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.