RESUMO
Crohn's disease, a chronic and recurrent gastrointestinal disease, frequently causes intestinal fibrosis. Transient receptor potential melastatin 2 (TRPM2), a non-selective cation channel, 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 (TRPM2KO) and wild-type (WT) mice through single and repeated intrarectal injections of 2,4,6-trinitrobenzene sulfonic acid (TNBS). Bone marrow-derived macrophages (BMDMs) from WT and TRPM2KO mice were stimulated using H2O2. In WT mice, a single TNBS injection induced acute colitis with upregulated inflammatory cytokines/chemokines and Th1/Th17-related cytokines, while repeated TNBS injections induced chronic colitis-associated fibrosis with upregulation of fibrogenic factors and Th2-related cytokines. Acute colitis and chronic colitis-associated fibrosis with cytokines/chemokine upregulation and fibrogenic factors were considerably suppressed in TRPM2KO mice. Treating BMDMs with H2O2 increased cytokine/chemokine expression and JNK, ERK, and p38 phosphorylation; however, these responses were significantly less in TRPM2KO than in WT mice. These findings 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-ß1-mediated fibrogenesis in addition to a consequence of acute colitis progression.
Assuntos
Colite , Canais de Cátion TRPM , Camundongos , Animais , Colo/metabolismo , Canais de Cátion TRPM/genética , Peróxido de Hidrogênio/metabolismo , Ácido Trinitrobenzenossulfônico/efeitos adversos , Ácido Trinitrobenzenossulfônico/metabolismo , Colite/induzido quimicamente , Colite/complicações , Colite/genética , Citocinas/metabolismo , Trinitrobenzenos/metabolismo , Quimiocinas/efeitos adversos , Quimiocinas/metabolismo , Fibrose , Modelos Animais de DoençasRESUMO
Transient receptor potential vanilloid type 2 (TRPV2) and type 1 (TRPV1) are originally identified as heat-sensitive TRP channels. We compared the expression patterns of TRPV2 and TRPV1 in the rat distal colon and extrinsic primary afferent neurons, and investigated their roles in visceral hypersensitivity in 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced colitis rats. Both TRPV2 and TRPV1 expressions in the colon, dorsal root ganglion (DRG), and nodose ganglion (NG) were significantly upregulated in the TNBS-induced colitis model. TRPV2 cell bodies co-localized with the intrinsic primary afferent marker NeuN and the inhibitory motor neuronal marker nNOS in the myenteric plexus. TRPV2 expressions were further detected in the resident macrophage marker ED2 in the mucosa. In contrast, no TRPV1-expressing cell bodies were detected in the myenteric plexus. Both TRPV2- and TRPV1-positive cell bodies in the DRG and NG were double-labeled with the neuronal retrograde tracer fluorescent fluorogold. Large- and medium-sized TRPV2-positive neurons were labeled with the A-fiber marker NF200, calcitonin gene-related peptide (CGRP), and substance P (SP) in the DRG while small-sized TRPV1-positive neurons were labeled with the C-fiber markers IB4, CGRP, and SP. TRPV2- and TRPV1-positive NG neurons were labeled with NF200 and IB4. TNBS treatment increased p-ERK1/2-positive cells in TRPV2 and TRPV1 neurons but did not affect the TRPV2 and TRPV1 subpopulations in the DRG and NG. Both TRPV2 and TRPV1 antagonists significantly alleviated visceral hypersensitivity in TNBS-induced colitis model rats. These findings suggest that intrinsic/extrinsic TRPV2- and extrinsic TRPV1-neurons contribute to visceral hypersensitivity in an experimental colitis model.
