Role of transient receptor potential vanilloid 4 channels in an ovalbumin-induced murine food allergic model.

The prevalence of food allergy (FA) has increased worldwide but an effective therapeutic strategy has not been established. Transient receptor potential vanilloid 4 (TRPV4), a mechanosensitive nonselective cation channel, is mainly expressed in the epithelium of various organs. The present study investigated the role of TRPV4 in the pathogenesis of an ovalbumin (OVA)-induced FA model in mice. Wild-type (WT) and TRPV4-deficient (TRPV4KO) mice were sensitized and challenged by OVA to establish FA model. Intestinal tissue samples were processed for biochemical, molecular, and image analyses. Intestinal permeability and antigen uptake assay were conducted using FITC-dextran and OVA-FITC, respectively. TRPV4 was expressed in the colonic epithelium in normal and OVA-treated WT mice. Repeated oral administration of OVA to mice induced systemic allergic symptoms, diarrhea, upregulation of T helper 2 cytokines, OVA-specific immunoglobulin, and FA-related inflammatory cells. These responses were significantly augmented in TRPV4KO mice compared with WT mice. After the induction of FA, the intestinal permeability was significantly increased in TRPV4KO mice compared with WT mice. The expressions of the tight junction protein occludin and adherence junction protein E-cadherin in the colon were significantly lower in TRPV4KO mice compared with WT mice under normal and FA conditions. In addition, the uptake of OVA by CD11c-positive cells was significantly increased in TRPV4KO mice compared with WT mice under FA conditions. These results suggest that epithelial TRPV4 protects against OVA-induced FA symptoms by suppressing the penetration of allergens by maintaining epithelial barrier functions.

d-Alanine as a biomarker and a therapeutic option for severe influenza virus infection and COVID-19.

Since the outbreak of coronavirus disease 2019 (COVID-19), biomarkers for evaluating severity, as well as supportive care to improve clinical course, remain insufficient. We explored the potential of d-amino acids, rare enantiomers of amino acids, as biomarkers for assessing disease severity and as protective nutrients against severe viral infections. In mice infected with influenza A virus (IAV) and in patients with severe COVID-19 requiring artificial ventilation or extracorporeal membrane oxygenation, blood levels of d-amino acids, including d-alanine, were reduced significantly compared with those of uninfected mice or healthy controls. In mice models of IAV infection or COVID-19, supplementation with d-alanine alleviated severity of clinical course, and mice with sustained blood levels of d-alanine showed favorable prognoses. In severe viral infections, blood levels of d-amino acids, including d-alanine, decrease, and supplementation with d-alanine improves prognosis. d-Alanine has great potentials as a biomarker and a therapeutic option for severe viral infections.

Intratracheal trimerized nanobody cocktail administration suppresses weight loss and prolongs survival of SARS-CoV-2 infected mice.

BACKGROUND: SARS-CoV-2 Omicron variants are highly resistant to vaccine-induced immunity and human monoclonal antibodies. METHODS: We previously reported that two nanobodies, P17 and P86, potently neutralize SARS-CoV-2 VOCs. In this study, we modified these nanobodies into trimers, called TP17 and TP86 and tested their neutralization activities against Omicron BA.1 and subvariant BA.2 using pseudovirus assays. Next, we used TP17 and TP86 nanobody cocktail to treat ACE2 transgenic mice infected with lethal dose of SARS-CoV-2 strains, original, Delta and Omicron BA.1. RESULTS: Here, we demonstrate that a novel nanobody TP86 potently neutralizes both BA.1 and BA.2 Omicron variants, and that the TP17 and TP86 nanobody cocktail broadly neutralizes in vitro all VOCs as well as original strain. Furthermore, intratracheal administration of this nanobody cocktail suppresses weight loss and prolongs survival of human ACE2 transgenic mice infected with SARS-CoV-2 strains, original, Delta and Omicron BA.1. CONCLUSIONS: Intratracheal trimerized nanobody cocktail administration suppresses weight loss and prolongs survival of SARS-CoV-2 infected mice.

Antibodies are made by the immune system to identify and inactivate infectious agents such as viruses. Alpacas produce a simple type of antibodies called nanobodies. We previously developed two nanobodies named P17 and P86 that inactivate SARS-CoV-2. In this study, we modified these nanobodies to create two nanobodies named TP17 and TP86. The cocktail of these nanobodies inactivated different types of SARS-CoV-2 viruses including Omicron BA.1 and BA.2. The cocktail also prolonged survival of mice infected with lethal doses of SARS-CoV-2.

