|Year : 2019 | Volume
| Issue : 3 | Page : 97-104
Preventive Effect of Arbutin on Isoproterenol-Induced Oxidative Stress, Mitochondrial Damage and Apoptosis in H9c2 Cells
Subramanian Sivasangari, Loordhurani Asaikumar, Lakshmanan Vennila, Natesan Vijayakumar
Department of Biochemistry and Biotechnology, Faculty of Science, Annamalai University, Annamalainagar, Tamil Nadu, India
|Date of Submission||06-Jun-2019|
|Date of Decision||09-Jul-2019|
|Date of Acceptance||30-Jul-2019|
|Date of Web Publication||25-Oct-2019|
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Aim: This study aims to investigate the potential mechanisms associated with cardioprotective effect of arbutin (ARB) on isoproterenol hydrochloride (ISO) induced cardiotoxicity in H9c2 cardiomyoblast cell lines. Materials and Methods: The effect of the drug on cell morphology was studied by using phase contrast microscope, cell viability was studied by using 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) staining, and reactive oxygen species (ROS) production was estimated by 20-7’dichlorofluorescein diacetate staining. H9c2 cells were treated with ISO to cause cell injury and the effect of the ARB on cell morphology, mitochondrial membrane potential, intracellular ROS generation, cell viability, and apoptosis were studied. Results and Conclusion: The results of this study showed that preadministration of ARB significantly prevented the ISO-induced toxic effects on cell morphology and enhanced the number of viable cells in dose-dependent manner. This study also demonstrates that ROS generation was significantly increased in ISO-administered cells and ISO-induced ROS production was found to be significantly reduced in preadministration of ARB on H9c2 cells. ISO-induced changes in mitochondrial membrane potential of H9c2 cells were remarkably improved with ARB pretreatment. These results clearly suggest that pretreatment of ARB protects the cells against ISO-induced injury through resuming mitochondrial function and regulating apoptosis.
Keywords: Arbutin, cardiotoxiciy, H9c2, isoproterenol, reactive oxygen species
|How to cite this article:|
Sivasangari S, Asaikumar L, Vennila L, Vijayakumar N. Preventive Effect of Arbutin on Isoproterenol-Induced Oxidative Stress, Mitochondrial Damage and Apoptosis in H9c2 Cells. Int J Nutr Pharmacol Neurol Dis 2019;9:97-104
|How to cite this URL:|
Sivasangari S, Asaikumar L, Vennila L, Vijayakumar N. Preventive Effect of Arbutin on Isoproterenol-Induced Oxidative Stress, Mitochondrial Damage and Apoptosis in H9c2 Cells. Int J Nutr Pharmacol Neurol Dis [serial online] 2019 [cited 2021 Jan 25];9:97-104. Available from: https://www.ijnpnd.com/text.asp?2019/9/3/97/270041
| Introduction|| |
Cardiovascular diseases (CVDs) are the number one cause of death globally and every year more people die from CVDs than from any other causes. Oxidative stress is the major reason for the pathogenesis of CVDs including myocardial ischemia, arteriosclerosis, congestive heart failure, and drug-induced cardiomyopathy. Isoproterenol hydrochloride (ISO) is a synthetic catecholamine and β-adrenergic agonist that causes severe stress in the cardiac muscle resulting in infarct-like necrosis of the myocardium. Out of several mechanisms proposed to explain the ISO-induced myocardial injury, extremely toxic-free radical generation during the autooxidation of ISO is a widely accepted one. Free radicals can cause severe stress in the myocardium and loss of myocardial integrity through hypoxia and calcium overload. Reactive oxygen species (ROS) have been reported as one of the major free radicals responsible for cardiotoxicity. The common ROS include hydrogen peroxide (H2O2), superoxide anion (O2), reactive hydroxyl radicals (OH), and peroxy radicals (ROO). ISO is reduced by nicotinamide adenine dinucleotide phosphate oxidase to a semiquinone and release free radicals, which induces oxidative stress. Oxidative stress is considered as the most important apoptotic stimulus in many CVDs, which trigger the apoptosis in cardiac cells by increasing the expression of proapoptotic genes. This destructive action of the free radicals can be inhibited or reversed by antioxidants and antioxidants are capable of preventing the oxidation of other biomolecules.
