Nutr Res Pract.  2015 Dec;9(6):586-591. 10.4162/nrp.2015.9.6.586.

Cytoprotective effect of rhamnetin on miconazole-induced H9c2 cell damage

• Lee KP
• Kim JE
• Park WH

• KMID: 2313881
• DOI: http://doi.org/10.4162/nrp.2015.9.6.586

Abstract

BACKGROUND/OBJECTIVES
Reactive oxygen species (ROS) formation is closely related to miconazole-induced heart dysfunction. Although rhamnetin has antioxidant effects, it remained unknown whether it can protect against miconazole-induced cardiomyocyte apoptosis. Thus, we investigated the effects of rhamnetin on miconazole-stimulated H9c2 cell apoptosis.
MATERIALS/METHODS
Cell morphology was observed by inverted microscope and cell viability was determined using a WelCount(TM) cell proliferation assay kit. Miconazole-induced ROS production was evaluated by fluorescence-activated cell sorting with 6-carboxy-2',7'-dichlorofluoroscein diacetate (H2DCF-DA) stain. Immunoblot analysis was used to determine apurinic/apyrimidinic endonuclease 1 (APE/Ref-1) and cleaved cysteine-aspartic protease (caspase) 3 expression. NADPH oxidase levels were measured using real-time polymerase chain reaction.
RESULTS
Miconazole (3 and 10 microM) induced abnormal morphological changes and cell death in H9c2 cells. Rhamnetin enhanced the viability of miconazole (3 microM)-treated cells in a dose-dependent manner. Rhamnetin (1 and 3 microM) treatment downregulated cleaved caspase 3 and upregulated APE/Ref-1 expression in miconazole-stimulated cells. Additionally, rhamnetin significantly reduced ROS generation.
CONCLUSIONS
Our data suggest that rhamnetin may have cytoprotective effects in miconazole-stimulated H9c2 cardiomyocytes via ROS inhibition. This effect most likely occurs through the upregulation of APE/Ref-1 and attenuation of hydrogen peroxide levels.

Keyword

Rhamnetin; miconazole; cardiomyocyte; apoptosis; APE/Ref-1

MeSH Terms

Antioxidants
Apoptosis
Caspase 3
Cell Death
Cell Proliferation
Cell Survival
Flow Cytometry
Heart
Hydrogen Peroxide
Miconazole
Myocytes, Cardiac
NADPH Oxidase
Reactive Oxygen Species
Real-Time Polymerase Chain Reaction
Up-Regulation
Antioxidants
Caspase 3
Hydrogen Peroxide
Miconazole
NADPH Oxidase
Reactive Oxygen Species

Figure

• 

  Fig. 1 Effect of miconazole on H9c2 cell viability. (A) The chemical structure of miconazole 1-(2,4-dichloro-β-[(2,4-dichlorobenzyl)oxy] phenethyl) imidazole; molecular weight 479.14. (B) Cell morphology was observed after 24 h miconazole (Mico) treatment (3 and 10 µM). (C) H9c2 cells were treated with miconazole (3 and 10 µM) for 24 h and cytotoxicity in the quiescent state was analyzed by XTT assay. The results represent the mean ± SE of three independent experiments. Values with the same superscript letter are not significantly different based on Tukey's multiple range test (P < 0.05).

• 

  Fig. 2 Effect of rhamnetin on miconazole-stimulated H9c2 cell viability. (A) The chemical structure of rhamnetin (Rham; molecular weight 316.26). (B) Cell morphology was observed after 24 h in the presence or absence of miconazole (Mico; 3 µM) and rhamnetin (1 and 3 µM). (C) H9c2 cells were co-treated with miconazole (3 µM) and rhamnetin (1, 3 and 5 µM) for 24 h, and cytotoxicity was evaluated in the quiescent state by XTT assay. The results represent the mean ± SE of three independent experiments. Values with the same superscript letter are not significantly different based on Tukey's multiple range test (P < 0.05).

• 

  Fig. 3 Effect of rhamnetin on miconazole-induced caspase3 cleavage in H9c2 cells. Cells were incubated in serum-free medium, followed by treatment with miconazole (Mico;3 µM) and rhamnetin (Rham;1 and 3 µM) for 24 h. (A) The cells were lysed, and proteins were resolved by SDS-PAGE, transferred to a PVDF membrane, and blotted with anti-cleaved capase3 antibodies to analyze protein expression. (B) Statistical results obtained from panel A. The basal levels of caspase3 were considered 100% (n = 3). The results represent the mean ± SE from three independent experiments. Values with the same superscript letter are not significantly different based on Tukey's multiple range test (P < 0.05).

• 

  Fig. 4 Effect of rhamnetin on miconazole-induced ROS production and signaling in H9c2 cells. Cells were cultured in serum-free medium, followed by miconazole (Mico;3 µM) and rhamnetin (Rham;1 and 3 µM) stimulation for 24 h. (A) Rhamnetin reduced miconazloe-induced ROS production in H9c2 cells as measured by FACS using H2DCF-DA stain (Left panel). Bar graph displays the the percentage of fluorescence cells on left panel. (B) mRNA expression levels of NOX family proteins (NOX1, NOX2, and NOX4) were measured by real-time polymerase chain reaction. The relative levels of mRNA were calculated by using 2-ΔΔCt values and normalizing GAPDH. (C) Proteins were separated and blotted with APE/Ref-1 antibodies to analyze protein expression. (D) Statistical results obtained from panel C. GAPDH was used for normalization. These results represent the mean ± SE from three independent experiments. Values with the same superscript letter are not significantly different based on Tukey's multiple range test (P < 0.05).

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