Neutrophil extracellular trap induction through peptidylarginine deiminase 4 activity is involved in 2,4,6-trinitrobenzenesulfonic acid-induced colitis.

Neutrophil extracellular traps (NETs) are induced in the innate immune response against infectious agents and are also implicated in the pathogenesis of various cancers and autoimmune diseases. Peptidylarginine deiminase 4 (PAD4), an enzyme that converts arginine to citrulline, is also involved in NET formation. In this study, we investigated the pathogenic effect of PAD4 on NETs in inflammatory bowel disease using a trinitrobenzene sulfonic acid (TNBS)-induced murine colitis model. PAD4-deficient (PAD4KO) mice were generated by CRISPR-Cas9-mediated genomic editing. NETs were triggered in peritoneal neutrophils obtained from wild-type mice by A23187 (a calcium ionophore), but these responses were completely abolished in the PAD4KO mice. Experimental colitis was induced in wild-type and PAD4KO mice via an intrarectal injection of TNBS. TNBS injection resulted in body weight loss, extensive colonic erosion, and ulceration in wildtype mice. However, these responses were significantly attenuated following the administration of Cl-amidine (an inhibitor of pan-PADs) and DNase I (an inhibitor of NET formation), in combination with PAD4KO in mice. TNBS-induced increases in myeloperoxidase activity, inflammatory cytokine expression, and NET formation in the colon were significantly reduced following the administration of Cl-amidine, DNase I injection, and PAD4KO. These findings suggest that NET formation contributes to the pathogenesis of TNBS-induced colitis via PAD4. Thus, PAD4 is a promising target for the treatment of inflammatory bowel disease.

Transient Receptor Potential Ankyrin 1 in Taste Nerve Contributes to the Sense of Sweet Taste in Mice.

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.

A quinazoline-based bromodomain inhibitor, CN210, ameliorates indomethacin-induced ileitis in mice by inhibiting inflammatory cytokine expression.

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.

Early functional recovery after two-stage surgery with an allogenic bone graft for baseplate loosening in reverse shoulder arthroplasty: a case report.

OBJECTIVES: Reverse shoulder arthroplasty (RSA) for cuff tear arthropathy results in good shoulder function. However, RSA is associated with several complications, including infection, dislocation of the shoulder joint, implant loosening, and axillary nerve palsy. Several problems may also occur on the glenoid side, including bone defects of the glenoid, baseplate loosening, and displacement of the sphere. Herein, we report a 79-year-old man who obtained early functional recovery following a two-stage operation with an allogenic bone graft to treat baseplate loosening and a glenoid bone defect after RSA. CASE REPORT: The patient presented with pain during motion and limited active shoulder joint movement 5 weeks after undergoing RSA for cuff tear arthropathy. CT revealed baseplate loosening and a glenoid bone defect; these complications were treated via a two-stage operation. The first stage comprised the removal of all implants and the grafting of allogenic bone from the femoral head into the glenoid defect. Six months later, CT confirmed complete union of the grafted bone and glenoid. The second stage comprised the re-insertion of all implants. Two months after the last operation, the active shoulder range of motion of the affected side was almost identical to that of the contralateral side. CONCLUSION: Good early functional recovery was obtained using a two-stage operation for baseplate loosening after RSA. Allogenic bone grafting was effective in the reconstruction of the glenoid defect.

Risk Factors Affecting Cage Retropulsion into the Spinal Canal Following Posterior Lumbar Interbody Fusion: Association with Diffuse Idiopathic Skeletal Hyperostosis.