Medicinal plants and their metabolites are used as medicines for many diseases in worldwide. These products exert more pharmacological activity in the therapeutic treatment of various disorders. Arbutin (ARB) is a naturally occurring derivative of hydroquinone found in wheat, pears, and blueberry and highest concentration in bearberry. ARB-rich leaves of bearberry are internally used to treat the inflammation in the urinary tract and bladder. ARB has a variety of pharmacological and therapeutic properties, including antiinflammatory, antiviral, antihyperglycemic, and antioxidant activity.,,, The cultured primary cardiomyocytes are widely used for evaluating cardiotoxicity. In this study, ISO-induced H9c2 cell damage was used as an experimental model and the cardioprotective activity of ARB against ISO-induced oxidative stress and mitochondrial function in H9c2 cells was investigated. The structure of ARB and ISO are shown in [Figure 1]a and 1b.
| Materials and Methods|| |
Chemicals and reagents
ARB and ISO were purchased from Sigma–Aldrich (St. Louis, MO, USA), and Dulbecco’s Modified Eagle Medium (DMEM) media, fetal bovine serum (FBS), penicillin, MTT-3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide, acridine orange (AO), ethidium bromide (EB), 20-7’dichlorofluorescein diacetate (DCFH-DA), rhodamine-123 (Rh-123), Tween-20 and phosphate buffer saline (PBS), acetone, ethanol, and dimethyl sulfoxide (DMSO) were obtained from E. Merck and HIMEDIA, India.
Cell culture and assessment of growth and viability
Cardiomyoblast cells of the H9c2 rats were obtained from National Centre for Cell Science (NCCS) Pune, India. H9c2 cells were cultured in DMEM media supplemented with 10% heat-inactivated FBS, 100 U/mL of penicillin, and 100 mg/mL of streptomycin at 37°C in a humidified 95% air and 5% CO2 incubator. Cells were seeded in 6 or 12-well plates prior to the addition of ARB. The H9c2 cells were incubated with ARB (10–50 μg/mL of H9c2 cells) for 24 hours.
Preparation of ARB
A stock solution of ARB (1:1) was prepared in 1 mL distilled water (w/v) and stored at 4°C. Further dilution was made in DMEM culture media to obtain the desired concentrations (control, 10, 20, 30, 40, and 50 μM). The working solution was diluted using sterile distilled water so that the final concentrations of ARB in the culture medium were not more than 0.01% (v/v); 0.01% DMSO was used as a vehicle control; and 5, 10, 15, 20, and 25 μM of ISO was dissolved in physiological saline.
Cell viability assay
Cell viability was measured by MTT reduction assay. H9c2 cells were preincubated with DMEM containing 10% FBS overnight in 96-well plates at a density of 5 × 104 cells/well. After cells were grown to 90% confluence, all cells were washed twice with PBS and serum-starved for 2 hours. According to the experimental design, the culture medium was replaced (with or without ISO or the other compounds) and then cultured for 6 hours. The medium was then removed and a solution containing 10% MTT and DMEM was added to each well. The cells were incubated at 37°C for 4 hours, the supernatants were removed, and the 150 μL DMSO was applied to dissolve in the blue formazan crystals. Absorbance was recorded at a wavelength of 490 nm using microtiter plate reader (Bio-Rad, Laboratories, Hercules, CA, USA).
Cell morphology − unstained live morphological assay
The H9c2 cells were placed on glass coverslip (22 × 22 mm) and allowed to settle for 24 hours. The medium was subsequently removed after 24 hours from the well of the control and ARB-treated H9c2 cells and a coverslip was inverted and placed over the slide. The morphological changes in the ARB-treated and vehicle control cells were observed using a differential interference phase contrast light microscope (Axio Scope A1, Carl Zeiss, Jena, Germany) and photographed (40×).
Determination of mitochondrial membrane potential
Mitochondrial membrane potential (MMP) has developed an emphasis of apoptotic analysis. The possible effect of ARB in disturbing MMP was evaluated using the lipophilic cationic fluorescent probe Rh-123. H9c2 cells (2 × 104 cells/well in 96-well plates) were seeded in DMEM medium and incubated for 48 hours at 37°C with 95% air and 5% CO2 to allow the cells to become semiconfluent. After this period, the cells were preadministered with ARB (20 and 40 μM, respectively) for 24 hours. Cells were then rinsed with PBS and fresh media containing Rh-123 solution (10 μg/mL) was added to treated wells and the plates were incubated in the dark at 37°C for 20 to 30 minutes. Subsequently, the cells were washed twice with PBS and the cell images were taken using a fluorescence microscope.