Assuntos
Peptídeo Relacionado com Gene de Calcitonina , Colite , Ratos , Animais , Ácido Trinitrobenzenossulfônico/efeitos adversos , Peptídeo Relacionado com Gene de Calcitonina/efeitos adversos , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Colite/induzido quimicamente , Neurônios/metabolismo , Canais de Cátion TRPV/metabolismo , Gânglios EspinaisRESUMO
Transient receptor potential (TRP) channels play a significant role in taste perception. TRP ankyrin 1 (TRPA1) is present in the afferent sensory neurons and is activated by food-derived ingredients, such as Japanese horseradish, cinnamon, and garlic. The present study aimed to investigate the expression of TRPA1 in taste buds, and determine its functional roles in taste perception using TRPA1-deficient mice. In circumvallate papillae, TRPA1 immunoreactivity colocalised with P2X2 receptor-positive taste nerves but not with type II or III taste cell markers. Behavioural studies showed that TRPA1 deficiency significantly reduced sensitivity to sweet and umami tastes, but not to salty, bitter, and sour tastes, compared to that in wild-type animals. Furthermore, administration of the TRPA1 antagonist HC030031 significantly decreased taste preference to sucrose solution compared to that in the vehicle-treated group in the two-bottle preference tests. TRPA1 deficiency did not affect the structure of circumvallate papillae or the expression of type II or III taste cell and taste nerve markers. Adenosine 5'-O-(3-thio)triphosphate evoked inward currents did not differ between P2X2- and P2X2/TRPA1-expressing human embryonic kidney 293T cells. TRPA1-deficient mice had significantly decreased c-fos expression in the nucleus of the solitary tract in the brain stem following sucrose stimulation than wild-type mice. Taken together, the current study suggested that TRPA1 in the taste nerve contributes to the sense of sweet taste in mice.
Assuntos
Papilas Gustativas , Percepção Gustatória , Camundongos , Humanos , Animais , Paladar/fisiologia , Anquirinas/metabolismo , Papilas Gustativas/metabolismo , SacaroseRESUMO
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 (BLT1KO) on ovalbumin (OVA)-induced allergic enteritis in mice to determine the pathogenic role of LTB4/BLT1 in allergic enteritis, a gastrointestinal form of food allergy. Repeated oral OVA challenges after sensitization with OVA and aluminium potassium sulphate 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 increased serum OVA-specific IgE levels. The severity of enteritis was significantly attenuated in BLT1KO 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 accumulation was significantly lower in BLT1KO 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 accumulation in the allergic colonic mucosa. Therefore, BLT1 is a promising drug target for treating food allergies.
Assuntos
Leucotrieno B4 , Receptores do Leucotrieno B4 , Camundongos , Animais , Ovalbumina , Receptores do Leucotrieno B4/genética , Receptores do Leucotrieno B4/metabolismo , Leucotrieno B4/metabolismo , Camundongos Knockout , Inflamação , Imunoglobulina ERESUMO
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.
Assuntos
Benzamidas/administração & dosagem , Compostos de Benzil/administração & dosagem , Dispepsia/tratamento farmacológico , Motilidade Gastrointestinal , Isotiocianatos/efeitos adversos , Morfolinas/administração & dosagem , Neostigmina/administração & dosagem , Fitoterapia , Extratos Vegetais/administração & dosagem , Tiazóis/administração & dosagem , Wasabia/química , Animais , Benzamidas/farmacologia , Compostos de Benzil/farmacologia , Modelos Animais de Doenças , Dispepsia/fisiopatologia , Motilidade Gastrointestinal/efeitos dos fármacos , Isotiocianatos/isolamento & purificação , Masculino , Camundongos Endogâmicos , Morfolinas/farmacologia , Neostigmina/farmacologia , Panax , Extratos Vegetais/farmacologia , Tiazóis/farmacologia , Zanthoxylum , ZingiberaceaeRESUMO
Gastroesophageal reflux disease (GERD) is a common gastrointestinal disorder. In the present study, we investigated TRP vanilloid subfamily member 2 (TRPV2) expression in lower oesophageal sphincter (LES) and its involvement in acid reflux oesophagitis in rats. Expression of TRPV2 and nerve growth factor mRNAs was significantly enhanced in LES of rats with reflux oesophagitis compared with normal rats. TRPV2 was mainly expressed in inhibitory motor neurons, and partly in intrinsic and extrinsic primary afferent neurons, and macrophages in LES of normal and reflux oesophagitis rats. Number of TRPV2-immunopositive nerve fibres was significantly increased, but that of nNOS-, CGRP-, and PGP9.5-nerve fibres was not changed in reflux oesophagitis compared with normal group. Probenecid produced nitric oxide production and relaxation in LES and this response was significantly enhanced in oesophagitis compared with normal group. Probenecid-induced relaxant effect was blocked by a TRPV2 inhibitor, tranilast, and a NOS inhibitor, NG-nitro-l-arginine methyl ester, in reflux oesophagitis rats. Oral administration of tranilast significantly improved body weight loss, oesophageal lesions, and epithelial thickness in oesophagitis model. These results suggest that up-regulation of TRPV2 in inhibitory motor neurons is involved in LES relaxation in oesophagitis model. TRPV2 inhibition might be beneficial for treatment of GERD.
