Natsuko Hayashi

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.

Carbon Monoxide Liberated from Carbon Monoxide-Releasing Molecule Exerts an Anti-inflammatory Effect on Dextran Sulfate Sodium-Induced Colitis in Mice

BackgroundEndogenous carbon monoxide (CO) is one of the three products of heme degradation by heme oxygenase-1 (HO-1) and exerts novel anti-inflammatory and anti-apoptotic effects as a gaseous second messenger. The purpose of this investigation was to determine whether exogenous CO could modulate intestinal inflammation.MethodsAcute colitis was induced with 2% DSS in male C57BL/6 mice. CO-releasing molecule-2 (CORM-2; tricarbonyldichlororuthenium(II) dimer) was intraperitoneally administered twice daily and the disease activity index (DAI) was determined. We measured tissue-associated myeloperoxidase (MPO) activity as an index of neutrophil infiltration, and the production of keratinocyte chemoattractant (KC) and tumor necrosis factor-α (TNF-α) protein in the intestinal mucosa. In an in-vitro study, young adult mouse colonic epithelial (YAMC) cells were incubated with TNF-α, and KC mRNA/protein expression and nuclear translocation of nuclear factor-kappa B (NF-κB) were measured with or without CORM-2 treatment.ResultsAfter DSS administration, DAI score increased in a time-dependent manner, and this increase was ameliorated by CORM-2 treatment. Increases in MPO activity and in the production of KC and TNF-α after DSS administration were significantly inhibited by CORM-2. TNF-α-induced KC production in YAMC cells was also inhibited by CORM-2 treatment. Further, nuclear translocation of NF-κB in YAMC cells was inhibited by CORM-2.ConclusionCORM-liberated CO significantly inhibited inflammatory response in murine colitis by inhibition of cytokine production in the colonic epithelium. These results suggest that CO could become a new therapeutic molecule for inflammatory bowel disease.

Involvement of reactive oxygen species in indomethacin-induced apoptosis of small intestinal epithelial cells

BackgroundThe precise pathogenic mechanism of nonsteroidal antiinflammatory drug-induced small intestinal injury is still unknown. In the present study, we investigated the mechanism by which indomethacin induced mucosal injury by using an in vitro model of small intestine.MethodsThe colon cancer cell line Caco-2, exhibiting a small intestinal phenotype starting as a crypt cell and differentiating to a villous phenotype, and RIE, a rat intestinal epithelial cell line, were employed. Indomethacin was added to differentiated the Caco-2 and RIE monolayer, and cell death was quantified by MTT assay and LDH release in the cell culture supernatant. Indomethacin-induced cell death was also qualified by fluorescent probes under the fluorescent microscope. As a functional study, the permeability of the Caco-2 monolayer was assessed by measuring transepithelial electrical resistance (TEER) and the flux of FITC-conjugated dextran across the monolayer. Indomethacin-induced reactive oxygen species production in Caco-2 and RIE was evaluated by redoxsensitive fluorogenic probes using a fluorometer. In some experiments, antioxidants were used to clarify the role of reactive oxygen species on indomethacin-induced Caco-2 cell death.ResultsIndomethacin caused cell death (mainly apoptosis) of Caco-2 and RIE in a dose-and time-dependent manner that was correlated with increased permeability of the Caco-2 monolayer. Exposure of Caco-2 and RIE with indomethacin also resulted in a significant reactive oxygen species production that was inhibited by the pretreatment of these cells with antioxidants.ConclusionsTaken together, reactive oxygen species production is one of the mechanisms by which indomethacin induced small intestinal injury.

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.

Heat-Shock Protein 70-Overexpressing Gastric Epithelial Cells Are Resistant to Indomethacin-Induced Apoptosis

Background/Aims: Protecting intestinal mucosa from nonsteroidal anti-inflammatory drugs is still an unsolved problem. It has been revealed that apoptosis in epithelial cells as a result of mitochondrial injury is an important pathogenesis in indomethacin-induced gastric mucosal injury. In this study, we revealed the effect of overexpressed heat-shock protein 70 (HSP70) in indomethacin-induced apoptosis and oxidative stress. Methods: HSP70-overexpressing rat gastric mucosal cells (7018-RGM-1 cells) and control cells (pBK-CMV-12 cells) were used and treated with 0–500 μM of indomethacin for 24 h. Cell viability and cytotoxity were measured by a WST-8 assay and a lactate dehydrogenase release assay, respectively. Apoptosis was observed by fluorescence microscopy staining with Hoechst 33342 and propidium iodide. The expression of Bcl-2 family proteins, activation of caspase-3, and 4-hydroxy-2-nonenal (4-HNE)-modified proteins were assessed by Western blot analysis. Results: Indomethacin caused apoptosis of gastric epithelial cells. The 7018-RGM-1 cells survived significantly after indomethacin treatment compared to the control cells. The increase in pro-apoptotic Bad proteins, the decrease in anti-apoptotic Bcl-2 proteins, and caspase activation were all suppressed in the 7018-RGM-1 cells. A lower level of indomethacin-induced 4-HNE-modification was detected in the 7018-RGM-1 cells than in the control cells. Conclusion: Overexpressed HSP70 may potentiate resistance to apoptosis and oxidative stress in indomethacin-induced gastric epithelial cell 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.

