018): 6697-6704.
Chicago
Fang, Z., Luo, W., Luo, Y."Protective effect of α-mangostin against CoCl2-induced apoptosis by suppressing oxidative stress in H9C2 rat cardiomyoblasts". Molecular Medicine Reports 17, no. 5 (2018): 6697-6704. https://doi.org/10.3892/mmr.2018.8680