Assuntos
Esfíncter Esofágico Inferior/metabolismo , Refluxo Gastroesofágico/tratamento farmacológico , Refluxo Gastroesofágico/genética , Expressão Gênica/genética , Canais de Cátion TRPV/genética , Canais de Cátion TRPV/metabolismo , Animais , Modelos Animais de Doenças , Esfíncter Esofágico Inferior/efeitos dos fármacos , Masculino , Relaxamento Muscular/efeitos dos fármacos , Óxido Nítrico/metabolismo , Probenecid/antagonistas & inibidores , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos Sprague-Dawley , Canais de Cátion TRPV/antagonistas & inibidores , ortoaminobenzoatos/farmacologia , ortoaminobenzoatos/uso terapêuticoRESUMO
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.
Assuntos
Anti-Inflamatórios não Esteroides , Indometacina , Canais de Cátion TRPM/genética , Anilidas/farmacologia , Animais , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Intestino Delgado/efeitos dos fármacos , Intestino Delgado/lesões , Intestino Delgado/metabolismo , Intestino Delgado/patologia , Masculino , Mentol/análogos & derivados , Mentol/farmacologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios Aferentes/efeitos dos fármacos , Neurônios Aferentes/metabolismo , Substância P/metabolismo , Canais de Cátion TRPM/agonistas , Canais de Cátion TRPM/metabolismoRESUMO
Inhibitors of bromodomain and extra-terminal motif (BET) proteins are emerging epigenetic therapeutics that suppress gene expressions that drive cancer and inflammation. The present study examined anti-inflammatory effects of a quinazoline-based BET inhibitor, CN210, in a murine ileitis model. CN210 was given orally 30 min before and 24 h after a subcutaneous administration of indomethacin. Macroscopic and histological evidences of ileitis, mucosal myeloperoxidase (MPO) activity and cytokine expressions were evaluated 48 h after the indomethacin administration. To further characterize the anti-inflammatory pathways modulated by CN210, its effects on RAW264 cells treated with lipopolysaccharide (LPS) were investigated. Competitive ligand binding and docking studies of CN210 to CREB-binding protein (CBP) and p300 were also performed. Oral administration of CN210 significantly reduced the severity of ileitis, normalized both proinflammatory MPO activity and concomitant cytokine expressions induced by indomethacin administration. Furthermore, CN210 attenuated the expression of cytokines and reversed the activation of nuclear factor κB (NF-κB) and mitogen-activated protein kinases (MAPK) induced by LPS. Competitive ligand binding assays showed that CN210 bound to the bromodomains of two paralogous histone acetyltransferases, CBP and p300, in addition to the bromodomains of BET proteins. Docking studies of CN210 to the bromodomains of CBP and p300 showed a similarity to the binding mode of SGC-CBP30, a specific CBP/p300 inhibitor. CN210 ameliorates indomethacin-induced ileitis by inhibiting the expression of inflammatory cytokines through the attenuation of NF-κB and MAPK pathways. CN210 thus represents a new mode of therapy for non-steroidal anti-inflammatory drug-induced ileitis and inflammatory bowel disease.
