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Background/Aim: Transient receptor potential ankyrin 1 (TRPA1) locates in the cell membrane of enterochromaffin cells (EC), and TRPA1 stimulation on EC is proposed to modulate intestinalmotility via release of serotonin (5-HT). Although a previous study has demonstrated that TRPA1 agonists produce a contractile response in isolated intestinal muscle strips in vitro, apparently contradictory results have been reported for gastrointestinal (GI) transit in vivo. The Japanese traditional medicine daikenchuto (TU-100), which is being investigated as a new drug in the US and is used for the treatment of postoperative ileus (POI) in Japan, was reported to accelerate GI transit in animals and humans. TU-100 has recently been shown to increase intestinal blood flow via stimulation of TRPA1 located on the epithelial cells of the small intestine. The present study investigates the effects of TU-100, TU-100 ingredients, and TRPA1 agonists on dysregulated intestinal transit in an established murine POI model. Methods: The POI model, which involves manipulation of colon and small intestinal transit, was evaluated by calculating the geometric center of distribution of fluorescent-labeled dextran markers. The in vitro motility of isolated mouse small intestine in an organ bath was evaluated by video imaging and intraluminal pressure was measured by a strain gauge transducer. 5-HT release from isolated small intestinal segments or a rat pancreatic endocrine cell line RIN-14B, which is supposed to have common properties with EC, was measured by ELISA and/or HPLC. The tested compounds included the well known TRPA1 agonists aryl isothiocyanate (AITC) and cinnamaldehyde (CA), as well as TU-100 and its ingredients shogaol and hydroxy-α-sanshool, which stimulate TRPA1. TRPA1 KO mice were also studied. Results: Orally administered TRPA1 agonists, TU-100, and TU100 ingredients improved small intestinal transit in the POI model. Incubation of small intestinal segments with TRPA1 agonists and TU-100 ingredients resulted in an increase in the level of 5-HT in the culture supernatant. The effects of AITC and TU-100 ingredients, but not CA, were abrogated in TRPA1 KO mice. All agents induced the release of 5-HT from RIN14B cells Conclusion: TRPA1 stimulation by TU-100 may be involved in its beneficial effect on POI. The role of TRPA1 in GI motility deserves further extensive investigation with the aim of developing a novel therapeutic strategy to treat GI motility disorders.

Background and Aim: Agonists of transient receptor potential vanilloid (TRPV1) and transient receptor potential ankyrin 1 (TRPA1) have been reported to induce contraction of intestinal smooth muscle In Vitro, but it is unclear whether activation of TRPV1 or TRPA1 affects gastrointestinal (GI) transit In Vivo. In this study, we investigated the effects of capsaicin (TRPV1 agonist), allyl isothiocyanate (AITC, TRPA1 agonist), cinnamaldehyde (CA, TRPA1 agonist), daikenchuto (TU-100, TRPA1/TRPV1 agonist), and 6-shogaol (6SG, TRPA1/TRPV1 agonist), the main constituent of TU-100, on murine postoperative ileus (POI) induced by surgical manipulation of mouse intestines. TU-100, a traditional Japanese medicine that has been prescribed to improve paralytic POI in Japan, is currently undergoing clinical trials on various intestinal diseases such as postoperative ileus, functional constipation, and Crohn's disease in the USA (http://clinicaltrials.gov/ct2/results?term=TU-100). Methods: Male C57Bl/ 6 mice underwent laparotomy and large bowel manipulation. Fluorescein isothiocyanatedextran (FD70) was administered orally at 23.5 h postoperatively, and the mice were killed at 30 min post gavage. GI transit was estimated by calculating the geometric center (GC) value. Serotonin release from RIN-14B cell, an enterochromaffin-like cell line, was measured by EIA. Results: Colonic manipulation decreased small intestinal transit to 70.2±2.7% of control. TRPA1 agonists (AITC, CA, TU-100, and 6SG) significantly improved the delayed GI transit induced by intestinal manipulation by 68.3±12.9%, 83.6±9.8%, 65.7±13.8%, and 26.3±6.7%, respectively. On the other hand, the TRPV1 agonist capsaicin had no effect. Freshly isolated intestinal epithelial cells and RIN-14B cells expressed abundant TRPA1 mRNA but no TRPV1 mRNA. POI was induced in TRPV1-deficient mice to a similar degree as in wild-type mice, and TU-100 was effective in POI of TRPV1-deficient mice. AITC, CA, TU-100, and 6SG stimulated RIN-14B cell to release serotonin. Conclusions: These results suggest that TRPA1 is essential to induction and alleviation of POI while TRPV1 stimulation may play little role. Stimulation of TRPA1 may be a novel therapeutic approach for the treatment of POI.