Japanese Cedar Pollen Allergens in Japan.

Pollen from members of the Cupressaceae tree family is one of the most important causes of allergic disease in the world. Cryptomeria japonica (Japanese cedar) and Chamaecyparis obtusa (Japanese cypress) are Japan's most common tree species. The pollen dispersal season is mainly from February to May. The major allergens of Japanese cedar and Japanese cypress exhibit high amino acid sequence similarity due to the phylogenetic relationship between the two species. An epidemiological study has shown that the prevalence of Japanese cedar pollinosis is approximately 40%. Younger children (5 to 9 years old) showed a high prevalence of Japanese cedar pollinosis as 30% in 2019, indicating that season pollinosis is getting worse. Pharmacotherapy is the most common treatment for pollinosis induced by Japanese cedar and Japanese cypress. Patients' satisfaction with pharmacotherapy is low due to insufficient experienced effect and daytime somnolence. Unlike pharmacotherapy, allergy immunotherapy (AIT) addresses the basic immunological mechanisms of allergic disease and activates protective allergen-reactive pathways of the immune system. AIT is now recognized as the only treatment option with the potential to provide long-term post-treatment benefits and alter the natural course of the allergic disease, including Japanese cedar pollinosis.

Correlation between sensitization to house dust mite major allergens, age, and symptoms in Japanese house dust mite allergic subjects.

Der p 23 has recently been recognized as a new house dust mite (HDM) major allergen that may be linked to the development of asthma in HDM allergic patients. This study aimed to investigate the frequency of sensitization to HDM major allergen components including Der p 23 and to examine the correlation between HDM-sensitization and AR symptom score in Japanese HDM allergic rhinitis (AR) patients without allergic asthma. Serum samples (n = 120) collected from Japanese HDM AR patients (12 to 64 years) without asthma were assessed for allergen-specific IgE (s-IgE) by ImmunoCAP (Dermatophagoides pteronyssinus (D. pteronyssinus; Der p) extract, Der p 23) or immunosolid-phase allergen chip (Der p 1, Der p 2). Japanese HDM AR patients without asthma showed a high prevalence of allergic sensitization to the HDM major allergens Der p 1 (94.2%), Der p 2 (97.5%) and Der p 23 (71.7%). No difference in the prevalence was detected for Der p 1 and Der p 2 s-IgE among three age groups. However, the prevalence of Der p 23 s-IgE was significantly higher in the younger group compared to the elderly group. No significant correlation was found between AR symptom scores and concentration of s-IgE towards Der p extract and any of the three HDM major allergens. Although the prevalence of sensitization towards D. pteronyssinus major allergens is high in Japanese AR patients without asthma, there was no correlation between allergen specific IgE including IgE towards Der p 23 and AR symptom in this population.

ACE2-like carboxypeptidase B38-CAP protects from SARS-CoV-2-induced lung injury.

Angiotensin-converting enzyme 2 (ACE2) is a receptor for cell entry of SARS-CoV-2, and recombinant soluble ACE2 protein inhibits SARS-CoV-2 infection as a decoy. ACE2 is a carboxypeptidase that degrades angiotensin II, thereby improving the pathologies of cardiovascular disease or acute lung injury. Here we show that B38-CAP, an ACE2-like enzyme, is protective against SARS-CoV-2-induced lung injury. Endogenous ACE2 expression is downregulated in the lungs of SARS-CoV-2-infected hamsters, leading to elevation of angiotensin II levels. Recombinant Spike also downregulates ACE2 expression and worsens the symptoms of acid-induced lung injury. B38-CAP does not neutralize cell entry of SARS-CoV-2. However, B38-CAP treatment improves the pathologies of Spike-augmented acid-induced lung injury. In SARS-CoV-2-infected hamsters or human ACE2 transgenic mice, B38-CAP significantly improves lung edema and pathologies of lung injury. These results provide the first in vivo evidence that increasing ACE2-like enzymatic activity is a potential therapeutic strategy to alleviate lung pathologies in COVID-19 patients.

Reduced anaphylactic potential of denatured pullulan-conjugated Cry j 1.

