We examined the effect of lafutidine, a histamine H(2) receptor antagonist with a mucosal protective action mediated by capsaicin-sensitive sensory neurons (CSN), on intestinal lesions produced by loxoprofen administration in rats.Animals were given loxoprofen (10-100 mg/kg p.o.) and killed 24 h later. Lafutidine (10 and 30 mg/kg), cimetidine (100 mg/kg) or famotidine (30 mg/kg) was given twice p.o. at 0.5 h before and 6 h after loxoprofen. Omeprazole (100 mg/kg) was given p.o. once 0.5 h before. Ampicillin (800 mg/kg) was given p.o. twice at 24 h and 0.5 h before loxoprofen, while 16,16-dimethyl prostaglandin E(2) (dmPGE(2); 0.01 mg/kg) was given i.v. twice at 5 min before and 6 h after.Loxoprofen dose-dependently produced hemorrhagic lesions in the small intestine, accompanied by invasion of enterobacteria and increased inducible nitric oxide synthase (iNOS) expression as well as myeloperoxidase activity in the mucosa. The ulcerogenic response to loxoprofen (60 mg/kg) was significantly prevented by lafutidine (30 mg/kg), similar to dmPGE(2) and ampicillin, and the effect of lafutidine was totally attenuated by ablation of CSN. Neither cimetidine, famotidine nor omeprazole had a significant effect against these lesions. Lafutidine alone increased mucus secretion and reverted the decreased mucus response to loxoprofen, resulting in suppression of bacterial invasion and iNOS expression. In addition, loxoprofen downregulated Muc2 expression, and this response was totally reversed by lafutidine mediated by CSN.Lafutidine protects the small intestine against loxoprofen-induced lesions, essentially mediated by the CSN, and this effect may be functionally associated with increased Muc2 expression/mucus secretion, an important factor in the suppression of bacterial invasion.
Crohn's disease, a chronic and recurrent gastrointestinal disease, frequently causes intestinal fibrosis. Transient receptor potential melastatin 2 (TRPM2) belonging to TRP channel family is activated by reactive oxygen species. This study investigated the role of TRPM2 in acute colitis and chronic colitis-associated fibrosis progression. Acute colitis and chronic colitis-associated fibrosis were induced in TRPM2-deficient (KO) and wild-type (WT) mice through single and repeated intrarectal injections of trinitrobenzene sulfonic acid (TNBS). Bone marrow-derived macrophages (BMDMs) were created by M-CSF stimulation. In WT, a single TNBS injection induced acute colitis and upregulated inflammatory cytokines/chemokines, Th1 and Th17-related cytokines, and their transcription factors. In contrast, repeated TNBS injections induced chronic colitis-associated fibrosis and upregulation of fibrogenic factors, Th2-related cytokines, and its transcription factor. However, these increases were considerably suppressed in KO. Treating BMDMs with H2O2 increased inflammatory, Th1 and Th17-related cytokines expression, and JNK and ERK phosphorylation, but these responses were significantly less in KO than those in WT. These finding suggest that TRPM2 contributes to acute colitis progression via Th1/Th17-mediated immune responses. Furthermore, TRPM2 may be directly involved in colitis-associated fibrosis induction, likely due to the regulation of Th2/TGF-β1-mediated fibrogenesis in addition to a consequence of acute colitis progression.
Leukotriene B4 receptor type 1 (BLT1), a high-affinity receptor for leukotriene B4 (LTB4), plays an important role in inflammatory responses, including allergic airway inflammation. In this study, we examined the effect of genetic BLT1 deletion (KO) on ovalbumin (OVA)-induced allergic enteritis, a gastrointestinal form of food allergy in mice. Repeated oral OVA challenges after sensitization with OVA/alum induced allergic enteritis, characterized by systemic allergic symptoms (scratching, immobility, and swelling), diarrhoea, colonic oedema, and colonic goblet cell hyperplasia, accompanied by increased colonic peroxidase activity, colonic inflammatory cytokine expression, and serum OVA-specific IgE levels. The severity of enteritis was significantly attenuated in KO mice compared with wild-type (WT) mice, without an increase in serum OVA-specific IgE levels. The accumulation of neutrophils, eosinophils, M2-macrophages, dendritic cells, CD4+ T cells, and mast cells was observed in the colonic mucosa of allergic enteritis, and such accumulations were significantly lower in KO mice than in WT mice. BLT1 expression was upregulated and colocalized mostly in neutrophils and partly in eosinophils and dendritic cells in the colonic mucosa of allergic enteritis. These findings indicate that BLT1 deficiency ameliorates OVA-induced allergic enteritis in mice, and that LTB4/BLT1 contributes to neutrophil and eosinophil accumulations in the allergic colonic mucosa. BLT1 is therefore a promising drug target for the treatment of food allergies.
