Kohei Fukumoto

Suppression of indomethacin-induced apoptosis in the small intestine due to Bach1 deficiency

Abstract BTB and CNC homologue 1 (Bach1) is a transcriptional repressor of heme oxygenase-1 (HO-1). This study hypothesized that Bach1 plays an important role in the indomethacin-induced apoptosis in the case of small-intestinal mucosal injury. Eight-week-old male C57BL/6 (wild-type) and homozygous Bach1-deficient C57BL/6 mice were included in this study. Mucosal injuries induced by subcutaneously administering indomethacin were evaluated macroscopically, histologically and biochemically. Indomethacin-induced injuries were improved in Bach1-deficient mice. Immunohistochemistry showed an increase in the number of HO-1-positive cells, which were mainly F4/80 positive macrophages, in Bach1-deficient mice. Indomethacin administration increased the expression of HO-1 mRNA and protein in the small intestine in Bach1-deficient mice. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) staining showed that the extent of apoptosis was suppressed in Bach1-deficent mice. In conclusion, deficiency of the Bach1 gene inhibited apoptosis and thus suppressed mucosal injury, indicating that Bach1 is a novel therapeutic target for indomethacin-induced intestinal injury.

Serpin B1 protects colonic epithelial cell via blockage of neutrophil elastase activity and its expression is enhanced in patients with ulcerative colitis

Serpin B1 is a monocyte neutrophil elastase (NE) inhibitor and is one of the most efficient inhibitors of NE. In the present study, we investigated the role of serpin B1 in the pathogenesis of ulcerative colitis by using clinical samples and an experimental model. The colonic expression of serpin B1 was determined by real-time polymerase chain reaction (PCR), Western blot analysis, and immunohistological studies in both normal and inflamed mucosa from patients with ulcerative colitis. Serpin B1 mRNA expression was determined by real-time PCR in the mouse dextran sodium sulfate (DSS)-induced colitis model. Young adult mouse colonic epithelial (YAMC) cells were used to determine the role of serpin B1. Serpin B1 gene transfected YAMC cells were treated with H(2)O(2) to measure cell viability. The expression of NE was determined in YAMC cells treated with H(2)O(2). NE-silenced YAMC cells were also treated with H(2)O(2) and then measured for viability. Upregulated expression of serpin B1 in colonic mucosa was confirmed from patients with active ulcerative colitis. Immunohistochemical studies showed that serpin B1 expression was localized not only in inflammatory infiltration cells but also in epithelial cells. Serpin B1 mRNA expression was also increased in colonic mucosa of mouse DSS-induced colitis. Serpin B1-transfected YAMC cells were resistant against the treatment of H(2)O(2). H(2)O(2) treatment significantly induced NE in YAMC cells, and NE-silenced YAMC cells were also resistant against the treatment of H(2)O(2). These results suggest that serpin B1 may be a novel marker of active ulcerative colitis and may play an important role in the pathogenesis of inflammatory bowel disease.

Reduced small-intestinal injury induced by indomethacin in interleukin-17A-deficient mice

Background and Aims:  The pathogenesis of enteropathy induced by non‐steroidal anti‐inflammatory drugs (NSAIDs) is still unclear, and there are no established treatments. Interleukin‐17A (IL‐17A) is a pro‐inflammatory cytokine that has been associated with the development of chronic inflammatory diseases, including autoimmune diseases. To define the role of IL‐17A in small intestinal injury and inflammation, we studied the effects of indomethacin administration in mice with targeted deletions of the IL‐17A gene.

Rebamipide ameliorates indomethacin-induced small intestinal injury in rats via the inhibition of matrix metalloproteinases activity.

The pathogenesis of non‐steroidal anti‐inflammatory drugs (NSAIDs)‐induced small intestinal lesions remains unclear, although it is considered to be quite different from that of upper gastrointestinal tract ulcers due to the absence of acid and the presence of bacteria and bile in the small intestine. The aim of this study was to characterize specific gene expression profiles of intestinal mucosa in indomethacin‐induced small intestinal injury, and to investigate the effects of rebamipide on the expression of these genes.