Japanese Cedar (JC) pollinosis is the most common seasonal allergic rhinitis in Japan. Throughout the JC pollen season, patients suffer from the allergic symptoms, resulting in a reduction of quality of life. Allergy immunotherapy (AIT) is an established treatment option for a wide range of allergens that unlike symptomatic treatments (e.g. antihistamines) may provide sustained immune tolerance. However, AIT, especially subcutaneous immunotherapy (SCIT) has a fatal anaphylaxis risk due to the use of crude allergen extracts. Consequently, development of allergen derivatives with substantially reduced anaphylactic potential is desirable. An allergen derivative that showed reduced IgE-binding and anaphylactic potential was developed through conjugation of native Cry j 1 (n Cry j 1), a major JC allergen, to the polysaccharide pullulan followed by chemical but non-covalent denaturation. The resulting Cry j 1 allergen derivative, Dn p-Cry j 1, showed reduced IgE-binding and IgE-mediated effector cell activation in vitro using an ELISA competition assay and a mast cell activation model (EXiLE). Reduced anaphylactic potential of Dn p-Cry j 1 in vivo was demonstrated using the rat passive cutaneous anaphylaxis (PCA) assay. The difference in anaphylactic potential of Dn p-Cry j 1 compared to n Cry j 1 in wild-type rats was of the same magnitude as the difference seen in the anaphylaxis reactions obtained with n Cry j 1 in wild-type rats and mast-cell deficient rats, indicating a dramatic reduction in anaphylactic potential of Dn p-Cry j 1. These results indicate that Dn p-Cry j 1 is a promising candidate for next-generation JC AIT.

Highly susceptible SARS-CoV-2 model in CAG promoter-driven hACE2-transgenic mice.

COVID-19, caused by SARS-CoV-2, has spread worldwide with dire consequences. To urgently investigate the pathogenicity of COVID-19 and develop vaccines and therapeutics, animal models that are highly susceptible to SARS-CoV-2 infection are needed. In the present study, we established an animal model highly susceptible to SARS-CoV-2 via the intratracheal tract infection in CAG promoter-driven human angiotensin-converting enzyme 2-transgenic (CAG-hACE2) mice. The CAG-hACE2 mice showed several severe symptoms of SARS-CoV-2 infection, with definitive weight loss and subsequent death. Acute lung injury with elevated cytokine and chemokine levels was observed at an early stage of infection in CAG-hACE2 mice infected with SARS-CoV-2. Analysis of the hACE2 gene in CAG-hACE2 mice revealed that more than 15 copies of hACE2 genes were integrated in tandem into the mouse genome, supporting the high susceptibility to SARS-CoV-2. In the developed model, immunization with viral antigen or injection of plasma from immunized mice prevented body weight loss and lethality due to infection with SARS-CoV-2. These results indicate that a highly susceptible model of SARS-CoV-2 infection in CAG-hACE2 mice via the intratracheal tract is suitable for evaluating vaccines and therapeutic medicines.

G protein-coupled receptor 40 activation ameliorates dextran sulfate sodium-induced colitis in mice via the upregulation of glucagon-likepeptide-2.

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.

Transient receptor potential vanilloid 4 mediates sour taste sensing via type III taste cell differentiation.

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 ß-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.

Role of transient receptor potential melastatin 2 in surgical inflammation and dysmotility in a mouse model of postoperative ileus.

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.

Transient receptor potential vanilloid 1 and transient receptor potential ankyrin 1 contribute to the progression of colonic inflammation in dextran sulfate sodium-induced colitis in mice: Links to calcitonin gene-related peptide and substance P.

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.

Transient receptor potential vanilloid 4 channel regulates vascular endothelial permeability during colonic inflammation in dextran sulphate sodium-induced murine colitis.