STUDY DESIGN: This was a retrospective observational study. PURPOSE: We identify risk factors, including physical and surgical factors, and comorbidities affecting cage retropulsion following posterior lumbar interbody fusion (PLIF). OVERVIEW OF LITERATURE: Diffuse idiopathic skeletal hyperostosis (DISH) is considered a risk factor for reoperation after PLIF. We evaluated the effect of DISH on cage retropulsion into the spinal canal, which may require surgical revision for severe neurological disorders. METHODS: A total of 400 patients (175 men, 225 women) who underwent PLIF were observed for >1 year. Factors investigated included the frequency of cage retropulsion and surgical revision. In addition, physical (age, sex, disease), surgical (fusion and PLIF levels, cage number, grade 2 osteotomy), and comorbid (DISH, existing vertebral fracture) factors were compared between patients with and without cage retropulsion. Factors related to surgical revision during the observation period were also considered. RESULTS: Cage retropulsion occurred in 15 patients and surgical revision was performed in 11. Revisions included the replacement of pedicle screws (PSs) with larger screws in all patients and supplementary implants in 10. Among the patients with cage retropulsion, the average PLIF level was 2.7, with DISH present in nine patients and existing vertebral fractures in six. Factors affecting cage retropulsion were diagnoses of osteoporotic vertebral fracture, multilevel fusion, single-cage insertion, grade 2 osteotomy, presence of DISH, and existing vertebral fracture. Multivariable analysis indicated that retropulsion of a fusion cage occurred significantly more frequently in patients with DISH and multilevel PLIF. CONCLUSIONS: DISH and multilevel PLIF were significant risk factors affecting cage retropulsion. Revision surgery for cage retropulsion revealed PS loosening, suggesting that implant replacement was necessary to prevent repeat cage retropulsion after revision.

Activities of Daily Living after Surgical Treatment for Osteoporotic Vertebral Fracture with or without Diffuse Idiopathic Skeletal Hyperostosis: A Retrospective Single-Institutional Study.

STUDY DESIGN: This investigation was a retrospective observational study. PURPOSE: The aim of this study was to evaluate whether having diffuse idiopathic skeletal hyperostosis (DISH) as a comorbidity affects the patient's ability to perform activities of daily living (ADL) after surgical treatment for osteoporotic vertebral fracture (OVF). OVERVIEW OF LITERATURE: A few studies have extensively evaluated elderly patients with comorbidities such as DISH and OVFinduced persistent back pain and their ability to perform ADL postoperatively. METHODS: In this study, 63 patients (21 men and 42 women) who underwent surgical treatment for OVF were enrolled. Of these patients, 26 had DISH (D+) and 37 did not have DISH (D-). Patient demographic characteristics and surgical, clinical, and radiological findings were compared between those with and without DISH. The change in their ability to perform ADL after surgery was also evaluated. RESULTS: Age, number of comorbidities, and 1-year mortality rate were significantly higher in the D+ group (p<0.05). Postoperative Visual Analog Scale (VAS) scores were significantly higher in patients with impaired (n=6, p=0.04) abilities to perform ADL, and improvements in VAS scores were significantly higher in patients with unchanged abilities to perform ADL (n=54, p=0.03) after surgery. The average postoperative VAS scores were 2.2 for the D+ group and 2.3 for the D- group, which were not significantly different. CONCLUSIONS: The frequency of OVF with DISH was higher in elderly men with multiple comorbidities and contributed to a higher 1-year mortality rate than those in patients without DISH. However, preoperative and postoperative VAS scores and improvements in VAS scores were similar between those with and without DISH. Postoperative impaired ability to perform ADL was associated with old age, high postoperative VAS scores, and little improvements in VAS scores, which were limitedly influenced by DISH. Surgical treatment of OVF combined with DISH is effective and appropriate for elderly patients.

Surgical Treatment of Osteoporotic Vertebral Fracture Associated with Diffuse Idiopathic Skeletal Hyperostosis along with Comparative Assessment of the Levels of Affected Vertebra or Anterior Column Reconstruction.

INTRODUCTION: Surgical treatment of osteoporotic vertebral fracture (OVF) often involves older patients with various comorbidities; thus, attending physicians must pay special attention to the invasiveness of surgical procedures and possible perioperative complications. In this retrospective observational study, we investigated the relationship between OVF and diffuse idiopathic skeletal hyperostosis (DISH) by examining the clinical characteristics and surgical outcomes. METHODS: Subjects comprised 26 patients (14 men, 12 women) who underwent surgical treatment for OVF complicated by DISH. Vertebral injuries affected the thoracolumbar transitional vertebrae in 18 patients and the middle and lower lumbar vertebrae in eight patients. The clinical characteristics, surgical results, radiological assessments, and outcomes were evaluated on the basis of the levels of affected vertebrae and whether anterior column reconstruction (ACR) was performed. RESULTS: Visual Analog Scale (VAS) measurements improved from an average of 69.7 mm before surgery to 21.3 mm after surgery. 14 patients had neurological deficits, who exhibited improvements by one or more steps on the Frankel scale after surgery. Activities of daily living (ADLs) were maintained during the six-month period following surgery in 23 patients. Comorbidity was observed in 22 patients. 14 patients had perioperative complications, and six required additional surgery. Both operating time and blood loss volume were significantly higher in patients in the middle and lower lumbar vertebrae and ACR groups. Postoperative correction loss was also significantly lower in the ACR group. CONCLUSIONS: Favorable degrees of improvement in neurological deficits and VAS were observed following surgery in patients with OVF complicated by DISH, and postoperative ADLs were maintained in 92% of the patients. Elderly men frequently presented with comorbidities, and the frequencies of patients with perioperative complications and those requiring additional surgery were high.