Successful Endoscopic Injection Sclerotherapy of High-Risk Gastroesophageal Varices in a Cirrhotic Patient with Hemophilia A

A 68-year-old man with hemophilia A and liver cirrhosis caused by hepatitis C virus was referred to our hospital to receive prophylactic endoscopic treatment for gastroesophageal varices (GOV). He had large, tense, and winding esophageal varices (EV) with cherry red spots extending down to lesser curve, predicting the likelihood of bleeding. Esophageal endoscopic injection sclerotherapy (EIS) was performed with a total 15 mL of 5% ethanolamine oleate with iopamidol (EOI). Radiographic imaging during EIS demonstrated that 5% EOI reached the afferent vein of the varices. He was administered sufficient factor VIII concentrate before and after EIS to prevent massive bleeding from the varices. Seven days after EIS, upper gastrointestinal endoscopy (UGIE) showed that the varices were eradicated almost completely. Eighteen months after EIS, the varices continued to diminish. We report a successful case of safe and effective EIS for GOV in a high-risk cirrhotic patient with hemophilia A.

SUCCESSFUL ENDOSCOPIC HEMOSTASIS FOR RUPTURED DUODENAL VARICES AFTER BALLOON-OCCLUDED RETROGRADE TRANSVENOUS OBLITERATION

A 75‐year‐old man with general malaise and appetite loss was transferred to our hospital for assessment and treatment of liver failure. Laboratory findings on admission showed anemia, and gastroduodenoscopy (GDS) revealed linear esophageal varices and tensive duodenal varices (DV) in the second portion of the duodenum. Systemic examinations did not reveal any significant lesion capable of explaining his anemia, except for DV. Balloon‐occluded retrograde transvenous obliteration was carried out to prevent DV bleeding. Good pooling of sclerosant was observed using two balloon catheters. However, contrast‐enhanced computed tomography after the procedure revealed no thrombosis in DV, and the patient complained of tarry stools before additional therapy. Emergent GDS revealed ruptured DV with fresh blood and erosions on the surface. Emergent endoscopic obliteration using the tissue adhesive N‐butyl‐2‐cyanoacrylate was carried out and complete hemostasis was achieved. Although no rebleeding episodes were observed after emergent obliteration, the patient died of sepsis following spontaneous bacterial peritonitis 53 days after admission. Autopsy revealed that DV dropped out, and the deep vein was replaced by granulation tissue. No signs of thrombi were detected, except varices. This autopsy case revealed the difficulty in DV management.

Usefulness of a novel observation method using a small-diameter rigid telescope through the gastrostomy catheter at exchange.

Background and Aim:  During catheter exchange for percutaneous endoscopic gastrostomy (PEG), endoscopic or radiological observation is widely used to confirm that the catheter is placed correctly. However, to carry out these procedures in all patients at every catheter exchange costs time and money. It is therefore important to develop a reliable and safe method, which can also be used outside the clinic, to check the exchanged catheter. We examined the usefulness and safety of intragastric observation using a small‐diameter rigid telescope, which can be inserted through the catheter lumen of a PEG tube.

Pirfenidone Regulates Intestinal Fibrosis Through the Inhibition of HSP47 Expression in a Rat Colitis Model

Pirfenidone Regulates Intestinal Fibrosis Through the Inhibition of HSP47 Expression in a Rat Colitis Model Ken Inoue, Yuji Naito, Tomohisa Takagi, Natsuko Hayashi, Yasuko Hirai, Katsura Mizushima, Toshifumi Tsuji, Hiroyuki Yoriki, Munehiro Kugai, Ryusuke Horie, Kohei Fukumoto, Shinya Yamada, Akihito Harusato, Naohisa Yoshida, Kazuhiko Uchiyama, Takeshi Ishikawa, Osamu Handa, Hideyuki Konishi, Naoki Wakabayashi, Nobuaki Yagi, Hiroshi Ichikawa, Satoshi Kokura, Toshikazu Yoshikawa

Deficiency of BACH1 Ameliorates TNBS-Induced Colitis in Mice Thorough the Anti-Inflammatory Function of HO-1 Highly Expressed Macrophages

