Hikaru Nishio

Non-steroidal anti-inflammatory drug-induced small intestinal damage is Toll-like receptor 4 dependent

Background: Enterobacteria and cytokines both play roles in the pathophysiology of NSAID-induced enteropathy. Toll-like receptor (TLR) 4 recognises lipopolysaccharide (LPS), resulting in activation of an inflammatory cascade via the accessory protein MyD88. Aims: To investigate role of TLR4 in inflammatory responses in indomethacin-induced enteropathy. Methods: Indomethacin was administered p.o. to non-fasting rats and mice to induce small intestinal damage. The extent of such damage was evaluated by measuring the injured area stained dark blue with Evans blue. Rats were given antibiotics (ampicillin, aztreonam or vancomycin) p.o., or intraperitoneal LPS (a TLR4 ligand) or neutralising antibodies against neutrophils, tumour necrosis factor (TNF)-α, or monocyte chemotactic protein (MCP)-1. Furthermore, the intestinal ulcerogenicity of indomethacin was examined in TLR4-mutant, TLR4−/−, and MyD88−/− mice. Results: Indomethacin induced small intestinal damage with an increase in expression of TNF-α and MCP-1 in both rats and mice. Antibodies against neutrophils, TNF-α and MCP-1 inhibited the damage by 83%, 67% and 63%, respectively, in rats. Ampicillin and aztreonam also inhibited this damage, and decreased the number of Gram-negative bacteria in the small intestinal contents of the rat. However, vancomycin, which exhibited no activity against Gram-negative bacteria, had no preventive effect against this damage. Administration of LPS 1 h after indomethacin aggravated the damage, whereas LPS pretreatment inhibited it with reduction of expression of TLR4 and cytokines. In TLR4-mutant mice, the damage and cytokine expression were markedly inhibited. TLR4−/− and MyD88−/− mice were also resistant to the damage. Conclusions: Indomethacin may injure the small intestine through a TLR4/MyD88-dependent pathway.

Probiotic Lactobacillus casei strain Shirota prevents indomethacin-induced small intestinal injury: involvement of lactic acid

Inflammatory responses triggered by activation of the lipopolysaccharide (LPS)/Toll-like receptor (TLR) 4 signaling pathway are a key mechanism in nonsteroidal anti-inflammatory drug-induced enteropathy. The aim of this study was to investigate the probiotic effect of Lactobacillus casei strain Shirota (LcS) on indomethacin-induced small intestinal injury. Rats pretreated with viable LcS or heat-killed LcS once or once daily for a week were administered indomethacin by gavage to induce injury. Anti-inflammatory effects of L-lactic acid (1-15 mM) were evaluated in vitro by use of THP-1 cells. One-week treatment with viable LcS prevented indomethacin-induced intestinal injury with increase in the concentration of lactic acid in small intestinal content and inhibited increases in myeloperoxidase activity and expression of mRNA for tumor necrosis factor-alpha (TNF-alpha) while affecting neither TLR4 expression nor the number of gram-negative bacteria in intestinal content, whereas neither heat-killed LcS nor a single dose of viable LcS inhibited intestinal injury. Prevention of this injury was also observed in rats given l-lactic acid in drinking water. Both L-lactic acid and LcS culture supernatant containing 10 mM lactic acid inhibited NF-kappaB activation and increases in TNF-alpha mRNA expression and TNF-alpha protein secretion in THP-1 cells treated with LPS. Western blot analyses showed that both L-lactic acid and LcS culture supernatants suppressed phosphorylation and degradation of I-kappaB-alpha induced by LPS without affecting expression of TLR4. These findings suggest that LcS exhibits a prophylactic effect on indomethacin-induced enteropathy by suppressing the LPS/TLR4 signaling pathway and that this probiotic effect of LcS may be mediated by L-lactic acid.