Assuntos
Anti-Inflamatórios/farmacologia , Citocinas/antagonistas & inibidores , Ileíte/tratamento farmacológico , Indometacina/efeitos adversos , Proteínas/antagonistas & inibidores , Animais , Citocinas/biossíntese , Proteína p300 Associada a E1A/metabolismo , Ileíte/induzido quimicamente , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/fisiologia , Peroxidase/metabolismo , Fosfoproteínas/metabolismo , Quinazolinas/farmacologia , Células RAW 264.7RESUMO
Leukotriene B4 receptor type 2 (BLT2) is a low-affinity leukotriene B4 receptor that is highly expressed in intestinal epithelial cells. Previous studies demonstrated the protective role of BLT2 in experimentally induced colitis. However, its role in intestinal lesion repair is not fully understood. We investigated the role of BLT2 in the healing of indomethacin-induced intestinal lesions in mice. There was no significant different between wild-type (WT) and BLT2-deficient (BLT2KO) mice in terms of the development of indomethacin-induced intestinal lesions. However, healing of these lesions was significantly impaired in BLT2KO mice compared with WT mice. In contrast, transgenic mice with intestinal epithelium-specific BLT2 overexpression presented with superior ileal lesion healing relative to WT mice. An immunohistochemical study showed that the number of Ki-67-proliferative cells was markedly increased during the healing of intestinal lesions in WT mice but significantly attenuated in BLT2KO mice. Exposure of cultured mouse intestinal epithelial cells to CAY10583, a BLT2 agonist, promoted wound healing and cell proliferation in a concentration-dependent manner. Nevertheless, these responses were abolished under serum-free conditions. The CAY10583-induced proliferative effect was also negated by Go6983, a protein kinase C (PKC) inhibitor, U-73122, a phospholipase C (PLC) inhibitor, LY255283, a BLT2 antagonist, and pertussis toxin that inhibits G protein-coupled receptor signaling via Gi/o proteins. Thus, BLT2 plays an important role in intestinal wound repair. Moreover, this effect is mediated by the promotion of epithelial cell proliferation via the Gi/o protein-dependent and PLC/PKC signaling pathways. The BLT2 agonists are potential therapeutic agents for the treatment of intestinal lesions. SIGNIFICANCE STATEMENT: The healing of indomethacin-induced Crohn's disease-like intestinal lesions was impaired in mice deficient in low-affinity leukotriene B4 receptor type 2 (BLT2). They presented with reduced epithelial cell proliferation during the healing. In contrast, healing was promoted in mice overexpressing intestinal epithelial BLT2. In cultured intestinal epithelial cells, the BLT2 agonist CAY10583 substantially accelerated wound repair by enhancing cell proliferation rather than migration. Thus, BLT2 plays an important role in the intestinal lesions via acceleration of epithelial cell proliferation.
Assuntos
Proliferação de Células/fisiologia , Colite/metabolismo , Mucosa Intestinal/metabolismo , Receptores do Leucotrieno B4/metabolismo , Animais , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Colite/induzido quimicamente , Colite/patologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Indometacina/toxicidade , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/patologia , Antagonistas de Leucotrienos/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Receptores do Leucotrieno B4/antagonistas & inibidores , Receptores do Leucotrieno B4/deficiência , Tetrazóis/farmacologiaRESUMO
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.
Assuntos
Compostos de Bifenilo/farmacologia , Compostos de Bifenilo/uso terapêutico , Colite/tratamento farmacológico , Colite/genética , Sulfato de Dextrana/efeitos adversos , Peptídeo 2 Semelhante ao Glucagon/metabolismo , Oxidiazóis/farmacologia , Oxidiazóis/uso terapêutico , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/metabolismo , Regulação para Cima/genética , Animais , Compostos de Bifenilo/administração & dosagem , Colite/induzido quimicamente , Colite/metabolismo , Citocinas/metabolismo , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Células Enteroendócrinas/metabolismo , Mediadores da Inflamação/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Terapia de Alvo Molecular , Oxidiazóis/administração & dosagem , Receptores Acoplados a Proteínas G/fisiologiaRESUMO
In this study, we investigated the role of transient receptor potential melastatin 2 (TRPM2), a nonselective cation channel abundantly expressed in inflammatory cells such as macrophages, in the development of postoperative ileus, a complication of abdominal surgery characterized by gastrointestinal dysmotility. In wild-type mice, we found that intestinal manipulation, a maneuver that elicits symptoms typical of postoperative ileus, delays the transit of fluorescein-labeled dextran, promotes the infiltration of CD68+ macrophages, Ly6B.2+ neutrophils, and MPO+ cells into intestinal muscles, boosts expression of IL-1ß, IL-6, TNF-α, iNOS, and CXCL2 in intestinal muscles and peritoneal macrophages, enhances phosphorylation of ERK and p38 MAPK in intestinal muscles, and amplifies IL-1ß, IL-6, TNF-α, iNOS, and CXCL2 expression in resident and thioglycolate-elicited peritoneal macrophages following exposure to lipopolysaccharide. Remarkably, TRPM2 deficiency completely blocks or diminishes these effects. Indeed, intestinal manipulation appears to activate TRPM2 in resident muscularis macrophages and elicits release of inflammatory cytokines and chemokines, which, in turn, promote infiltration of macrophages and neutrophils into the muscle, ultimately resulting in dysmotility. NEW & NOTEWORTHY Activation of transient receptor potential melastatin 2 (TRPM2) releases inflammatory cytokines and chemokines, which, in turn, promote the infiltration of inflammatory cells and macrophages into intestinal muscles, ultimately resulting in dysmotility. Thus TRPM2 is a promising target in treating dysmotility due to postoperative ileus, a complication of abdominal surgery.