Intracellular ROS generation
Intracellular ROS level was measured by using a cell permeable fluorescent probe 2,7-dichlorofluorescin diacetate (DCFH-DA). DCFH-DA diffuses through the cell membrane where it is hydrolyzed by an intracellular esterase to the nonfluorescent dichlorofluorescin (DCFH) that is rapidly oxidized by ROS to fluorescent dichlorofluoresin. H9c2 cells (8 × 104 cells/well in 24-well plates) were seeded in DMEM medium and incubated for 48 hours at 37°C with 95% air and 5% CO2 to allow the cells to become semiconfluent. Semiconfluent H9c2 cells were treated with ARB and ISO (25 μM) for 6 hours. After the treatment period, DCFH-DA (in absolute DMSO) was added to treated plates at a final concentration of 10 μM and incubated in the dark at 37°C for 30 minutes. Poststaining, plates were rinsed twice with PBS and images were taken on a fluorescent microscope using the appropriate band pass Fluorescein isothiocyanate (FITC) filter and relative fluorescence was measured using Image J 1.48 software.
Acridine orange (AO)/ethidium bromide (EB) dual staining assay
DNA-binding dyes AO/EB were used for the morphological apoptotic and necrotic cells. Thereafter, the various concentration of ARB, 20 and 40 μM for 48 hours, the H9c2 cells were detached, washed by cold PBS, and then stained with a mixture of AO (100 mg/mL) and EB (100 mg/mL) at room temperature for 5 minutes. The stained H9c2 cells were observed by a fluorescence microscope at 40× magnifications. The H9c2 cells were separated into four classes as follows: living cells normal green nucleus appeared, early apoptotic condensed or fragmented form of bright green nucleus with chromatin, late apoptotic chromatin condensation, or fragmentation orange-stained nuclei and necrotic cells (uniformly orange-stained cell nuclei). In each experiment, 300 cells/concentration were counted.
Statistical analysis was performed by one-way analysis of variance (ANOVA) followed by Duncan’s Multiple range test using Software Package for the Social Science (SPSS) software package version 17. Results were expressed as mean ± standard deviation for six rats in each group and the values of P < 0.05 were considered significant.
| Results|| |
In vitro cytotoxicity assay
The cardiotoxicity effect of various concentrations of ISO and ARB on H9c2 cardiomyoblast cells were evaluated by MTT assay [Figure 2]a. ISO administration increases cardiotoxicity in a dose-dependent manner. The IC50 value of ISO was found to be at 25 μM and this concentration was set as the working drug concentration for all further experiments. The effect of different concentrations of ARB on H9c2 cell viability was also examined. The cells were incubated with different concentrations of ARB (0–50 μM) for 72 hours and the percentage of cell viability with different concentrations of ARB has been shown in [Figure 2]b. The results clearly showed that treatment with ARB alone does not cause any changes in cell viability, which indicates that ARB was not cytotoxic toward the relevant cell line at all tested concentrations, and cell viability was slightly decreased in 50 μg/mL of ARB treatment. Therefore, we have chosen 40 μg/mL concentration of ARB as an effective drug for the further experimental analysis.
|Figure 2 Cardiotoxicity effect of different concentrations of ISO and ARB against H9c2 cells. Values are expressed as ±SD of three individual experiments. (a) Different concentrations of ISO. (b) Different concentrations of ARB. *Significant compared to control (P < 0.05). ARB, arbutin; ISO, isoproterenol hydrochloride; SD, standard deviation.|
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ISO-induced morphological changes in H9c2 cells
The degree of morphological changes of H9c2 cells was evaluated and cytomorphology was performed using a phase-contrast inverted biological microscope. H9c2 cells treated with 25 μM of ISO showed significant morphological changes and the results are displayed in [Figure 3]. ISO-pronounced cell damage as displayed by the loss of their adherent property and the cells appeared to be rounded off.