Aggravation by Selective COX-1 and COX-2 Inhibitors of Dextran Sulfate Sodium (DSS)-Induced Colon Lesions in Rats

We examined the effect of cyclooxygenase (COX) inhibitors on dextran sulfate sodium (DSS)-induced ulcerative colitis in rats and investigated the role of COX isozymes in the pathogenesis of this model. Experimental colitis was induced by treatment with 2.5% DSS in drinking water for 6 days. Indomethacin (a nonselective COX inhibitor), SC-560 (a selective COX-1 inhibitor), or celecoxib (a selective COX-2 inhibitor) was given PO twice daily for 6 days, during the first 3 or last 3 days of the experimental period. Daily treatment with 2.5% DSS for 6 days caused damage to the colon, with a decrease in body weight gain and colon length as well as an increase of myeloperoxidase (MPO) activity. All COX inhibitors given for 6 days significantly worsened the severity of DSS-induced colonic damage with increased MPO activity. The aggravation was also observed by SC-560 given for the first 3 days or by celecoxib given for the last 3 days. The expression of COX-2 mRNA in the colon was upregulated on day 3 during DSS treatment, with significant increase of prostaglandin E2 PGE2 production. The PGE2 content on day 3 during DSS treatment was inhibited by both indomethacin and SC-560, but not by celecoxib; on day 6 it was suppressed by both indomethacin and celecoxib, but not SC-560. These results suggest that endogenous prostaglandins (PGs) afford protection against colonic ulceration, yet the COX isozyme responsible for the production of PGs differs depending on the stage of ulceration; COX-1 in the early stage and COX-2 in the late stage.

Essential Role of Pepsin in Pathogenesis of Acid Reflux Esophagitis in Rats

Pepsin, a protease activated by gastric acid, is a component of the refluxate, yet the role of pepsin in the pathogenesis of reflux esophagitis has not been well studied. In the present study, we examined the effect of pepstatin, a specific inhibitor of pepsin, on acid reflux esophagitis. Acid reflux esophagitis was induced in rats by ligating both the pylorus and the forestomach for 3 or 4 hr. Pepstatin, ecabet Na (the anti-ulcer drug), and L-glutamine were administered intragastrically after the ligation. Pepstatin or ecabet Na, given intragastrically, significantly prevented esophageal lesions, even though they did not affect basal acid secretion in pylorus-ligated rats. Pepstatin significantly inhibited pepsin activity in vivo and in vitro, while ecabet Na inhibited this activity in vitro. By contrast, L-glutamine given intragastrically aggravated the lesions in a dose-dependent manner, but even in the presence of L-glutamine the development of esophageal lesions was totally prevented by coadministration of pepstatin or ecabet Na. L-Glutamine increased the pH of gastric contents to approximately 2.0, the optimal pH for the proteolytic activity of pepsin in vitro. In addition, intragastric administration of exogenous pepsin worsened the severity of esophageal damage. These results suggest that pepstatin is highly effective against acid reflux esophagitis, without influencing acid secretion, while L-glutamine aggravated these lesions by increasing the pepsin activity by shifting the intraluminal pH to the optimal pH range for proteolytic action. It is assumed that pepsin plays a major pathogenic role in the development of acid reflux esophagitis.

Healing impairment effect of cyclooxygenase inhibitors on dextran sulfate sodium-induced colitis in rats.

We examined the effects of various cyclooxygenase (COX) inhibitors on the healing of colonic lesions induced by dextran sulfate sodium (DSS) in the rat. Colonic lesions were induced by 2.5% DSS in the drinking water for 7 days, and then the animals were fed with tap water for subsequent 7 days. Indomethacin (a nonselective COX inhibitor), SC-560 (a selective COX-1 inhibitor), or rofecoxib (a selective COX-2 inhibitor) was given orally twice daily after termination of the DSS treatment. DSS treatment caused severe colonic lesions with a decrease in body weight gain and colon length as well as an increase in myeloperoxidase activity and thiobarbituric acid reactant levels. The severity of colitis gradually reduced, with an improvement of morphological and histological alterations. Daily administration of indomethacin and rofecoxib significantly delayed the healing of colitis with deleterious influences on histological restitution as well as mucosal inflammation, while SC-560 had no effect. Although COX-1 mRNA was expressed in the colon without much alteration during the test period, the expression of COX-2 was upregulated with a peak on day 3 and decreased thereafter. The mucosal prostaglandin E2 content in the colon showed a biphasic change, in parallel with that of the COX-2 expression. The increased prostaglandin E2 production in the injured mucosa was attenuated by indomethacin and rofecoxib, but not by SC-560. These results suggest that endogenous prostaglandins produced by COX-2 play an important role in the healing of DSS-induced colonic lesions. Caution should be paid to the use of selective COX-2 inhibitors as well as nonsteroidal anti-inflammatory drugs in patients with colitis.

