Korean J Physiol Pharmacol.  2012 Oct;16(5):313-320. 10.4196/kjpp.2012.16.5.313.

The Protective Effect of Eupatilin against Hydrogen Peroxide-Induced Injury Involving 5-Lipoxygenase in Feline Esophageal Epithelial Cells

• Lim JC
• Park SY
• Nam Y
• Nguyen TT
• Sohn UD

• KMID: 1493963
• DOI: http://doi.org/10.4196/kjpp.2012.16.5.313

Abstract

In this study, we focused to identify whether eupatilin (5,7-dihydroxy-3',4',6-trimethoxyflavone), an extract from Artemisia argyi folium, prevents H2O2-induced injury of cultured feline esophageal epithelial cells. Cell viability was measured by the conventional MTT reduction assay. Western blot analysis was performed to investigate the expression of 5-lipoxygenase by H2O2 treatment in the absence and presence of inhibitors. When cells were exposed to 600 microM H2O2 for 24 hours, cell viability was decreased to 40%. However, when cells were pretreated with 25~150 microM eupatilin for 12 hours, viability was significantly restored in a concentration-dependent manner. H2O2-treated cells were shown to express 5-lipoxygenase, whereas the cells pretreated with eupatilin exhibited reduction in the expression of 5-lipoxygenase. The H2O2-induced increase of 5-lipoxygenase expression was prevented by SB202190, SP600125, or NAC. We further demonstrated that the level of leukotriene B4 (LTB4) was also reduced by eupatilin, SB202190, SP600125, NAC, or nordihydroguaiaretic acid (a lipoxygenase inhibitor) pretreatment. H2O2 induced the activation of p38MAPK and JNK, this activation was inhibited by eupatilin. These results indicate that eupatilin may reduce H2O2-induced cytotoxicity, and 5-lipoxygenase expression and LTB4 production by controlling the p38 MAPK and JNK signaling pathways through antioxidative action in feline esophageal epithelial cells.

Keyword

5-lipoxygenase; Esophageal epithelial cell; Eupatilin; Flavonoid; Hydrogen peroxide

MeSH Terms

Anthracenes
Arachidonate 5-Lipoxygenase
Artemisia
Blotting, Western
Cell Survival
Epithelial Cells
Flavonoids
Hydrogen
Hydrogen Peroxide
Imidazoles
Leukotriene B4
Lipoxygenase
MAP Kinase Signaling System
Nordihydroguaiaretic Acid
p38 Mitogen-Activated Protein Kinases
Pyridines
Anthracenes
Arachidonate 5-Lipoxygenase
Flavonoids
Hydrogen
Hydrogen Peroxide
Imidazoles
Leukotriene B4
Lipoxygenase
Nordihydroguaiaretic Acid
Pyridines
p38 Mitogen-Activated Protein Kinases

Figure

• 

  Fig. 1 Effect of H2O2 on the cell viability of feline EECs and Effect of eupatilin on the H2O2-induced cell viability. Serum-starved EECs were incubated with H2O2 for 24 hours at the indicated concentration. (A) The cell viability was estimated using MTT assay. (B) The morphologic changes of EECs were observed (magnification: 100×). (C) Serumstarved EECs were incubated in the presence of eupatilin alone for 12 hours at the indicated concentration. (D) the cells were incubated in the 600 µM H2O2 with or without eupatilin 12 hours before and during 24 hours, and then their survival was estimated using the MTT assay and the morphologic changes of cells were observed (E) (magnification: 100×). Data are expressed as Means±S.E of four experiments (*; p<0.05 vs. control, **; p<0.01 vs. control, #; p< 0.05 vs. H2O2 alone, ##; p<0.01 vs. H2O2 alone).

• 

  Fig. 2 Effects of eupatilin on the H2O2-induced 5-LOX expression. (A) Serum-starved EECs were treated with H2O2 for 24 hours at each dose. (B) Serum-starved cells were preincubated in the presence of eupatilin for 12 hours at the indicated concentration and then stimulated with 300 µM H2O2 for 24 hours. 5-LOX expression was estimated by Western blot. Data are expressed as Means±S.E of three experiments (*; p<0.05 vs. control, #; p<0.05 vs. H2O2 alone).

• 

  Fig. 3 The effect of eupatilin, SB202190, SP600125 or NAC on the H2O2-induced 5-LOX expression and LTB4 production. Serum-starved EECs were preincubated in the presence of eupatilin (150 µM, 12 hours), SB202190 (30 µM, 1 hours), SP600125 (30 µM, 1 hours), or NAC (5 mM, 1 hours). The cells were then stimulated with H2O2 (300 µM, 24 hours). (A) The change level of 5-LOX expression was estimated by Western blot analysis. (B) The production level of LTB4 was estimated by LTB4EIA kit. Data are expressed as Means±S.E of three experiments (*; p<0.05 vs. control, #; p<0.05, ##; p<0.01 vs. H2O2 alone).

• 

  Fig. 4 p38 MAPK and JNK phosphorylation by H2O2. Serum-starved EECs were stimulated with H2O2 for 24 hr at each dose. (A) The change in the level of phosphorylated p38 MAPK was estimated by Western blot analysis. (B) The change of phosphorylated JNK level was estimated by Western blot analysis. Data are expressed as Means±S.E of three experiments (*; p<0.05 vs. control, **; p<0.001 vs. control).

• 

  Fig. 5 The effect of eupatilin, SB202190, SP600125, NAC on p38 MAPK (A) and (B) JNK phosphorylation in EECS. Serum-starved EECs were preincubated in the presence of eupatilin (150 µM, 12 hr), SB202190 (p38 MAPK inhibitor, 30 µM, 1 hr), SP600125 (JNK inhibitor, 30 µM, 1hr), or NAC (N-acetyl-L-cysteine, ROS scavenger, 5 mM, 1 hr). EECs were then stimulated with H2O2 (300 µM, 24 hr). The change of phosphorylated p38MAPK and JNK was estimated by Western blot analysis. Data are expressed as Means±S.E of three experiments (#; p<0.05 vs. control, *; p<0.05 vs. H2O2 alone).

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