|Figure 3 Effect of ARB on ISO-induced morphological changes in H9c2 cells. (a) Untreated H9c2 cells. (b) ISO (25 μM) block arrow indicates cells that appeared rounded off. (c) ARB (40 μM). (d) ARB (40 μM) + ISO (25 μM). ARB, arbutin; ISO, isoproterenol hydrochloride.|
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Effect of ARB on MMP in H9c2 cells
The MMP in H9c2 cells were evaluated to investigate the effects of ARB on the electrical potential across the inner mitochondrial membrane of ISO-administered H9c2 cells and Rh-123, a lipophilic and cationic indicator, were used for this study. Exposure of cells to ISO for 24 hours induced a significant loss of MMP in H9c2 cells and the results are shown in [Figure 4]. Significant decrease in MMP of ISO-administered H9c2 cells were indicated by the reduction in Rh-123 fluorescence. There were no changes observed in ARB alone cells and administration of ARB before the administration of ISO showed a significant restoration of MMP.
|Figure 4 (a) Effect of ARB on MMP was evaluated with H9c2 cells using Rh-123. (i) Control. (ii) ISO (25 μM) yellow arrow indicated reduction in Rh-123 florescence. (iii) ARB (40 μM). (iv) ARB 40 μM + ISO (25 μM) white arrows indicate restoration of MMP for 24 hours and fluorescence intensity decreased as indicated by collapsed mitochondria matrix. (b) Cells-depicted fluorescence intensity was detected by spectrofluorometer. All experiments were performed in triplicate and all values were expressed as mean ± standard deviation of the mean. ARB, arbutin; ISO, isoproterenol hydrochloride; MMP, mitochondrial membrane potential; Rh-123, rhodamine 123.|
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Examination of intracellular ROS generation
[Figure 5] showed the effect of ARB on ROS generation that was investigated by DCFH-DA. The results show that the treatment of cultured H9c2 cells with ISO for 24 hours significantly increase the level of ROS and it was observed by increased green fluorescence. ARB-alone treated H9c2 cells were depicted weak background fluorescence. These results also showed that the fluorescence of DCFH-DA observed in the ISO group was effectively suppressed following treatment with ARB as compared to ISO-alone treated cells.
|Figure 5 (a) Analysis of ROS formation using DCFH-DA on H9c2 cells treated with ARB and ISO. (i) Control. (ii) ISO (25 μM) yellow arrow indicates increased green fluorescence and increased ROS formation. (iii) ARB (40 μM). (iv) ARB 40 μM + ISO. *Significant compared to control (P < 0.05). (b) Cells-depicted fluorescence intensity was detected by spectrofluorometer. **Significant compared to ISO-administered cells. *Significant compared to normal H9c2 cells (P < 0.05). ARB, arbutin; DCFH-DA, dichlorofluorescein diacetate; ISO, isoproterenol hydrochloride; ROS, reactive oxygen species.|
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Effects of ARB on ISO induce apoptosis in H9c2 cells
Apoptosis is considered to be one of the important pathogenesis of free radical-mediated injury. The preventive effect of ARB on apoptosis was confirmed by measuring the level of apoptosis through AO/EB staining assay. The numbers of apoptotic cells were significantly elevated in ISO-administered cells compared to the control group. ARB preadministration significantly prevented the generation of apoptotic cells by ISO when compared to ISO-alone administered cells [Figure 6]. AO is taken up by both viable and nonviable cells, intercalated into double-stranded DNA, and emits green fluorescence. EB is only taken up by nonviable cells, intercalated into DNA, and emits red fluorescence. Thus, viable cells have a normal green nucleus, whereas the apoptotic cells have green nucleus.
|Figure 6 (a) Effect of ARB on apoptosis. (i) Untreated normal H9c2 cells, white arrows indicate green fluorescence. (ii) ISO (25 μM) red arrows indicate necrotic cells. (iii) ARB (40 μM) blue arrows indicate apoptotic cells. (iv) ARB 40 μM + ISO 25 μM. (b) Percentage of apoptotic cells were calculated by scoring apoptotic and viable cells. The values are given as mean ± SD of six experiments in each group, ANOVA followed by DMRT. *Significant compared to control (P < 0.05). **Significant compared to ISO (P < 0.05). ANOVA, analysis of variance; ARB, arbutin; DMRT, Duncan’s Multiple range test; ISO, isoproterenol hydrochloride; SD, standard deviation.|
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| Discussion|| |
The present investigation reveals that the ARB has cardioprotective activity against ISO-induced oxidative stress and mitochondrial abnormalities in rat H9c2 cell line. Cell death and cell injury are the most critical events in the evaluation of many diseases. Necrosis and apoptosis are the important mediator of cell death, which differ in their morphology, molecular mechanism, and role in physiology and disease. ISO-induced cardiotoxicity in H9c2 cells involves many similarities with human. An autooxidation of ISO results in the generation of highly cytotoxic free radicals. Furthermore, free radicals could initiate the peroxidation of membrane-bound polyunsaturated fatty acids, leading to both functional and structural myocardial injury. Excess amount of free radicals generated by ISO causes extensive damage to tissues and biomolecules such as proteins and nucleic acids leading to apoptosis that can be overcome by using many synthetic drugs which are associated with severe adverse side effects. The purpose of this study is to identify the natural antioxidant for preventing the cardiotoxicity with devoid of any side effects.