Assuntos
Motilidade Gastrointestinal/imunologia , Íleus , Laparotomia/efeitos adversos , Complicações Pós-Operatórias/imunologia , Canais de Cátion TRPM/metabolismo , Animais , Quimiocina CXCL2/metabolismo , Modelos Animais de Doenças , Íleus/etiologia , Íleus/imunologia , Inflamação/metabolismo , Interleucina-1beta/metabolismo , Macrófagos Peritoneais/metabolismo , Camundongos , Músculo Liso/metabolismo , Neutrófilos/metabolismo , Canais de Cátion TRPC/metabolismoRESUMO
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.
Assuntos
Peptídeo Relacionado com Gene de Calcitonina/genética , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Colite/induzido quimicamente , Colite/genética , Sulfato de Dextrana/efeitos adversos , Substância P/genética , Substância P/metabolismo , Canal de Cátion TRPA1/fisiologia , Canais de Cátion TRPV/fisiologia , Animais , Capsaicina/efeitos adversos , Modelos Animais de Doenças , Progressão da Doença , Masculino , Camundongos Endogâmicos C57BL , Fibras Nervosas/metabolismo , Dor/genética , Regulação para Cima/efeitos dos fármacosRESUMO
Contact dermatitis is a form of delayed-type hypersensitivity characterized by localized thickening, papules, redness and vesicles of the skin. A model of contact dermatitis involving repeated challenge of a hapten is adapted to assess dermatitis as characterized by skin thickening. Recently, it was reported that neutrophils have crucial roles in contact hypersensitivity. We thus examined the involvement of CXC chemokines bearing the glutamic acid-leucine-arginine (ELR) motif ("ELR+ chemokines") and neutrophils in the ear swelling induced by 2,4,6-trinitrochlorobenzene (TNCB) challenges in the present study. Mice were sensitized by application of TNCB on their abdominal skin. They were then challenged thrice with TNCB to the ear. The CXCR2 antagonist SB225002 (9 mg/kg, i.p.) was administered before each TNCB challenge. Gene expressions and protein levels of the ELR+ chemokines CXCL1, 2 and 5 was increased markedly in mouse ear after the final TNCB challenge. In addition, we indicated that gene expression of CXCL1 was enhanced in the epidermis and dermis upon TNCB challenge. Expression of the CXCL2 gene was enhanced in the epidermis, and that of the CXCL5 gene was enhanced in the dermis. The swelling induced by TNCB challenges was significantly attenuated by SB225002. Furthermore, the increases in myeloperoxidase activity, and expression of myeloperoxidase and neutrophil elastase induced by TNCB challenge in mouse ear were inhibited by SB225002. These data suggest that ear swelling resulting from TNCB challenges might be concerned by upregulated ELR+ chemokine-induced neutrophil recruitment.