Psychological stress is a major contributing factor in the onset and exacerbation of gastrointestinal disease through poorly understood mechanisms. Our previous studies in a porcine model showed that stress-induced disturbances in mucosal barrier function are regulated by peripheral activation of CRFr subtypes CRFr1 and CRFr2. Furthermore, results from these studies suggested that CRFr1 mediates deleterious mucosal barrier responses to stress while CRFr2 may play a protective role. The objective of this study was to further elucidate the role of CRFr subtypes in stress intestinal barrier dysfunction. Porcine ileum was mounted on Ussing Chambers and exposed to CRFr1 and CRFr2 agonist treatments: 1) Control 2) CRF;(1μM;CRFr1/r2 agonist) 3) UCN2;(0.1μM CRFr2 agonist) 4) Stressin 1 (2nM;CRFr1 agonist). Intestinal barrier function was measured in terms of mucosal-to-serosal flux of 4 kDa-FITC dextran (FD4) over a 180-minute period on Ussing chambers. CRF treatment increased (p < 0.005) paracellular flux of FD4 compared with controls. Ileal mucosa treated with the CRFr1 agonist Stressin 1 exhibited the greatest FD4 flux compared with all other treatments (p< 0.001) whereas treatment with the CRFr2 agonist UCN2 had no effect on baseline ileal barrier function. However, when added in combination with Stressin 1, UCN2 inhibited (p<0.05) Stressin 1-induced increases in FD4 flux. To determine if the effects of CRFr activation on barrier function were mediated via intestinal mast cell activation, ileal tissue was pre-treated with the mast cell stabilizer cromolyn (10 -4M) and then CRFr agonists were applied to tissues. Blockade of mast cell activation prevented increases in paracellular flux induced by CRF and Stressin 1 (p <0.05). Overall, these data demonstrate CRFr1 and CRFr2 play distinct roles in regulation of stress-induce mucosal barrier dysfunction with CRFr1 mediating intestinal barrier dysfunction and CRFr2 mediating protection, potentially through negative regulation of CRFr1 pathways. In addition, these data show that CRFr1mediated intestinal barrier dysfunction is mediated through activation of intestinal MCs.

The Therapeutic Effect of Heme Oxygenase-1 in the Indomethacin-Induced Intestinal Injury in Mice

Background: Previously, we have reported that inflammatory mediators such as lipopolysaccharide promote indomethacin (Indo)-induced small intestinal damage via Toll-like receptor (TLR) 4 (Watanabe T et al, GUT 2008 ). High-mobility group box-1 (HMGB1), originally identified as a DNA binding protein, also has potent proinflammatory properties as an alarmin. HMGB1 exerts proinflammatory effect via the receptor for advanced glycation endproducts (RAGE) and Toll-like receptors (TLRs). AIM: To investigate the role of HMGB1 and the receptor responsible for HMGB1 in Indo-induced small intestinal damage.Methods: Indo (10 mg/kg body weight) was administered orally to C57BL/6J mice, and the small intestines were removed at 3, 6, 12, and 24 h after the administration of Indo.The mRNA expression of tumor necrosis factor-α (TNF-α), keratinocyte chemoattractant (KC), monocyte chemotactic protein-1 (MCP1), TLR4, RAGE, and HMGB1 were assessed by real-time reverse transcription-polymerase chain reaction. Localization of TLR4, RAGE, and HMGB1 in the small intestine was determined by immunohistochemical examination. To clarify the role of HMGB1 in Indo-induced small intestinal damage, mouse recombinant HMGB1 (rHMGB1), anti-HMGB1 neutralizing antibodies, ethyl pyruvate, which inhibits secretion of HMGB1, or nonspecific chicken IgY were intraperitoneally administered to the mice after administration of Indo and the small intestinal damage was evaluated. To investigate the involvement of RAGE and TLR4 in HMGB-1-mediated Indo-induced small intestinal damage, RAGE knockout (KO) or TLR4 KO or wild-type mice were intraperitoneally administered rHMGB1 accompanied by Indo administration and the damage was evaluated. Results: Indo-induced small intestinal damage occurred 3 h after the administration of Indo and the damage was accompanied by an increase in the expression of TNF-α and KCmRNAs in the small intestine. HMGB-1, TLR4, and RAGE mRNAs were constitutively expressed. Immunoreactivity for HMGB1 was mainly observed in the nuclei of the intestinal epithelial cells and inflammatory cells. Immunoreactivity for RAGE and TLR4 was mainly observed in macrophage cells. Inhibition of HMGB1 by neutralizing antibodies and ethyl pyruvate reduced the small intestinal damage, accompanied by reduction in the expression of TNF-α, KC, and MCP1 mRNAs, while rHMGB1 further increased the Indo-induced small intestinal damage. The Indo-induced small intestinal damage observed in RAGE KO mice was almost of the same level as that observed in wild-type mice, while gene disruption of TLR4 resulted in decrease in Indo-induced small intestinal damage. Conclusion: These results suggest that HMGB1 acts as a factor promoting the Indo-induced small intestinal damage by upregulating the proinflammatory cytokines through TLR4.