Orally administered L-arginine and glycine are highly effective against acid reflux esophagitis in rats

Summary Background Reflux esophagitis is caused mainly by excessive exposure of the mucosa to gastric contents. In the present study, we examined the effect of several amino acids on acid reflux esophagitis in rats. Material/Methods After 18 h of fasting, acid reflux esophagitis was induced by ligating both the pylorus and the transitional region between the forestomach and the corpus under ether anesthesia, and the animals were killed 4 h later. The severity of esophagitis was reduced by the oral administration of omeprazole, a proton pump inhibitor, or pepstatin, a specific pepsin inhibitor. Results The development of esophageal lesions was dose-dependently prevented by L-arginine and glycine, given intragastrically (i.g.) after the ligation, with complete inhibition obtained at 250 mg/kg and 750 mg/kg, respectively, and these effects were not influenced by the prior s.c. administration of indomethacin or L-NAME. By contrast, both L-alanine and L-glutamine given i.g. after the ligation aggravated these lesions in a dose-dependent manner. These amino acids had no effect on acid secretion but increased the pH of the gastric contents to 1.8~2.3 due to their buffering action. Conclusions The results confirmed an essential role for acid and pepsin in the pathogenesis of acid reflux esophagitis in the rat model and further suggested that various amino acids affect the severity of esophagitis in different ways, due to yet unidentified mechanisms; L-alanine and L-glutamine exert a deleterious effect on the esophagitis, while L-arginine and glycine are highly protective, independent of endogenous prostaglandins and nitric oxide.

Protective Effect of Intra-Rectal Administration of Rebamipide on Dextran Sulfate Sodium-Induced Rat Colitis

Background/Aim: Rebamipide, an anti-ulcer drug, has various actions including radical scavenging and mucus-stimulating as well as anti-inflammatory effects, and exhibits both mucosal protective and healing promoting actions in the stomach. In the present study, we examined the effect of rebamipide on an animal model of colitis induced by dextran sulfate sodium (DSS). Methods: Experimental colitis was induced in rats by daily treatment with 3% DSS in drinking water for 7 days. Rebamipide (3–30 mg/kg), 5-aminosalicylic acid (5-ASA: 150 mg/kg) or metronidazole (10 and 30 mg/kg) was administered intra-rectally once daily for 6 days. The ulceration area, colon length, and mucosal myeloperoxidase (MPO) activity as well as thiobarbituric acid-reactive substance (TBARS) were measured on the 7th day after the onset of DSS treatment. The effects of rebamipide on the secretion of mucus in the colon was also examined. Results: DSS treatment caused severe lesions in the colon, accompanied by an increase in MPO activity and TBARS as well as a decrease in body weight gain and colon length. Repeated administration of rebamipide dose-dependently suppressed the colon lesions and improved the pathological changes induced by DSS treatment. Rebamipide significantly increased the mucus contents in the colon. Both 5-ASA and metronidazole also reduced the severity of DSS-induced lesions. Conclusion: These results suggest that intra-rectal administration of rebamipide is effective against DSS-induced colitis. The protective effect of rebamipide may be attributable to both the radical scavenging action and the increase in the production of mucus in the colon, the latter presumably suppressing the process of intestinal bacterial infiltration.

Involvement of prostacyclin/IP receptors in decreased acid response of damaged stomachs — Mediation by somatostatin/SST2 receptors