Assuntos
Quimiocinas CXC/química , Quimiocinas CXC/metabolismo , Dermatite de Contato/imunologia , Dermatite de Contato/metabolismo , Infiltração de Neutrófilos/efeitos dos fármacos , Cloreto de Picrila/efeitos adversos , Motivos de Aminoácidos , Animais , Dermatite de Contato/etiologia , Feminino , Camundongos Endogâmicos BALB C , Receptores de Interleucina-8B/antagonistas & inibidoresRESUMO
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.
Assuntos
Movimento Celular/fisiologia , Colo/citologia , Células Epiteliais/fisiologia , Mucosa Intestinal/metabolismo , Receptores Acoplados a Proteínas G/agonistas , Cicatrização/fisiologia , Animais , Linhagem Celular , Colite/induzido quimicamente , Colite/tratamento farmacológico , Colite/metabolismo , Colite/patologia , Colo/metabolismo , Colo/patologia , Citocinas/genética , Sulfato de Dextrana , Células Epiteliais/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Fibronectinas/metabolismo , Furanos/farmacologia , Integrinas/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Naftóis/uso terapêutico , Nitrilas/farmacologia , Peroxidase/metabolismo , Purinonas/farmacologia , RNA Mensageiro/metabolismo , Ratos , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Receptores Acoplados a Proteínas G/metabolismoRESUMO
OBJECTIVES: Contact dermatitis model involving repeated application of hapten is used as a tool to assess dermatitis, as characterized by thickening. Involvement of cell proliferation, elicited by repeated hapten-stimulation, in this swelling has been unclear. Curcumin is reported to reduce inflammation. We examined involvement of cell proliferation and the role of extracellular regulated kinase (ERK) in 2,4,6-trinitrochlorobenzene (TNCB) challenge-induced ear swelling. We also examined the effects of curcumin in this model. METHODS: Mice were sensitized with TNCB to the abdominal skin. Then, they were challenged with TNCB to the ear three times. The ERK activation inhibitor U0126 or curcumin was applied 30 min before each TNCB challenge. RESULTS: TNCB challenge-induced increased epidermal cell number and dermal thickening. Gene expressions of epithelial mitogen (EPGN), amphiregulin (AREG) and heparin-binding-epidermal growth factor (HB-EGF) were increased in the ears after the last TNCB challenge. Ki-67 immunoreactivity was increased in the dermis in TNCB-challenged ears. TNCB-induced swelling was inhibited by U0126 and curcumin. Curcumin also attenuated TNCB-induced ERK phosphorylation and expression of EPGN and AREG genes. CONCLUSION: Ear swelling induced by TNCB challenge might be mediated, in part, by the EPGN- and AREG-ERK proliferation pathway and was inhibited by curcumin.
Assuntos
Anfirregulina/metabolismo , Curcumina/farmacologia , Dermatite Alérgica de Contato/metabolismo , Epigen/metabolismo , Animais , Citocinas/genética , Dermatite Alérgica de Contato/patologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Haptenos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Antígeno Ki-67/genética , Antígeno Ki-67/metabolismo , Camundongos Endogâmicos BALB C , Fosforilação/efeitos dos fármacos , Cloreto de Picrila , Pele/efeitos dos fármacos , Pele/metabolismo , Pele/patologia , Regulação para Cima/efeitos dos fármacosRESUMO
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.
