Transient receptor potential melastatin 8 (TRPM8) is a non-selective cation channel activated by mild cooling and chemical agents including menthol. Nonsteroidal anti-inflammatory drugs have ...antipyretic, analgesic effects, and they can cause stomach and small intestinal injury. The current study investigated the role of TRPM8 in the pathogenesis of indomethacin-induced small intestinal injury. In male TRPM8-deficient (TRPM8KO) and wild-type (WT) mice, intestinal injury was induced via the subcutaneous administration of indomethacin. In addition, the effect of WS-12, a specific TRPM8 agonist, was examined in TRPM8KO and WT mice with indomethacin-induced intestinal injury. TRPM8KO mice had a significantly higher intestinal ulcerogenic response to indomethacin than WT mice. The repeated administration of WS-12 significantly attenuated the severity of intestinal injury in WT mice. However, this response was abrogated in TRPM8KO mice. Furthermore, in TRPM8-enhanced green fluorescent protein (EGFP) transgenic mice, which express EGFP under the direction of TRPM8 promoter, the EGFP signals in the indomethacin-treated intestinal mucosa were upregulated. Further, the EGFP signals were commonly found in calcitonin gene-related peptide (CGRP)-positive sensory afferent neurons and partly colocalized with substance P (SP)-positive neurons in the small intestine. The intestinal CGRP-positive neurons were significantly upregulated after the administration of indomethacin in WT mice. Nevertheless, this response was abrogated in TRPM8KO mice. In contrast, indomethacin increased the expression of intestinal SP-positive neurons in not only WT mice but also TRPM8KO mice. Thus, TRPM8 has a protective effect against indomethacin-induced small intestinal injury. This response may be mediated by the upregulation of CGRP, rather than SP.
G protein-coupled receptor (GPR) 40 is a receptor for long-chain free fatty acids that enhances glucagon-like peptide (GLP)-2 production in intestinal L-cells. GLP-2 and its analogs have reported to ...increase remission rates in patients with Crohn's disease and improve experimental colitis in rodents. In the present study, we investigated the ameliorative effect of GPR40 activation in a dextran sulfate sodium (DSS)-induced murine colitis model using a specific GPR40 agonist, AS2034178. The daily administration of AS2034178 attenuated DSS-induced increases in the disease activity index, the shortening of the colon length, and the histological colonic injury, and increased the myeloperoxidase (MPO) activity and expression of inflammatory cytokines, in a dose-dependent manner. These effects were abolished by treatment with DC260126, a GPR40 antagonist, or GLP-2 (3-33), a GLP-2 antagonist. GPR40 was expressed in the colonic mucosa, which was colocalized with proglucagon, a precursor of GLP-2. AS2034178 significantly increased the amount of GLP-2 in the colonic tissue, which was abolished by DC260126 but not GLP-2 (3-33). Furthermore, AS2034178 significantly promoted the healing of DSS-induced colitis. These findings suggest that GPR40 activation ameliorates DSS-induced colitis in mice by enhancing GLP-2 production. Thus, GPR40 is a potential target for the treatment of IBD.
Abstract
Irritable bowel syndrome (IBS) is the most common functional gastrointestinal disorder. Traumatic stress during adolescence increases the risk of IBS in adults. The aim of this study was to ...characterize the juvenile social defeat stress (SDS)-associated IBS model in mice. Juvenile mice were exposed to an aggressor mouse for 10 min once daily for 10 consecutive days. Behavioral tests, visceral sensitivity, immune responses, and fecal bacteria in the colon were evaluated in 5 weeks after SDS exposure. Social avoidance, anxiety- and depression-like behavior, and visceral hypersensitivity were observed. Juvenile SDS exposure significantly increased the number of 5-HT-containing cells and calcitonin gene-related peptide-positive neurons in the colon. The gut microbiota was largely similar between the control and juvenile SDS groups. The alterations in fecal pellet output, bead expulsion time, plasma corticosterone concentration, and colonic 5-HT content in response to restraint stress were exacerbated in the juvenile SDS group compared with the control group. The combination of juvenile SDS and restraint stress increased the noradrenaline metabolite 3-Methoxy-4-hydroxyphenylglycol (MHPG) content and MHPG/noradrenaline ratio in the amygdala when compared with restraint stress in control mice. These results suggest that juvenile SDS exposure results in later onset of IBS-like symptoms.