Although evidence of several lines show that polyphenols and flavonoids, including quercetin, resveratrol, and curcumin extracted from natural products, have antiinflammatory effects in several diseases such as rheumatoid arthritis and neurological disorders, an antiinflammatory effect of ARB has not been established., Hence, this study was designed to evaluate the in vitro antioxidant and antiapoptotic effect of ARB on H9c2 cardiomyoblast treated with ISO. ISO is a norepinepherine analog and it activates beta adrenergic receptors, leading to cardiomyocytes dysfunction by inducing apoptosis and affecting MMP. ARB is a glycosylated compound widely distributed in plants and used in many cosmetics particularly in lotions, creams, cleansers, and serums used for the skin lightning.
The results of the present study on cell viability showed that the viability of the cells was significantly decreased on ISO treatment with concomitant loss of their adherent nature and appeared to be rounded indicating progressive cell death. The cells pretreated with ARB showed a significant increase in cell viability with normal morphology when compared to the ISO-alone treated cells. Previous studies showed that H9c2 cell, when exposed to higher concentrations of ISO, causes more profound alterations, including mitochondrial swelling, extensive cytoplasm vacuolization, and nuclear swelling with disruption of the nuclear membrane.
The effect of ARB on MMP was detected by using Rh-123. The MMP was highly affected by the production of ROS by ISO. Previous studies have demonstrated that the disturbance of MMP could impact the generation of ROS, which initiate the release of proapoptotic factors, leading to the loss of MMP and induce apoptosis. In this study, ISO showed increased the level of ROS generation with decreased MMP as evidenced by the higher fluorescent intensity and while cells treated with ARB showed reduced levels of ROS with less fluorescent intensity when compared to the ISO alone treated cells, which may be due to the ability of ARB to prevent the loss of MMP by quenching the free radicals generated by ISO.
The cardioprotective effect of ARB on the reduction of ROS in the myocardium was evaluated by using DCFH-DA. ISO significantly increased the formation of ROS in H9c2 cells as evidenced from the higher fluorescent intensity, whereas cells treated with ARB showed reduced levels of ROS in comparison to the ISO-alone administered cells. ARB significantly reduced the ROS formation in H9c2 cells, which might be due to the antioxidant activity.The cell line treated with ISO and ARB was also stained with AO/EB to determine the apoptotic effect. Results of this study clearly showed that the cells treated with ISO undergo cell death as indicated by the presence of a large amount of red-colored cells in comparison to the untreated cells. Cells pretreated with ARB showed a significant reduction in cell death as indicated by the reduced number of red-colored cells. There are several studies suggesting that cardiomyocytes undergo apoptosis following ISO treatment both in vivo and in vitro, indicating that apoptosis is the major mechanism in ISO-mediated cardiac dysfunction. The cardioprotective effect of ARB might be due to its antiapoptotic activity.,
| Conclusion|| |
In conclusion, our data demonstrated that ARB protects H9c2 cardiomyoblast cells from ISO-induced cardiotoxicity. This effect was mediated by the attenuated production of ROS, enhanced antioxidant activity, increased cell viability, increased membrane potential, and inhibited apoptosis, which could be useful for the treatment of oxidative stress-mediated cardiac diseases, and further studies are needed to investigate the cardioprotective mechanism of ARB in the animal model.
Financial support and sponsorship
The department of Biochemistry and Biotechnology, Annamalai University is supported by University Grants Commission-Special Assistance Programme (UGC-SAP), Government of India. Financial support from University Grants Commission, New Delhi in the form of RGNF (research fellow) to Ms.S.Sivasangari is gratefully acknowledged.Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]