Heat shock protein 70-dependent protective effect of polaprezinc on acetylsalicylic acid-induced apoptosis of rat intestinal epithelial cells.

Protection of the small intestine from mucosal injury induced by nonsteroidal anti-inflammatory drugs including acetylsalicylic acid is a critical issue in the field of gastroenterology. Polaprezinc an anti-ulcer drug, consisting of zinc and L-carnosine, provides gastric mucosal protection against various irritants. In this study, we investigated the protective effect of polaprezinc on acetylsalicylic acid-induced apoptosis of the RIE1 rat intestinal epithelial cell line. Confluent rat intestinal epithelial cells were incubated with 70 µM polaprezinc for 24 h, and then stimulated with or without 15 mM acetylsalicylic acid for a further 15 h. Subsequent cellular viability was quantified by fluorometric assay based on cell lysis and staining. Acetylsalicylic acid-induced cell death was also qualified by fluorescent microscopy of Hoechst33342 and propidium iodide. Heat shock proteins 70 protein expression after adding polaprezinc or acetylsalicylic acid was assessed by western blotting. To investigate the role of Heat shock protein 70, Heat shock protein 70-specific small interfering RNA was applied. Cell viability was quantified by fluorometric assay based on cell lysis and staining and apoptosis was analyzed by fluorescence-activated cell sorting. We found that acetylsalicylic acid significantly induced apoptosis of rat intestinal epithelial cells in a dose- and time-dependent manner. Polaprezinc significantly suppressed acetylsalicylic acid-induced apoptosis of rat intestinal epithelial cells at its late phase. At the same time, polaprezinc increased Heat shock protein 70 expressions of rat intestinal epithelial cells in a time-dependent manner. However, in Heat shock protein 70-silenced rat intestinal epithelial cells, polaprezinc could not suppress acetylsalicylic acid -induced apoptosis at its late phase. We conclude that polaprezinc-increased Heat shock protein 70 expression might be an important mechanism by which polaprezinc suppresses acetylsalicylic acid-induced small intestinal apoptosis, a hallmark of acetylsalicylic acid-induced enteropathy.

FGF19 Protects Colonic Epithelial Cells against Hydrogen Peroxide

Background/Aims: The apoptosis induced by hydrogen peroxide (H2O2) in colonic epithelial cells is very important for the pathogenesis of inflammatory bowel disease (IBD). Fibroblast growth factor (FGF) 15, the human ortholog of FGF19, is reported to be secreted from colonic myofibroblasts and enhances colonic epithelial restitution, but little is known about the function of FGF19 to colonic epithelial cells. In the present study, we investigate the anti-apoptosis effect of FGF19 in colonic epithelial cells treated with H2O2. Methods: Young adult mouse colonic epithelial (YAMC) cells are used to investigate the protective role of FGF19. Cellular viability was determined by WST-8 assay, and apoptosis was measured by Hoechst staining and Western blotting of cleaved caspase-3. YAMC cells were pretreated by FGF19 and H2O2 was used for cellular damage. Results: We demonstrated that pretreatment of FGF19 (50 ng/ml) significantly protects YAMC cells treated with H2O2 assessed by WST-8. We also demonstrated Hoechst staining of YAMC cells and that H2O2-induced apoptosis is significantly reduced by FGF19 treatment via inhibition of the caspase-3 pathway. Conclusion: These results indicate FGF19 protects YAMC cells against H2O2 and might be related to the pathogenesis of IBD. Even further studies are needed – FGF19 may be one of the possible therapeutic strategies of IBD.

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

Non-Toxic Concentration of Acetyl Salicylic Acid Can Induce ROS-Dependent Increase of Small Intestinal Epithelial Cell Permeability

G A A b st ra ct s condition, and normalized to that of β-actin. Results: Increased CPP was observed in colon frommice submitted tomild colitis when comparedwith healthy colonic segments (1.92±0.20 vs 1.34±0.25 nmol.h-1.cm-2 of FD4, p<0.05). In healthy colonic segments, CMCF strongly increased CPP (3.47±0.54 vs 1.34±0.25 nmol.h-1.cm-2 of FD4, p<0.01), whereas CMPAT had no effect. In contrast, exposure to CMCF of colonic segments from mice submitted to mild colitis did not exacerbate the increased CPP previously observed in these mildly inflamed tissues (1.8±0.26 vs 1.92±0.20 nmol.h-1.cm-2 of FD4). Accordingly, the expression of occludin in T84 cells was decreased in presence of IFNγ/TNFα (26.6±1.4 vs 16.3±1.1, p<0.05), and this effect was not exacerbated by CMCF. T84 treatment with CMCF increased their ability to proliferate (113% of controls), migrate (182.8% of controls) and improved wound healing (206.2% of controls) in comparison with T84 cells exposed to CMPAT (p<0.05) or control medium (p<0.01). Conclusion: CF secretions do not exacerbate the primary increase of colonic permeability observed in mild inflammatory tissue, nor worsen occludin downexpression. Moreover, CF secretions lead to increased intestinal cell proliferation and migration promoting intestinal epithelial barrier healing.