Leukotriene B4 Receptor Type 2 Accelerates the Healing of Intestinal Lesions by Promoting Epithelial Cell Proliferation.

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.

NOX1/NADPH oxidase in bone marrow-derived cells modulates intestinal barrier function.

The involvement of reactive oxygen species (ROS) has been suggested in the development of inflammatory bowel disease (IBD). An impaired intestinal barrier function is common in IBD patients. Here, we report the central role of NOX1/NADPH oxidase, a major source of ROS in nonphagocytic cells, in intestinal barrier dysfunction. By in vivo imaging using L-012 as a probe, a time-dependent increase in ROS was demonstrated in the abdomen of wild-type mice (WT) administered lipopolysaccharide (LPS: 6 mg/kg i.p.), but it was almost completely abolished in mice deficient in Nox1 (Nox1-KO) or the inducible nitric oxide synthase gene (iNOS-KO). By ex vivo imaging, increased ROS production was mainly shown in the ileum, where enhanced immunostaining of NOX1 was observed on the apical side of the epithelium. On the other hand, a punctate staining pattern of 3-nitrotyrosine, a marker of peroxynitrite production, was demonstrated in the lamina propria. When LPS-induced intestinal hyperpermeability was assessed by the oral administration of fluorescein isothiocyanate-conjugated dextran (FD-4), it was significantly suppressed in Nox1-KO as well as iNOS-KO. When Nox1-KO adoptively transferred with WT bone marrow were treated with LPS, the serum level of FD-4 was significantly elevated, whereas it remained unchanged in WT receiving bone marrow derived from Nox1-KO. Concomitantly, the activation of matrix metalloproteinase-9 induced by LPS was alleviated not only in intestinal tissue but also in peritoneal macrophages of Nox1-KO. Up-regulation of iNOS by LPS was significantly inhibited in macrophages deficient in Nox1, illustrating a functional hierarchy in NOX1/iNOS signaling. Together, these findings suggest that NOX1 in bone marrow-derived cells, but not epithelial cells, perturbs intestinal barrier integrity during endotoxemia.

Role of transient receptor potential vanilloid subtype 4 in the regulation of azoymethane/dextran sulphate sodium-induced colitis-associated cancer in mice.

Ca2+-permeable ion channels, such as transient receptor channels, are one of the potential therapeutic targets in cancer. Transient receptor potential vanilloid subtype 4 (TRPV4) is a nonselective cation channel associated with cancer progression. This study investigates the roles of TRPV4 in the pathogenesis of colitis-associated cancer (CAC) in mice. The role of TRPV4 was examined in azoxymethane (AOM)/dextran sulphate sodium (DSS)-induced murine CAC model. The formation of colon tumours induced by AOM/DSS treatment was significantly attenuated in TRPV4-deficient mice (TRPV4KO). TRPV4 was co-localised with markers of angiogenesis and macrophages. AOM/DSS treatment upregulated the expression of CD105, vascular endothelial growth factor receptor 2, and TRPV4 in wildtype, but the upregulation of CD105 was significantly attenuated in TRPV4KO. Bone marrow chimera experiments indicated that TRPV4, expressed in both vascular endothelial cells and bone marrow-derived macrophages, played a significant role in colitis-associated tumorigenesis. There was no significant difference in the population of hematopoietic cells, neutrophils, and monocytes between untreated and AOM/DSS-treated WT and TRPV4KO on flow cytometric analysis. TRPV4 activation by a selective agonist induced TNF-α and CXCL2 release in macrophages. Furthermore, TRPV4 activation enhanced the proliferation of human umbilical vein endothelial cells. These results suggest that TRPV4 expressed in neovascular endothelial cells and bone marrow-derived macrophages contributes to the progression of CAC 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.

Changes in serum interleukin-6 levels as possible predictor of efficacy of tocilizumab treatment in rheumatoid arthritis.