Assuntos
Bifidobacterium bifidum/fisiologia , Disbiose/complicações , Fluoruracila/efeitos adversos , Enteropatias/microbiologia , Mucosite/microbiologia , Probióticos/farmacologia , Animais , Apoptose/efeitos dos fármacos , Peso Corporal/efeitos dos fármacos , Diarreia/complicações , Inflamação/complicações , Enteropatias/induzido quimicamente , Enteropatias/complicações , Enteropatias/patologia , Intestino Delgado/efeitos dos fármacos , Intestino Delgado/patologia , Masculino , Camundongos , Microbiota/efeitos dos fármacos , Mucosite/induzido quimicamente , Mucosite/complicações , Mucosite/patologia , Tamanho do Órgão/efeitos dos fármacosRESUMO
(E)-Methyl 2-((2S,3S,7aS,12bS)-3-ethyl-7a-hydroxy-8-methoxy-1,2,3,4,6,7,7a,12b-octahydroindolo[2,3-a]quinolizin-2-yl)-3-methoxyacrylate (7-hydroxymitragynine), a main active constituent of the traditional herbal medicine Mitragyna speciosa, is an indole alkaloid that is structurally different from morphine. 7-Hydroxymitragynine induces a potent antinociceptive effect on mouse acute pain through µ-opioid receptors. In this study, we developed dual-acting µ- and δ-opioid agonists MGM-15 and MGM-16 from 7-hydroxymitragynine for the treatment of acute and chronic pain. MGM-16 showed a higher potency than that of 7-hydroxymitragynine and MGM-15 in in vitro and in vivo assays. MGM-16 exhibited a high affinity for µ- and δ-opioid receptors, with K(i) values of 2.1 and 7.0 nM, respectively. MGM-16 showed µ- and δ-opioid full agonistic effects in a guanosine 5'-O-(3-[(35)S]thiotriphosphate) binding assay and in a functional test using electrically elicited guinea pig ileum and mouse vas deferens contractions. Systemic administration of MGM-16 produced antinociceptive effects in a mouse acute pain model and antiallodynic effects in a chronic pain model. The antinociceptive effect of MGM-16 was approximately 240 times more potent than that of morphine in a mouse tail-flick test, and its antiallodynic effect was approximately 100 times more potent than that of gabapentin in partial sciatic nerve-ligated mice, especially with oral administration. The antinociceptive effect of MGM-16 was completely and partially blocked by the µ-selective antagonist ß-funaltrexamine hydrochloride (ß-FNA) and by the δ-selective antagonist naltrindole, respectively, in a tail-flick test. The antiallodynic effect of MGM-16 was completely blocked by ß-FNA and naltrindole in a neuropathic pain model. These findings suggest that MGM-16 could become a class of a compound with potential therapeutic utility for treating neuropathic pain.
Assuntos
Hiperalgesia/tratamento farmacológico , Neuralgia/tratamento farmacológico , Receptores Opioides delta/agonistas , Receptores Opioides mu/agonistas , Alcaloides de Triptamina e Secologanina/farmacologia , Administração Oral , Animais , Células CHO , Cricetinae , Cricetulus , Hiperalgesia/fisiopatologia , Íleo/efeitos dos fármacos , Íleo/fisiopatologia , Injeções Subcutâneas , Masculino , Camundongos , Contração Muscular , Músculo Liso/efeitos dos fármacos , Músculo Liso/fisiologia , Neuralgia/fisiopatologia , Estimulação Física , Coelhos , Ensaio Radioligante , Receptores Opioides delta/antagonistas & inibidores , Receptores Opioides mu/antagonistas & inibidores , Neuropatia Ciática/tratamento farmacológico , Neuropatia Ciática/fisiopatologia , Alcaloides de Triptamina e Secologanina/química , Alcaloides de Triptamina e Secologanina/uso terapêutico , Estereoisomerismo , Tato , Ducto Deferente/efeitos dos fármacos , Ducto Deferente/fisiopatologiaRESUMO
Transient receptor potential cation channel subfamily V member 1 (TRPV1) plays a role in esophageal function. However, the distribution of TRPV1 nerve fibers in the esophagus is currently not well understood. In the present study, we investigated the distribution of TRPV1 and neurotransmitters released from TRPV1 nerve fibers in the mouse lower esophagus. Furthermore, we investigated changes in the presence of TRPV1 in the mouse model of esophagitis. Numerous TRPV1-immunoreactive nerve fibers were seen in both the submucosal layer and myenteric plexus of the lower esophagus and colocalized with calcitonin gene-related peptide (CGRP). TRPV1 colocalized with substance P in axons in the submucosal layer and myenteric plexus. TRPV1 colocalized with neuronal nitric oxide synthase in the myenteric plexus. We observed some colocalization of CGRP with the vesicular acetylcholine (ACh) transporter, packaging of ACh into synaptic vesicles after its synthesis in terminal cytoplasm, in the submucosal layer and myenteric plexus. In the esophagitis model, the number of the TRPV1 nerve fibers did not change, but their immunoreactive intensity increased compared with sham-operated mice. Inhibitory effect of exogenous capsaicin on electrically stimulated twitch contraction significantly increased in esophagitis model compared with the effect in sham-operated mice. Overall, these results suggest that TRPV1 nerve fibers projecting to both the submucosal and muscle layer of the esophagus are extrinsic spinal and vagal afferent neurons. Furthermore, TRPV1 nerve fibers contain CGRP, substance P, nitric oxide, and ACh. Therefore, acid influx-mediated TRPV1 activation may play a role in regulating esophageal relaxation.