Peptidylarginine deiminase (PAD) is an enzyme, which citrullinates arginine residues of proteins. PAD4 is known to be an important factor in the induction of neutrophil extracellular traps (NETs), which are implicated in the pathogenesis of various inflammatory diseases. PAD2 is also reportedly involved in the pathogenesis of inflammatory diseases, but the details are not fully understood. In this study, we investigated the pathogenic roles of PAD2 and PAD4 in inflammatory bowel disease using a trinitrobenzene sulfonic acid (TNBS)-induced murine colitis model. PAD2- and PAD4-deficient (PAD2KO and PAD4KO) mice were generated by CRISPR-Cas9-mediated genomic editing. Colitis was induced by an intrarectal injection of TNBS. TNBS injection produced severe colitis, accompanied by body weight loss, increase in myeloperoxidase activity, and inflammatory cytokine expression in wild-type (WT) mice. In contrast, the severity of colitis with these inflammatory responses was significantly reduced in either PAD2KO or PAD4KO mice. NETs formation in peritoneal neutrophils was significantly suppressed in PAD4KO but not PAD2KO mice compared with WT mice. In contrast, macrophage extracellular traps formation in peritoneal macrophages was affected neither PAD2KO nor PAD4KO mice. In conclusion, PAD2 and PAD4 contribute to the pathogenesis of TNBS-induced colitis. PAD4 is involved in NET formation while PAD2 is not involved in the NET formation, suggesting that PAD2 may contribute to the progression of colitis via a mechanism different from NETs formation and PAD4.
Orphan G protein-coupled receptor GPR35, which can be activated by lysophosphatidic acid and kynurenic acid, is highly expressed in gastrointestinal tracts. Previous studies demonstrate the implication of GPR35 in the pathogenesis of inflammatory bowel disease but this role remains undefined. The present study investigated the pathogenic role of GPR35 in murine ileitis and colitis models. GPR35-deficient (GPR35KO) mice were generated by CRISPR-Cas9-mediated genome editing on C57BL/6 background. Colitis and ileitis were induced in GPR35KO and its wild-type (WT) mice by 7-days treatment with dextran sulfate sodium (DSS) and single injection of indomethacin, respectively. DSS-treatment produced body weight loss with diarrhea and blood feces, and caused severe colitis, characterized by shortening colon length and histological injury 7 days later. The severity of colitis with systemic symptoms was significantly augmented in GPR35KO mice compared with WT mice. Indomethacin injection produced intestinal injury 48 h later and the severity was also more higher in GPR35KO mice than WT mice. These findings suggest that GPR35 plays an anti-inflammatory role in DSS-induced colitis and indomethacin-induced ileitis in mice. Thus, GPR35 may be a promising target for treatment of inflammatory bowel disease.
The effect of subtype-selective phosphodiesterase (PDE) inhibitors on acid secretion was examined in mouse stomachs to investigate which PDE isozymes are involved in the local regulation of this secretion. Male DDY mice were used after 18 h fasting. An isolated stomach was incubated in an organ bath containing buffered solution gassed with 95% O(2)/5% CO(2), while the lumen was perfused with unbuffered solution gassed with 100% O(2). Acid secretion was measured at pH 5.4 using a pH-stat method. Histamine or pituitary adenylate cyclase activating polypeptide (PACAP) was added to the serosal solution. PDE inhibitors were added to the serosal solution 30 min before histamine or PACAP. The secretion of acid in the isolated stomach was increased by histamine or PACAP, and these responses were totally inhibited by famotidine. IBMX alone increased basal acid secretion and significantly enhanced the acid responses to histamine and PACAP. Among the PDE inhibitors tested, only rolipram (PDE4 inhibitor) significantly increased basal acid secretion and potentiated the acid responses to histamine and PACAP. The latter peptide increased histamine release into the medium, and this response was also enhanced by rolipram. Furthermore, rolipram significantly increased cAMP production induced in the isolated stomach by histamine and PACAP. These results suggest that PDE4 is involved in the local regulation of gastric acid secretion via the degradation of cAMP and that the PDE4 inhibitor rolipram increases the secretion of acid by potentiating acid production in parietal cells and enhancing histamine release from enterochromaffin-like cells.
