Korean J Physiol Pharmacol.  2011 Dec;15(6):319-326. 10.4196/kjpp.2011.15.6.319.

The Protective Effect of Quercetin-3-O-beta-D-Glucuronopyranoside on Ethanol-induced Damage in Cultured Feline Esophageal Epithelial Cells

• Cho JH
• Park SY
• Lee HS
• Whang WK
• Sohn UD

• KMID: 2285417
• DOI: http://doi.org/10.4196/kjpp.2011.15.6.319

Abstract

Quercetin-3-O-beta-D-glucuronopyranoside (QGC) is a flavonoid glucoside extracted from Rumex Aquaticus Herba. We aimed to explore its protective effect against ethanol-induced cell damage and the mechanism involved in the effect in feline esophageal epithelial cells (EEC). Cell viability was tested and 2',7'-dichlorofluorescin diacetate assay was used to detect intracellular H2O2 production. Western blotting analysis was performed to investigate MAPK activation and interleukin 6 (IL-6) expression. Exposure of cells to 10% ethanol time-dependently decreased cell viability. Notably, exposure to ethanol for 30 min decreased cell viability to 43.4%. When cells were incubated with 50 microM QGC for 12 h prior to and during ethanol treatment, cell viability was increased to 65%. QGC also inhibited the H2O2 production and activation of ERK 1/2 induced by ethanol. Pretreatment of cells with the NADPH oxidase inhibitor, diphenylene iodonium, also inhibited the ethanol-induced ERK 1/2 activation. Treatment of cells with ethanol for 30 or 60 min in the absence or presence of QGC exhibited no changes in the IL-6 expression or release compared to control. Taken together, the data indicate that the cytoprotective effect of QGC against ethanol-induced cell damage may involve inhibition of ROS generation and downstream activation of the ERK 1/2 in feline EEC.

Keyword

Flavonoid; Hydrogen peroxide; ERK; Esophageal epithelial cell; Ethanol

MeSH Terms

Blotting, Western
Cell Survival
Epithelial Cells
Ethanol
European Union
Fluoresceins
Hydrogen Peroxide
Interleukin-6
NADPH Oxidase
Onium Compounds
Quercetin
Rumex
Ethanol
Fluoresceins
Hydrogen Peroxide
Interleukin-6
NADPH Oxidase
Onium Compounds
Quercetin

Figure

• 

  Fig. 1 Time course analysis for the effect of 10% ethanol in the absence or presence of QGC on the viability of cultured EECs. (A) Serum-starved EECs were stimulated with ethanol for the indicated times and their survival was estimated using the MTT assay. (B) Serum-starved EECs were pre-incubated in the presence of QGC for 12 h at indicated concentrations. EECs were then stimulated with 10% ethanol for 30 min and their survival was estimated using MTT assay. Data are expressed as means±SEM of three experiments. Student's t-test; *p<0.05, ***p<0.001 vs. control, #p<0.05 vs. cells in 10% ethanol alone.

• 

  Fig. 2 Effect of QGC on intracellular H2O2 levels induced by ethanol in EECs. Serum-starved EECs were pre-incubated in the presence of 50 µM QGC for 4 h. EECs were then stimulated with ethanol for 10 min and ROS production was estimated using DCF-DA assay. Data are expressed as means±SEM of three experiments. Student's t-test; *p<0.05 vs. control, ##p<0.001 vs. cells in 10% ethanol alone.

• 

  Fig. 3 Time course of MAPK phosphorylation induced by ethanol. Serum-starved EECs were incubated with ethanol for indicated time periods. Phosphorylation of ERK 1/2 (A), p38 MAPK (B) and JNK (C) were estimated by western blotting analysis. Data are expressed as means±SEM of three experiments. Student's t-test; *p<0.05 vs. control.

• 

  Fig. 4 Effects of QGC and DPI on ethanol-induced ERK 1/2 phosphorylation. Serum-starved EECs were incubated with QGC (50 µM, 12 h), DPI (a NADPH oxidase inhibitor, 10 µM, 0.5 h), PD98059 (a MEK inhibitor, 30 µM, 1 h) prior to ethanol treatment for 10 min. Phosphorylation of ERK 1/2 was estimated by western blot analysis. Data are expressed as means±SEM of three experiments.Student's t-test; *p<0.05 vs. control, #p<0.05 vs. cells in 10% ethanol alone.

• 

  Fig. 5 Effect of ethanol in the absence or presence of QGC on IL-6 protein expression or release in EECs. Serum-starved EECs were stimulated with ethanol for 30 and 60 min in the absence or presence of 50 µM QGC for 12 h. (A) IL-6 expression was estimated by western blot analysis. (B) IL-6 production in culture medium was estimated by an EIA kit. Data are expressed as means±SEM of three experiments.

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