The chemotherapeutic agent 5‐fluorouracil (5‐FU) causes intestinal mucositis with severe diarrhoea, but the pathogenesis is not fully understood. In this study, we investigated the pathogenic effects ...of 5‐FU in mice, focusing on apoptosis, enterobacteria and inflammatory cytokines. Repeated administration of 5‐FU caused severe intestinal mucositis on day 6, accompanied by diarrhoea and body‐weight loss. TNF‐α expression increased 1 day after exposure to the drug, and spiked a second time on day 4, at which point myeloperoxidase activity and IL‐1β expression also increased. Apoptotic cells were observed in intestinal crypts only on day 1. 5‐FU also induced dysbiosis, notably decreasing the abundance of intestinal Firmicutes while increasing the abundance of Bacteroidetes and Verrucomicrobia. Twice‐daily co‐administration of oral antibiotics significantly reduced the severity of intestinal mucositis and dysbiosis, and blocked the increase in myeloperoxidase activity and cytokine expression on day 6, without affecting apoptosis and TNF‐α up‐regulation on day 1. In cultured colonic epithelial cells, exposure to 5‐FU also up‐regulated TNF‐α expression. Collectively, the data suggest that crypt apoptosis, dysbiosis and expression of inflammatory cytokines are sequential events in the development of intestinal mucositis after exposure to 5‐FU. In particular, 5‐FU appears to directly induce apoptosis via TNF‐α and to suppress intestinal cell proliferation, thereby resulting in degradation of the epithelial barrier, as well as in secondary inflammation mediated by inflammatory cytokines.
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G protein-coupled receptor 35 (GPR35), a receptor for lysophosphatidic acid, is highly expressed in the gastrointestinal tract. Recently, GPR35 has been implicated in the onset of ...inflammatory bowel disease (IBD), but its role in physiological and pathological processes in the colon remains undefined. In this study, we investigated the contribution of GPR35-mediated signalling to mucosal repair of colonic epithelium in IBD. GPR35 function was examined in a wound healing model, using young adult mouse colon epithelium (YAMC) cells, and in a dextran sulphate sodium (DSS)-induced mouse model of colitis. Cell proliferation, mRNA expression, extracellular signal-regulated kinase (ERK) activation, and protein localization were determined by MTT assay, quantitative RT-PCR, western blotting, and immunohistochemistry, respectively. GPR35 agonists (YE120, zaprinast, and pamoic acid) promoted wound repair in a concentration-dependent manner independently of cell proliferation, whereas a specific GPR35 antagonist CID2745687, forskolin, and pertussis toxin reversed the YE120-induced effect. YE120 increased the mRNA expression of fibronectin and its receptor integrin α5, and ERK1/2 phosphorylation, but these responses were attenuated by CID2745687 and forskolin. Furthermore, the severity of DSS-induced colitis was significantly reduced by daily injections of pamoic acid via upregulation of fibronectin and integrin α5 in the colonic epithelium. GPR35 signalling promotes mucosal repair by inducing fibronectin and integrin α5 expression, coupling to Gi protein, and activating ERK1/2 in colonic epithelial cells. These findings define GPR35 as a candidate therapeutic target in IBD.
Taste buds are comprised of taste cells, which are classified into types I to IV. Transient receptor potential (TRP) channels play a significant role in taste perception. TRP vanilloid 4 (TRPV4) is a ...non-selective cation channel that responds to mechanical, thermal, and chemical stimuli. The present study aimed to define the function and expression of TRPV4 in taste buds using Trpv4-deficient mice. In circumvallate papillae, TRPV4 colocalized with a type IV cell and epithelial cell marker but not type I, II, or III markers. Behavioural studies showed that Trpv4 deficiency reduced sensitivity to sourness but not to sweet, umami, salty, and bitter tastes. Trpv4 deficiency significantly reduced the expression of type III cells compared with that in wild type (WT) mice in vivo and in taste bud organoid experiments. Trpv4 deficiency also significantly reduced Ki67-positive cells and β-catenin expression compared with those in WT circumvallate papillae. Together, the present results suggest that TRPV4 contributes to sour taste sensing by regulating type III taste cell differentiation in mice.
Transient receptor potential (TRP) vanilloid 1 (TRPV1) and TRP ankyrin 1 (TRPA1), which are non-selective cation channels, play important roles in the sensation of pain. This study investigated the ...roles of TRPV1 and TRPA1 in dextran sulfate sodium (DSS)-induced murine colitis. DSS (2%) administered for 7 days caused severe colitis that was significantly less severe in TRPV1-deficient (TRPV1KO) and TRPA1-deficient (TRPA1KO) mice than that in wild-type (WT) mice. Similar colitis attenuations were observed in TRPV1KO and TRPA1KO mice but not in WT mice that had been transplanted with bone marrow cells from WT, TRPA1KO, or TRPV1KO mice. DSS treatment upregulated calcitonin gene-relative peptide (CGRP)- and substance P (SP)-positive nerve fibers in the colonic mucosa of WT mice. TRPV1KO and TRPA1KO mice showed significant reductions in the DSS-induced upregulation of SP, but the DSS-induced upregulation of CGRP was not reduced. Sensory deafferentation evoked by pretreatment with high doses of capsaicin markedly exacerbated DSS-induced colitis with reductions in DSS-induced upregulation of SP- and CGRP-positive nerve fibers. These findings suggest that neuronal TRPV1 and TRPA1 contribute to the progression of colonic inflammation. While these responses may be mediated by the upregulation of SP-mediated deleterious mechanisms, CGRP may be associated with protective mechanisms.