Background: Intestinal scarring is a major cause of morbidity in Crohn's disease. Current therapies treat inflammation, but do not alter the progression of fibrosis and bowel obstruction. Given the observation that late use of potent anti-inflammatory therapies does not reduce stenosis or obstruction, we hypothesized that intestinal fibrosis becomes self-propagating, despite removal of inflammatory stimuli. Methods: The Salmonella typhimurium murine model of inflammation and fibrosis in which clearance of commensal microbiota by streptomycin, followed by infection with S. typhimurium, produces chronic inflammation, culminating with fibrosis by day 21 post infection was used. The inflammatory stimulus (S. typhimurium) was removed with the oral antibiotic levofloxacin at day 2, 4, or 8 post-infection. Eradication of S.typhimurium was confirmed by stool plating and T-RFLP analysis. Results: By day 2 post-infection, the mouse cecae infected with S.typhimurium developed an inflammatory phenotype, characterized by a shrunken yet heavier cecum, expansion of the cecal submucosa, and induction of inflammatory genes (IL-1b, TNFa, IL-6, IL-17, IL12p40). However, fibrotic gene expression (CTGF, IGF-1, TGFb) was indistinguishable from uninfected controls. In addition, aSMA protein expression was not induced until day 8 postinfection. Early intervention (day 2 & 4) repressed inflammation as determined by gross pathology, histopathology, and repression of inflammatory genes. Fibrotic gene expression was partially repressed by day 2 levofloxacin treatment. However, day 8 levofloxacin treatment did not prevent induction of aSMA protein or pro-fibrotic genes (CTGF, IGF-1, TGFb) by day 8 and which continued to increase after levofloxacin treatment to day 21, remaining significantly higher than uninfected controls or matched S.typhimurium infected mice harvested at day 4 and 8 post-infection (p < 0.03) Conclusions: Early removal of the inflammatory stimulus reduces fibrosis, but fibrosis after initiation continues to propagate in the absence of an inflammatory stimulus. Since many Crohn's patients develop complications of fibrosis, understanding the mechanisms of auto-propagation of fibrosis and developing anti-fibrotic therapies is critical to improving outcomes in Crohn's disease.

Background&Aim: Small intestinal Paneth cells contnbute to mucosal innate immunity by sensing bactena and secreting microbicidaI peptides, particularly a-defensins (Nature Immunol l: 113, 2000). Our aims were to investigate the expression and function of Toll-like receptor (TLR)s and related molecules in crypts of human small intestine. Methods: Ileal mucosa were obtained from surgical specimens resected from patients with colon cancer with normal ileum under informed consent. Intact crypts were isolated by standard EDTA dissociation, and further collagenase dissociation was conducted to prepare crypt single cell s0spensions. RT-PCR experiments were performed on individual crypts or isolated Paneth cells to assay for TLRI-10, CD14, MD2 and MyD88 mRNAs. Specific TLRs were immunocolocalized in isolated crypts and in Paneth cells with a-defensin HD5 using anti-TLR-2 and TLR-4 antibodies and confocal microscopic analysis. Isolated intact crypts were exposed to I x l03 S. typhimurium CFU/crypt to induce Paneth cell secretion. Secretions were assayed for bactericidal activity against defensin sensitive S. typhimurium phoPand for HD5 secretion by western blot analysis. Inhibition assay was performed using anti-TLR2 neutralizing antibody in the secretion assay system Results: TLR1-8, TLR10, CD14 and MyD88, but not M02 mRNAs were detected in single isolated crypts and isolated Paneth cells. TLR2 and ]-LR4 expression was restricted to Paneth cells in the isolated crypts. TLR4 staining in single Paneth cells was localized at the membrane surface and also near secretory granules, aDefensin secretion by Paneth cells in response to bacteria was inhibited by TLR2 antibody pre-treatment as judged by the diminished bactencidal activity and reduced secretion of HD5 in western blots. Conclusion: Paneth cells in human small intestinal crypts express Toll-like receptors that appear to be implicated in ex vivo responses to bacterial exposure.