OBJECTIVES: We aimed to evaluate the association between the change in serum IL-6 during the clinical course of tocilizumab (TCZ) therapy and rheumatoid arthritis (RA) disease activity or occurrence of adverse events. METHODS: General laboratory data including serum IL-6 levels and physical findings were obtained every 4 weeks, and, in addition, at the time when any adverse events occurred. RESULTS: The proportion achieving Clinical Disease Activity Index (CDAI) remission at 52 weeks was significantly lower in 20 patients with serum IL-6 ≥ 30 pg/ml at 12 weeks than 24 patients with serum IL-6 < 30 pg/ml. In 17 patients with serum IL-6 ≥ 30 pg/ml at 24 weeks, the proportion achieving CDAI remission was also significantly lower than 27 patients with serum IL-6 < 30 pg/ml then. In these 17 patients, Disease Activity Score (DAS) 28-ESR and CDAI at 52 weeks were significantly higher than those with serum IL-6 < 30 pg/ml. Age- and sex-adjusted logistic regression analysis showed logIL-6 at 12 weeks to be a predictive factor for DAS28-ESR remission at 52 weeks. CONCLUSION: Serum IL-6 levels from 12 to 24 weeks after TCZ initiation better reflect the efficacy of TCZ at 52 weeks.

Probiotic Bifidobacterium bifidum G9-1 attenuates 5-fluorouracil-induced intestinal mucositis in mice via suppression of dysbiosis-related secondary inflammatory responses.

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.

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.

Apoptosis, Dysbiosis and Expression of Inflammatory Cytokines are Sequential Events in the Development of 5-Fluorouracil-Induced Intestinal Mucositis in Mice.

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-1ß 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.

Curcumin inhibits epigen and amphiregulin upregulated by 2,4,6-trinitrochlorobenzene associated with attenuation of skin swelling.

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.

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.

[Glutamate as a potential protective drug in the gastrointestinal mucosa].

Monosodium glutamate (MSG) is known to provide the umami taste in the food. We have recently reported that glutamate has the potential to protect the small intestine against non-steroidal anti-inflammatory drugs (NSAIDs)-induced lesions in rats. In this paper, we examined this protective effect using sodium loxoprofen, one of the NSAIDs frequently used in Asian countries, to determine whether MSG accelerates the healing of loxoprofen-induced small intestinal lesions in rats. Loxoprofen at 60 mg/kg caused hemorrhagic lesions in the small intestine, mainly in the jejunum and ileum. These lesions spontaneously healed within 7 days, but this healing process was delayed by repeated administration of loxoprofen at low doses (10, 30 mg/kg) for 5 d after lesion induction. The healing-impairment action of loxoprofen was accompanied by the down-regulation of vascular endothelium-derived growth factor (VEGF) expression and an angiogenic response. The impaired healing caused by loxoprofen was significantly restored by co-treatment with a diet containing 5% MSG for 5 d, accompanied by the enhancement of VEGF expression and angiogenesis. We suggest that daily intake of MSG not only protects the small intestine against NSAIDs-induced damage but also exerts healing-promoting effects on these lesions.

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.

Inorganic polyphosphate adsorbed onto hydroxyapatite for guided bone regeneration: an animal study.

Inorganic polyphosphate (poly(P)) is recognized as a therapeutic agent that promotes fibroblast growth factor and enhances osteogenic differentiation, and in vivo, when adsorbed onto interconnected porous calcium hydroxyapatite (IP-CHA) enhances bone regeneration. The present study focused on the effect of poly(P) adsorbed onto IP-CHA granules (Poly(P)/IP-CHA) in guided bone regeneration (GBR). Dental implants were placed into the edentulous mandibular areas of five Beagle-Labrador hybrid dogs with screw expose on the buccal side, and then bone defects were filled Poly(P)/IP-CHA (test) or IP-CHA (control). After 12 weeks, histological evaluation and histomorphometrical analysis were performed. Newly-bone formation around exposed implant screw was clearly detected in the test-group. The ratio for regenerated bone height in the test group versus the control-group was 85.6±20.2 and 62.6±23.8, respectively, with no significant difference, while, that for bone implant contact was significantly higher (67.9±11.8 and 48.8±14.1, respectively). These findings indicate that Poly(P)/IP-CHA enhances bone regeneration in GBR.