AIMS We examined the effect of a prostacyclin (PGI(2)) analog iloprost on histamine-induced acid secretion and investigated how endogenous PGI(2) mediated the decreased secretory response in the damaged stomach after exposure to taurocholate (TC). MAIN METHODS Male C57BL/6 mice, both wild-type (WT) and IP receptor knockout (IP-KO) animals, were used after 18 h of fasting. Under urethane anesthesia, the abdomen was incised, and an acute fistula was provided in the stomach. KEY FINDINGS Acid secretion in WT and IP-KO mice was similarly and dose-dependently increased by histamine. Iloprost decreased the histamine-stimulated secretion in WT but not IP-KO mice. The inhibitory effect of iloprost in WT mice was totally abrogated by the prior administration of CYN154806, a selective somatostatin SST2 receptor antagonist. On the other hand, the acid secretion in WT mice was decreased after exposure of the stomach to 20 mM TC for 20 min, with an increase in mucosal PGI(2) content, but the decrease was significantly less marked in IP-KO mice. The decreased acid response to TC in WT mice was totally prevented by the prior administration of CYN154806 as well as indomethacin. Somatostatin contents in the stomach were reduced after the administration of iloprost or the mucosal exposure to TC, while the blood levels increased. SIGNIFICANCE Somatostatin/SST2 receptors are involved in the decreased acid response of the damaged stomach, in addition to PGI(2)/IP receptors. It is assumed that PGI(2) releases somatostatin from D cells, which in turn decreases acid secretion via the activation of SST2 receptors.

Tu1864 A Novel Orally Active Phosphatidylinositol 3-Phosphate 5-Kinase (Pikfyve) Inhibitor Ameliorates Mouse Colitis by Inhibition of IL-12 and IL-23 Production From Macrophages

A Novel Orally Active Phosphatidylinositol 3-Phosphate 5-Kinase (Pikfyve) Inhibitor Ameliorates Mouse Colitis by Inhibition of IL-12 and IL-23 Production From Macrophages Ayatoshi Andou, Takashi Yamamoto, Eviryanti Agung, Yukie Seki, Hikaru Nishio, Manami Shuto, Misato Noguchi, Yoichiro Shima, Sen Takeshita, Ryohei Yokoyama, Nobuhiko Hayakawa, Masatsugu Noguchi, Shunsuke Kageyama, Hiroyuki Eda, Makoto Shiozaki, Itsuya Tanabe, Ryusuke Nakagawa, Kuniya Sakurai, Shoji Masataka

W1553 Glutamate Reduces Helicobacter pylori-Induced Gastric Atrophy in Rodents - Possible Involvement of Chief Cell Protection via Glutamate Metabolism-

The pH of the alkaline microclimate overlying the duodenal enterocyte brush border is regulated by an ecto-purinergic signaling system consisting of intestinal alkaline phosphatase (IAP), extracellular ATP and P2Y receptors. This system regulates the rate of duodenal bicarbonate secretion (DBS). IAP inhibition increases DBS and non-lytic ATP release into the lumen, the mechanism of which has not been clarified. Ecto-F1F0-ATP synthase has been localized to the plasma membrane of several non-epithelial cell types. We hypothesized that ecto-ATP synthase generates extracellular ATP, regulating DBS as part of the ectopurinergic signaling system. We measured DBS with flow-through pH and CO2 electrodes with the perfusate ATP content measured by luciferin-luciferase bioassay. We tested the effect of perfusion of the competitive AP inhibitor glycerol phosphate (GP, 10 mM) with or without the addition of ATP synthase inhibitors oligomycin (Om, 5 μg/ml), piceatannol (Pic, 20 μM) or resveratorol (Res, 20 μM), or the mitochondrial uncoupler carbonyl cyanide 3-chlorophenylhydrazone (CCCP, 1 μM) on DBS and ATP output. GP increased DBS and ATP output. Pic or Res inhibited GP-induced augmented DBS and ATP output. GP, or Pic or Res with GP had no effect on LDH release, confirming the absence of lytic cellular injury. Om or CCCP had a lesser effect on GP-induced DBS, but inhibited GP-induced ATP output accompanied by increased LDH release, consistent with cellular injury. Furthermore, α and β subunits of F1 complex of ATP synthase were immunolocalized to the brush border membranes (α β) in the lamina propria mucosa. Furthermore, phosphate buffer saline (pH 7.0, 10 mM) increased ATP output, suggesting that excess supply of inorganic phosphate (Pi) may enhance ATP synthesis and/or inhibit ATP degradation. Luminal ATP release unmasked by IAP inhibition is dependent on extracellular ATP synthesis, consistent with the presence of ecto-ATP synthase on the brush border. Extracellular ATP synthesis and ATP degradation by IAPmay coordinately regulate ATP-P2Y signaling for DBS as part of a novel ecto-purinergic signaling system.