Assuntos
Esôfago/metabolismo , Fibras Nervosas/metabolismo , Canais de Cátion TRPV/metabolismo , Animais , Capsaicina/farmacologia , Modelos Animais de Doenças , Esofagite Péptica/fisiopatologia , Esôfago/citologia , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Contração Muscular/efeitos dos fármacos , Óxido Nítrico Sintase/metabolismo , Fármacos do Sistema Sensorial/farmacologia , Canais de Cátion TRPV/químicaRESUMO
Diarrhea is a common side effect experienced by cancer patients undergoing clinical chemotherapy, such as with 5-fluorouracil (5-FU). However, the precise mechanisms underlying 5-FU-induced diarrhea remain unclear. In the present study, we examined the role of neutrophil in 5-FU-induced diarrhea. Mice were given 5-FU (50mg/kg, i.p.) daily for 4 days. Sivelestat sodium (100 or 300 mg/kg, i.p., neutorophil elastase inhibitor) or SB225002 (3 or 9 mg/kg, i.p., CXCR2 antagonist) was administered before the administration of 5-FU. Gene expression levels of aquaporin (AQP) 4 and 8, CXCL1, CXCL2, CXCL3, neutrophil elastase (Elane) and myeloperoxidase (MPO) in the colon were examined by real-time RT-PCR. The neutrophil (Ly-6G positive cell) number in the mucosa of colon was measured by flow-cytometric analysis. Administration of 5-FU induced diarrhea and decreased the expression levels of AQP 4 and 8 in the colon. Under the present conditions, the expression levels of CXCL1, CXCL2, CXCL3, the neutrophil markers Elane and MPO, as well as Ly-6G-positive neutrophils, in the colon were significantly increased by 5-FU. Neutrophil recruitment with decreased levels of AQP 4 and 8 were dramatically inhibited by either sivelestat sodium or SB225002. Furthermore, these reagents reduced the 5-FU-induced body weight loss and diarrhea. These findings provide evidence that neutrophil recruitment and neutrophil elastase may decrease the levels of AQP 4 and 8 in the colon of mice treated with 5-FU and contribute to the pathophysiology of 5-FU-induced body weight loss and diarrhea.
Assuntos
Antimetabólitos Antineoplásicos/efeitos adversos , Diarreia/induzido quimicamente , Fluoruracila/efeitos adversos , Animais , Aquaporina 4/genética , Aquaporinas/genética , Quimiocina CXCL1/genética , Quimiocina CXCL2/genética , Quimiocinas CXC/genética , Colo/efeitos dos fármacos , Colo/metabolismo , Diarreia/genética , Diarreia/imunologia , Elastase de Leucócito/genética , Masculino , Camundongos Endogâmicos C57BL , Infiltração de Neutrófilos , Peroxidase/genética , RNA Mensageiro/metabolismoRESUMO
Macrophages are the originators of inflammatory compounds, phagocytic purifiers in their local environment, and wound healing protectors in oxidative environments. They are molded by the tissue milieu they inhabit, with gastrointestinal (GI) muscularis macrophages (MMs) being a prime example. MMs are located in the muscular layer of the GI tract and contribute to muscle repair and maintenance of GI motility. MMs are often in close proximity to the enteric nervous system, specifically near the enteric neurons and interstitial cells of Cajal (ICCs). Consequently, the anti-inflammatory function of MMs corresponds to the development and maintenance of neural networks in the GI tract. The capacity of MMs to shift from anti-inflammatory to proinflammatory states may contribute to the inflammatory aspects of various GI diseases and disorders such as diabetic gastroparesis or postoperative ileus, functional disorders such as irritable bowel syndrome, and organic diseases such as inflammatory bowel disease. We reviewed the current knowledge of MMs and their influence on neighboring cells due to their important role in the GI tract.