Submitted on June 25, 2004
Accepted on September 20, 2004
Ecabet sodium inhibits Helicobacter pylori lipopolysaccharide-induced activation of NADPH oxidase 1 or apoptosis of guinea pig gastric mucosal cells
Kenji Kusumoto1, Tsukasa Kawahara2, Yuki Kuwano2, Shigetada Teshima-Kondo3, Kyoko Morita3, Kyoichi Kishi3, and Kazuhito Rokutan3*
1 COE Research Fellow, Institute of Health Biosciences, The University of Tokushima Graduate School, Tokushima, Tokushima Prefecture, Japan; Department of Nutritional Physiology, Institute of Health Biosciences, The University of Tokushima Graduate School, Tokushima, Tokushima Prefecture, Japan
2 Department of Nutritional Physiology, Institute of Health Biosciences, The University of Tokushima Graduate School, Tokushima, Tokushima Prefecture, Japan
3 Department of Stress Science, Institute of Health Biosciences, The University of Tokushima Graduate School, Tokushima, Tokushima Prefecture, Japan
* To whom correspondence should be addressed. E-mail: rokutan{at}basic.med.tokushima-u.ac.jp.
Helicobacter pylori (H. pylori) lipopolysaccharide (LPS) activates a homologue of gp91phox,
NADPH oxidase 1 (Nox1), in guinea pig gastric mucosal cells cultured in 10% fetal bovine
serum (FBS)-containing medium. Reverse transcriptase-PCR and Northern hybridization
demonstrated that H. pylori LPS stimulated expression of Nox1 and a novel p47phox homologue
(Noxo1) mRNAs with a peak at 4 h, followed by up-regulation of superoxide anion (O2
-)
generation. Pretreatment with 10 mg/ml of a non-absorbable anti-gastric ulcer drug, ecabet
sodium (ecabet), completely blocked these two mRNA expressions and the up-regulation of
O2
- production. Under low (0.1%)-FBS conditions, H. pylori LPS predominantly caused
apoptosis of the cells. Ecabet completely blocked the LPS-triggered phosphorylation of
transforming growth factor-
-activated kinase 1 (TAK1) and TAK1-binding protein 1,
activation of caspase 8, loss of mitochondrial membrane potential, release of cytochrome c,
activation of caspase 3, and appearance of apoptotic cells. In contrast, ecabet had no effect on
ethanol- or etoposide-initiated apoptosis. The ecabet-pretreated cells exhibited the
responsiveness to H. pylori LPS, similarly as untreated control cells did, when ecabet was
removed by washing prior to the addition of H. pylori LPS. Incubation of H. pylori LPS with
ecabet eliminated the toxic effects of the LPS, and non-denatured polyacrylamide gel
electrophoresis indicated the formation of higher molecular mass complexes between H. pylori
LPS and ecabet, suggesting that ecabet may interact with H. pylori LPS and block the
activation of Toll-like receptor 4 (TLR4). Our results suggest that ecabet may suppress
TLR4-mediated inflammation or accelerated apoptosis caused H. pylori infection.
