Ecabet sodium inhibitsHelicobacter pylorilipopolysaccharide-induced activation of NADPH oxidase 1 or apoptosis of guinea pig gastric mucosal cells

Helicobacter pylori LPS activates a homolog of gp91phox, NADPH oxidase 1 (Nox1), in guinea pig gastric mucosal cells cultured in 10% FBS-containing medium. RT-PCR and Northern hybridization demonstrated that H. pylori LPS stimulated expression of Nox1 and a novel p47phoxhomolog (Noxo1) mRNAs with a peak at 4 h, followed by upregulation of superoxide anion (O2−) generation. Pretreatment with 10 mg/ml of a nonabsorbable antigastric ulcer drug, ecabet sodium (ecabet), completely blocked these two mRNA expressions and the upregulation 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-β-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 before the addition of H. pylori LPS. Incubation of H. pylori LPS with ecabet eliminated the toxic effects of the LPS, and nondenatured 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.

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Helicobacter pylori lipopolysaccharide activates Rac1 and transcription of NADPH oxidase Nox1 and its organizer NOXO1 in guinea pig gastric mucosal cells

AJP Cell Physiology ◽

10.1152/ajpcell.00319.2004 ◽

2005 ◽

Vol 288 (2) ◽

pp. C450-C457 ◽

Cited By ~ 106

Author(s):

Tsukasa Kawahara ◽

Motoyuki Kohjima ◽

Yuki Kuwano ◽

Hisano Mino ◽

Shigetada Teshima-Kondo ◽

...

Keyword(s):

Helicobacter Pylori ◽

Guinea Pig ◽

Nadph Oxidase ◽

Primary Cultures ◽

Transcriptional Level ◽

Type I ◽

Gastric Mucosal Cells ◽

Mucosal Cells ◽

H Pylori ◽

Constitutively Active

Primary cultures of guinea pig gastric mucosal cells express NADPH oxidase 1 (Nox1), a homolog of gp91 phox, and produce superoxide anion (O2−) at a rate of ∼100 nmol·mg protein−1·h−1 in response to Helicobacter pylori ( H. pylori) lipopolysaccharide (LPS) from virulent type I strains. The upregulated O2− production also enhances H. pylori LPS-stimulated tumor necrosis factor-α or cyclooxygenase-2 mRNA expression, which suggests a potential role for Nox1 in the pathogenesis of H. pylori-associated diseases. The H. pylori LPS-stimulated O2− production in cultured gastric mucosal cells was inhibited by actinomycin D as well as cycloheximide, suggesting that the induction is regulated at the transcriptional level. The LPS treatment not only increased the Nox1 mRNA to a greater extent but also induced expression of the message-encoding, Nox-organizing protein 1 (NOXO1), a novel p47 phox homolog required for Nox1 activity. In addition, H. pylori LPS activated Rac1; i.e., it converted Rac1 to the GTP-bound state. A phosphoinositide 3-kinase inhibitor, LY-294002, blocked H. pylori LPS-induced Rac1 activation and O2− generation without interfering with the expression of Nox1 and NOXO1 mRNA. O2− production inhibited by LY-294002 was completely restored by transfection of an adenoviral vector encoding a constitutively active Rac1 but not an inactive Rac1 or a constitutively active Cdc42. These findings indicate that Rac1 plays a crucial role in Nox1 activation. Thus the H. pylori LPS-stimulated O2− production in gastric mucosal cells appears to require two distinct events: 1) transcriptional upregulation of Nox1 and NOXO1 and 2) activation of Rac1.

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Helicobacter pylorilipopolysaccharide induces apoptosis of cultured guinea pig gastric mucosal cells

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.2001.281.3.g726 ◽

2001 ◽

Vol 281 (3) ◽

pp. G726-G734 ◽

Cited By ~ 16

Author(s):

Tsukasa Kawahara ◽

Shigetada Teshima ◽

Yuki Kuwano ◽

Ayuko Oka ◽

Kyoichi Kishi ◽

...

Keyword(s):

Growth Factor ◽

Cytochrome C ◽

Guinea Pig ◽

Primary Cultures ◽

Specific Inhibitor ◽

Caspase 8 ◽

Cytochrome C Release ◽

Gastric Mucosal Cells ◽

Mucosal Cells ◽

H Pylori

Helicobacter pylori lipopolysaccharide (LPS) is generally accepted as a low-toxicity virulence. Primary cultures of guinea pig gastric mucosal cells expressed the Toll-like receptor 4 and were sensitive to H. pylori LPS as well as Escherichia coli LPS. H. pylori LPS stimulated phosphorylation of transforming growth factor-β-activated kinase 1 (TAK1), TAK1-binding protein 1 (TAB1), and c-Jun NH2-terminal kinase (JNK) 2. H. pylori LPS at >2.1 endotoxin unit/ml (>1 ng/ml) activated caspase-8, stimulated cytochrome c release from mitochondria, and subsequently activated caspases-9 and -3, leading to apoptosis. Epidermal growth factor blocked all of these apoptotic processes and inhibited apoptosis, whereas it did not modify the phosphorylation of TAK1, TAB1, and JNK2. A comparatively specific inhibitor of caspase-8 or -9 blocked apoptosis, whereas cytochrome c release was prevented only with a caspase-8-like inhibitor. Our results suggest that caspase-8 and mitochondria may play crucial roles in H. pylori LPS-induced apoptosis and that this accelerated apoptosis may be involved in abnormal cell turnover of H. pylori-infected gastric mucosa.

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