Functional dyspepsia (FD) is thought to be mainly based on gastric motility dysfunction and chronic hypersensitivity, yet FD animal models has been reported a few. We studied to establish the mouse ...model of impaired gastric motility induced by a pungent ingredient of wasabi allyl isothiocyanate (AITC), which is reliable to evaluate prokinetic agents. Male ddY mice were used. Gastric motility was measured by 13C-acetic acid breath test in conscious mice. AITC (80 mM) was given 60 min before the measurement of motility. Prokinetic agents including itopride (30, 100 mg/kg), mosapride (0.1–1 mg/kg), neostigmine (30 μg/kg), acotiamide (10–100 mg/kg), and daikenchuto (100–1000 mg/kg) were given 40 min before the measurement. AITC impaired gastric motility without mucosal damages, which reverted 24 h after AITC treatment. The decreased motility induced by AITC was restored by prokinetic agents such as itopride, mosapride, neostigmine, and acotiamide. In separate experiment, daikenchuto recovered the decreased motility induced by AITC, although daikenchuto had no effect on motility in normal condition. In conclusion, it is considered that the AITC-induced impaired gastric motility mouse model is useful to develop new prokinetic agents for treatment of FD, and to re-evaluate traditional Japanese herbal medicines.
Motilin is a gastrointestinal peptide hormone that stimulates gastrointestinal motility. Motilin is produced primarily in the duodenum and jejunum. Motilin receptors (MTLRs) are G protein-coupled ...receptors that may represent a clinically useful pharmacological target as they can be activated by erythromycin. The functions of motilin are highly species-dependent and remain poorly understood. As a functional motilin system is absent in rodents such as rats and mice, these species are not commonly used for basic studies. In this study, we examine the usefulness of human MTLR-overexpressing transgenic (hMTLR-Tg) mice by identifying the mechanisms of the gastric motor response to human motilin and erythromycin. The distribution of hMTLR was examined immunohistochemically in male wild-type (WT) and hMTLR-Tg mice. The contractile response of gastric strips was measured isometrically in an organ bath, while gastric emptying was determined using phenol red. hMTLR expression was abundant in the gastric smooth muscle layer. Interestingly, higher levels of hMTLR expression were observed in the myenteric plexus of hMTLR-Tg mice but not WT mice. hMTLR was not co-localized with vesicular acetylcholine transporter, a marker of cholinergic neurons in the myenteric plexus. Treatment with human motilin and erythromycin caused concentration-dependent contraction of gastric strips obtained from hMTLR-Tg mice but not from WT mice. The contractile response to human motilin and erythromycin in hMTLR-Tg mice was affected by neither atropine nor tetrodotoxin and was totally absent in Ca2+-free conditions. Furthermore, intraperitoneal injection of erythromycin significantly promoted gastric emptying in hMTLR-Tg mice but not in WT mice. Human motilin and erythromycin stimulate gastric smooth muscle contraction in hMTLR-Tg mice. This action is mediated by direct contraction of smooth muscle via the influx of extracellular Ca2+. Thus, hMTLR-Tg mice may be useful for the evaluation of MTLR agonists as gastric prokinetic agents.
Summary
Bifidobacterium, a major component of the intestinal microbiota, has been clinically used for the treatment of diarrhoea and constipation. 5‐Fluorouracil (5‐FU), widely used for cancer ...chemotherapy, is known to frequently induce intestinal mucositis accompanied by severe diarrhoea. The present study examined the effect of Bifidobacterium bifidum G9‐1 (BBG9‐1) on 5‐FU‐induced intestinal mucositis in mice. Intestinal mucositis was induced by repeated administration of 5‐FU for 6 days. BBG9‐1 was administered orally once daily for 9 days, beginning 3 days before the onset of 5‐FU treatment. Repeated administration of 5‐FU caused severe intestinal mucositis, characterised by shortening of villi and destruction of crypts, accompanied by increases in intestinal myeloperoxidase activity and inflammatory cytokine expression, body weight loss, and diarrhoea on day 6. Daily administration of BBG9‐1 significantly reduced the severity of intestinal mucositis and inflammatory responses and tended to attenuate clinical symptoms. In contrast, BBG9‐1 failed to prevent apoptosis induction on day 1 after the first 5‐FU administration. The structure of the intestinal microbiota, as analysed by weighted UniFrac distance, was largely altered by 5‐FU treatment, but this change was mitigated by daily administration of BBG9‐1. Moreover, 5‐FU treatment decreased the abundance of Firmicutes and increased the abundance of Bacteroidetes, but these responses were also significantly inhibited by daily administration of BBG9‐1. These results suggest that BBG9‐1 has an ameliorative effect against 5‐FU‐induced intestinal mucositis through the attenuation of inflammatory responses via improve dysbiosis. BBG9‐1 could be useful for the prevention of intestinal mucositis during cancer chemotherapy.