Akihito Harusato

Reactive oxygen species-quenching and anti-apoptotic effect of polaprezinc on indomethacin-induced small intestinal epithelial cell injury

BackgroundTo protect the small intestine from mucosal injury induced by nonsteroidal anti-inflammatory drugs is one of the critical issues in the field of gastroenterology. Polaprezinc (PZ), a gastric muco-protecting agent, has been widely used for the treatment of gastric ulcer and gastritis for its unique effects, such as its strong reactive oxygen species (ROS)-quenching effect. The aim of this study was to clarify the mechanism by which indomethacin-induced small intestinal mucosal injury occurs, by using a rat intestinal epithelial cell line (RIE-1). In addition, the protective role of PZ and the possible mechanism of its effect on indomethacin-induced small intestinal injury were investigated.MethodsCell death was evaluated by methyl thiazolyl tetrazolium (MTT) assay and a double-staining method with Hoechst33342 dye and propidium iodide. Indomethacin-induced ROS production was evaluated by detecting the oxidation of a redox-sensitive fluorogenic probe, RedoxSensor, and the oxidation of cysteine residues of proteins (protein S oxidation). The activation of cytochrome c, smac/DIABLO, and caspase-3 was assessed by western blotting. In some experiments, PZ or its components, l-carnosine and zinc, were used.ResultsWe found that indomethacin caused apoptosis in RIE-1 cells in a dose- and time-dependent manner. Indomethacin also induced ROS production and an increase in the protein S oxidation of RIE-1. Pretreatment of RIE-1 with PZ or zinc sulfate, but not l-carnosine, significantly reduced the indomethacin-induced apoptosis. PZ prevented ROS production and the increase in protein S-oxidation. PZ inhibited indomethacin-induced cytochrome c and smac/DIABLO release and subsequent caspase-3 activation.ConclusionsThe protective effect of PZ on indomethacin-induced small intestinal injury may be dependent on its ROS-quenching effect.

BTB and CNC homolog 1 (Bach1) deficiency ameliorates TNBS colitis in mice: role of M2 macrophages and heme oxygenase-1.

Background:BTB and CNC homolog 1 (Bach1) is a transcriptional repressor of heme oxygenase-1 (HO-1), which plays an important role in the protection of cells and tissues against acute and chronic inflammation. However, the role of Bach1 in the gastrointestinal mucosal defense system remains little understood. HO-1 supports the suppression of experimental colitis and localizes mainly in macrophages in colonic mucosa. This study was undertaken to elucidate the Bach1/HO-1 systems effects on the pathogenesis of experimental colitis. Methods:This study used C57BL/6 (wild-type) and homozygous Bach1-deficient C57BL/6 mice in which colonic damage was induced by the administration of an enema of 2,4,6-trinitrobenzene sulfonic acid (TNBS). Subsequently, they were evaluated macroscopically, histologically, and biochemically. Peritoneal macrophages from the respective mice were isolated and analyzed. Then, wild-type mice were injected with peritoneal macrophages from the respective mice. Acute colitis was induced similarly. Results:TNBS-induced colitis was inhibited in Bach1-deficient mice. TNBS administration increased the expression of HO-1 messenger RNA and protein in colonic mucosa in Bach1-deficient mice. The expression of HO-1 mainly localized in F4/80-immunopositive and CD11b-immunopositive macrophages. Isolated peritoneal macrophages from Bach1-deficient mice highly expressed HO-1 and also manifested M2 macrophage markers, such as Arginase-1, Fizz-1, Ym1, and MRC1. Furthermore, TNBS-induced colitis was inhibited by the transfer of Bach1-deficient macrophages into wild-type mice. Conclusions:Deficiency of Bach1 ameliorated TNBS-induced colitis. Bach1-deficient macrophages played a key role in protection against colitis. Targeting of this mechanism is applicable to cell therapy for human inflammatory bowel disease.

Carbon monoxide enhance colonic epithelial restitution via FGF15 derived from colonic myofibroblasts.

Carbon monoxide (CO) has been reported to ameliorate colonic inflammation and improve experimental colitis. It is well known that mucosal restitution is important to improve colitis as well as reduction of mucosal inflammation. However, it has not been clear whether CO effects to colonic mucosal restitution or not. In general, colonic myofibroblast (MF) has been reported to play an important role of colonic epithelial cell restitution via constitutive secretion of TGF-beta. In this study, we showed CO (supplied by CO-releasing molecule; CORM) treated MF conditioned medium enhanced colonic epithelial cell (YAMC) restitution and we determined gene expression in colonic MF treated with CO using microRNA. The microRNA array suggested that miR-710 was significantly reduced in MF by CO treatment and the target gene of miR-710 is determined to fibroblast growth factor (FGF)15. The CO treated MF conditioned medium which FGF15 expression was silenced extinguished the enhancement effect of epithelial cell restitution. Our findings demonstrate that CO treatment to MF increased FGF15 expression via inhibition of miR-710 and FGF15 enhanced colonic epithelial cell restitution.

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.

Hemin ameliorates indomethacin-induced small intestinal injury in mice through the induction of heme oxygenase-1

Although non‐steroidal anti‐inflammatory drugs can induce intestinal injury, the mechanisms are not fully understood, and treatment has yet to be established. Heme oxygenase‐1 (HO‐1) has recently gained attention for anti‐inflammatory and cytoprotective effects. This study aimed to investigate the effects of hemin, an HO‐1 inducer, on indomethacin‐induced enteritis in mice.

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.

Preventive effect of agaro‐oligosaccharides on non‐steroidal anti‐inflammatory drug‐induced small intestinal injury in mice

Non‐steroidal anti‐inflammatory drugs (NSAIDs), which are commonly used in clinical medicine, cause erosion, ulcers, and bleeding in the gastrointestinal tract. No effective agent for the prevention and treatment of small intestinal injury by NSAIDs has been established. This study investigates the effects of agaro‐oligosaccharides (AGOs) on NSAID‐induced small intestinal injury in mice.

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