BACKGROUND AND PURPOSE: The transient receptor potential vanilloid 4 (TRPV4) channel is a non-selective cation channel involved in physical sensing in various tissue types. The present study aimed to elucidate the function and expression of TRPV4 channels in colonic vascular endothelial cells during dextran sulphate sodium (DSS)-induced colitis. EXPERIMENTAL APPROACH: The role of TRPV4 channels in the progression of colonic inflammation was examined in a murine DSS-induced colitis model using immunohistochemical analysis, Western blotting and Evans blue dye extrusion assay. KEY RESULTS: DSS-induced colitis was significantly attenuated in TRPV4-deficient (TRPV4 KO) as compared to wild-type mice. Repeated intrarectal administration of GSK1016790A, a TRPV4 agonist, exacerbated the severity of DSS-induced colitis. Bone marrow transfer experiments demonstrated the important role of TRPV4 in non-haematopoietic cells for DSS-induced colitis. DSS treatment up-regulated TRPV4 expression in the vascular endothelia of colonic mucosa and submucosa. DSS treatment increased vascular permeability, which was abolished in TRPV4 KO mice. This DSS-induced increase in vascular permeability was further enhanced by i.v. administration of GSK1016790A, and this effect was abolished by the TRPV4 antagonist RN1734. TRPV4 was co-localized with vascular endothelial (VE)-cadherin, and VE-cadherin expression was decreased by repeated i.v. administration of GSK1016790A during colitis. Furthermore, GSK106790A decreased VE-cadherin expression in mouse aortic endothelial cells exposed to TNF-α. CONCLUSION AND IMPLICATIONS: These findings indicate that an up-regulation of TRPV4 channels in vascular endothelial cells contributes to the progression of colonic inflammation by increasing vascular permeability. Thus, TRPV4 is an attractive target for the treatment of inflammatory bowel diseases.

G protein-coupled receptor 35 contributes to mucosal repair in mice via migration of colonic epithelial cells.

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.

NOX1/NADPH Oxidase Expressed in Colonic Macrophages Contributes to the Pathogenesis of Colonic Inflammation in Trinitrobenzene Sulfonic Acid-Induced Murine Colitis.

NOX1/NADPH oxidase, a nonphagocytic isoform of reactive oxygen species-producing enzymes, is highly expressed in the colon, but the physiologic and pathophysiologic roles of this isoform are not fully understood. The present study investigated the role of NOX1 in the development of colonic inflammation in a trinitrobenzene sulfonic acid (TNBS)-induced murine colitis model. Intrarectal injection of TNBS caused severe colitis accompanied by body weight loss, diarrhea, and increased myeloperoxidase (MPO) activity in wild-type (WT) mice. In contrast, the severity of colitis was significantly attenuated in NOX1-deficient (NOX1KO) mice (the inhibitions of macroscopic damage score, body weight loss, diarrhea score, and MPO activity were 73.1%, 36.8%, 83.3%, and 98.4%, respectively). TNBS-induced upregulation of inflammatory cytokines (tumor necrosis factor (TNF)-α and interleukin (IL)-1ß), chemokines (CXCL1 and CXLC2), and inducible nitric oxide synthase (iNOS) was also significantly less in NOX1KO than in WT mice (the inhibitions were 100.8%, 89.0%, 63.5%, 96.7%, and 97.1%, respectively). Expression of NOX1 mRNA was detected not only in the lamina propria but also in peritoneal macrophages isolated from WT mice. Increased expression of TNF-α, IL-1ß, and iNOS in peritoneal macrophages exposed to lipopolysaccharide was significantly attenuated in macrophages isolated from NOX1KO mice (68.1%, 67.0%, and 79.3% inhibition, respectively). These findings suggest that NOX1/NADPH oxidase plays an important role in the pathogenesis of TNBS-induced colonic inflammation via upregulation of inflammatory cytokines, chemokines, and iNOS. NOX1 in colonic macrophages may become a potential target in pharmacologic intervention for inflammatory bowel disease.

5-HT3 receptors promote colonic inflammation via activation of substance P/neurokinin-1 receptors in dextran sulphate sodium-induced murine colitis.

