Eupatilin with heme oxygenase-1-inducing ability protects cultured feline esophageal epithelial cells from cell damage caused by indomethacin.

We previously reported that eupatilin (5,7-dihydroxy-3,4,6-trimethoxyflavone) extracted from Artemisia asiaitica, augmented the cellular antioxidant defense capacity through induction of the antioxidant protein heme oxygenase-1 (HO-1), thereby protecting ileal smooth muscle cells from nonsteroidal anti-inflammatory drug (NSAID)-induced intestinal toxicity. In the present study, we used cultured feline esophageal epithelial cells (EEC) to investigate the ability of eupatilin to induce expression of HO-1 and to analyze its cytoprotective effect against indomethacin-induced damage, since NSAID users have a higher risk of esophageal ulcers or esophagitis than non-NSAID users. A culture of EEC from cat was prepared. The identity of the cultures was confirmed by immunocytochemistry using cytokeratin antibodies. Western blot analysis showed a concentration- and time- dependent expression of HO-1 in response to eupatilin. Phosphorylation of extracellular regulating protein kinase (ERKs) and Akt, and nuclear translocation of nuclear related factor 2 (Nrf2) were induced by 150 microM eupatilin in a time-dependent manner. Eupatilin-induced HO-1 expression and Nrf2 were partly attenuated by MEK inhibitor PD98059 and almost completely by phosphatidyl-inactiol 3 kinase (PI3K) inhibitor LY294002, but not by c-Jun N-terminal kinase (JNK) inhibitor SP600125 or p38 mitogen activated protein kinase (MAPK) inhibitor SB202190. MTT assay showed that treatment with 2 mM indomethacin for 2 h decreased cell viability to about 41%. Pre-treatment of cells with eupatilin resulted in the dose-dependent inhibition of indomethacin-induced cell damage. We confirmed that ZnPP, an HO-1 inhibitor, repressed eupatilin-induced HO-1 activity and showed the protective effect of eupatilin against indomethacin-induced cell injury. The data suggested that HO-1 was partly responsible for the eupatilin-mediated protective action of esophageal epithelial cells against indomethacin via both ERKs and PI3K/Akt pathways as well as Nrf2 translocation.
AuthorsHyun Ju Song, Chang Yell Shin, Tae Young Oh, Young Sil Min, Eon Sub Park, Uy Dong Sohn
JournalBiological & pharmaceutical bulletin (Biol Pharm Bull) Vol. 32 Issue 4 Pg. 589-96 (Apr 2009) ISSN: 0918-6158 [Print] Japan
PMID19336889 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
Chemical References
  • Anti-Inflammatory Agents, Non-Steroidal
  • Culture Media, Serum-Free
  • Enzyme Inhibitors
  • Flavonoids
  • Indicators and Reagents
  • Nuclear Proteins
  • Protective Agents
  • eupatilin
  • Heme Oxygenase-1
  • Indomethacin
  • Animals
  • Anti-Inflammatory Agents, Non-Steroidal (toxicity)
  • Blotting, Western
  • Cats
  • Cell Survival (drug effects)
  • Cells, Cultured
  • Culture Media, Serum-Free
  • Enzyme Induction (drug effects)
  • Enzyme Inhibitors (pharmacology)
  • Epithelial Cells (drug effects)
  • Esophagus (cytology, drug effects)
  • Flavonoids (pharmacology)
  • Heme Oxygenase-1 (biosynthesis)
  • Immunohistochemistry
  • Indicators and Reagents
  • Indomethacin (antagonists & inhibitors, toxicity)
  • Nuclear Proteins (metabolism)
  • Protective Agents
  • Rats
  • Rats, Sprague-Dawley

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