BACKGROUND AND PURPOSE: 5-HT (serotonin) regulates various physiological functions, both directly and via enteric neurons. The present study investigated the role of endogenous 5-HT and 5-HT3 receptors in the pathogenic mechanisms involved in colonic inflammation, especially in relation to substance P (SP) and the neurokinin-1 (NK1 ) receptor. EXPERIMENTAL APPROACH: The effects of 5-HT3 and NK1 receptor antagonists were examined in dextran sulphate sodium (DSS)-induced colitis in mice. Inflammatory mediator expression and the distribution of 5-HT3 and NK1 receptors were also determined. KEY RESULTS: Daily administration of ramosetron and ondansetron (5-HT3 antagonists) dose-dependently attenuated the severity of DSS-induced colitis and up-regulation of inflammatory mediator expression. Immunohistochemical analysis showed 5-HT3 receptors are mainly expressed in vesicular ACh transporter-positive cholinergic nerve fibres in normal colon. DSS increased the number of colonic nerve fibres that were double positive for 5-HT3 receptors and SP but not of those that were double positive for 5-HT3 receptors and vesicular ACh transporter. DSS increased colonic SP levels and SP-positive nerve fibres; these responses were attenuated by ramosetron. DSS-induced colitis and up-regulation of inflammatory mediators were attenuated by aprepitant, an NK1 antagonist. Immunohistochemical studies further revealed that DSS treatment markedly increased NK1 receptor expression in CD11b-positive cells. CONCLUSIONS AND IMPLICATIONS: These findings indicate that the 5-HT/5-HT3 receptor and SP/NK1 receptor pathways play pathogenic roles in colonic inflammation. 5-HT acts via 5-HT3 receptors to up-regulate inflammatory mediators and promote colonic inflammation. These effects may be further mediated by activation of macrophage NK1 receptors via SP released from 5-HT3 receptor-positive nerve fibres.

Saireito (TJ-114), a Japanese traditional herbal medicine, reduces 5-fluorouracil-induced intestinal mucositis in mice by inhibiting cytokine-mediated apoptosis in intestinal crypt cells.

Clinical chemotherapy frequently causes intestinal mucositis as a side effect, which is accompanied by severe diarrhea. We recently showed that the cytokine-mediated apoptotic pathway might be important for the development of intestinal mucositis induced by 5-fluorouracil (5-FU). Saireito, the traditional Japanese herbal (Kampo) medicine, is widely used to treat diarrhea and various inflammatory diseases in Japan. In the present study, we investigated the effect of saireito on 5-FU-induced intestinal mucositis in mice, especially in relation to apoptosis in the intestinal crypt. Male C57BL/6 mice were given 5-FU (50 mg/kg), i.p. once daily for 6 days. Intestinal mucositis was evaluated histochemically. Saireito (100-1000 mg/kg) was administered p.o. twice daily for 6 days. Repeated 5-FU treatment caused severe intestinal mucositis including morphological damage, which was accompanied by body weight loss and diarrhea. Daily administration of saireito reduced the severity of intestinal mucositis in a dose-dependent manner. Body weight loss and diarrhea during 5-FU treatment were also significantly attenuated by saireito administration. The number of apoptotic and caspase-3-activated cells in the intestinal crypt was increased, and was accompanied by up-regulated tumor necrosis factor (TNF)-α and interleukin (IL)-1ß mRNA within 24 h of the first 5-FU injection. However, all of these measures were significantly lower after saireito administration. These results suggest that saireito attenuates 5-FU-induced intestinal mucositis. This action may come from the reduction of apoptosis in the intestinal crypt via suppression of the up-regulation of inflammatory cytokines. Therefore, saireito may be clinically useful for the prevention of intestinal mucositis during cancer chemotherapy.

Potential role of the NADPH oxidase NOX1 in the pathogenesis of 5-fluorouracil-induced intestinal mucositis in mice.

Although NADPH oxidase 1 (NOX1) has been shown to be highly expressed in the gastrointestinal tract, the physiological and pathophysiological roles of this enzyme are not yet fully understood. In the present study, we investigated the role of NOX1 in the pathogenesis of intestinal mucositis induced by the cancer chemotherapeutic agent 5-fluorouracil (5-FU) in mice. Intestinal mucositis was induced in Nox1 knockout (Nox1KO) and littermate wild-type (WT) mice via single, daily administration of 5-FU for 5 days. In WT mice, 5-FU caused severe intestinal mucositis characterized by a shortening of villus height, a disruption of crypts, a loss of body weight, and diarrhea. In Nox1KO mice, however, the severity of mucositis was significantly reduced, particularly with respect to crypt disruption. The numbers of apoptotic caspase-3- and caspase-8-activated cells in the intestinal crypt increased 24 h after the first 5-FU administration but were overall significantly lower in Nox1KO than in WT mice. Furthermore, the 5-FU-mediated upregulation of TNF-α, IL-1ß, and NOX1 and the production of reactive oxygen species were significantly attenuated in Nox1KO mice compared with that in WT mice. These findings suggest that NOX1 plays an important role in the pathogenesis of 5-FU-induced intestinal mucositis. NOX1-derived ROS production following administration of 5-FU may promote the apoptotic response through upregulation of inflammatory cytokines.