Ikuhiro Hirata

Increased expression of microRNA in the inflamed colonic mucosa of patients with active ulcerative colitis

Background and Aims:  MicroRNA (miRNA) are endogenous, approximately 22‐nucleotide non‐coding RNA that suppress gene expression at post‐transcriptional levels by binding to the 3′‐untranslated region of specific mRNA targets through base‐pairing. It has been recently reported that miRNA have critical functions in key biological processes such as cell proliferation and cell death in various cancer cells. However, the relationship between intestinal inflammation and miRNA expression remains unclear. In the present study, we used microarray technology to identify miRNA induced in the colonic mucosa of patients with active ulcerative colitis (UC).

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.

Increased intestinal expression of heme oxygenase-1 and its localization in patients with ulcerative colitis.

Background:  Heme oxygenase‐1 (HO‐1) is regarded as a sensitive and reliable indicator of cellular oxidative stress. Two end products of heme degradation, carbon monoxide (CO) and bilirubin, are involved in the protective role of HO‐1 against oxidative injury. We have demonstrated enhanced expression of this enzyme and increased concentration of CO in experimental models of colitis, but the role of HO‐1 in patients with ulcerative colitis (UC) has not been extensively investigated. The aim of the present study was to determine the intestinal levels and localization of ho‐1 mRNA and HO‐1 protein in patients with UC.

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.

Endogenous Hydrogen Sulfide Is an Anti-inflammatory Molecule in Dextran Sodium Sulfate-Induced Colitis in Mice

BackgroundEndogenous hydrogen sulfide (H2S) is increasingly being recognized as an important gaseous physiological mediator. Accumulating evidence shows the functions of H2S in various models of disease, but rarely in colitis. In this study, we investigated the role of endogenous H2S in a dextran sodium sulfate (DSS)-induced colitis model.MethodsAcute colitis was induced using 8% DSS in male BALB/c mice. The mRNA expression of cystathionine γ-lyase (CSE), the primary synthetase of H2S in the gastrointestinal tract, and cystathionine-β-synthetase (CBS) was measured by real-time RT-PCR. The amount of H2S in the colonic mucosa was measured by gas chromatography. Colitis severity was evaluated clinically, histologically, and biochemically under the condition of co-treatment with DL-propargylglycine (PAG), an irreversible CSE inhibitor, and sodium sulfide (Na2S), an H2S donor.ResultsThe mRNA expression levels of CSE and CBS, and the H2S content in the colonic mucosa were increased with time after DSS administration. The disease activity index, which was determined by weight loss, stool consistency, and intestinal bleeding, increased after DSS administration. PAG significantly enhanced the increase in the disease activity index scores. PAG also significantly increased tissue-associated myeloperoxidase activity and thiobarbituric acid-reactive substances in the inflamed mucosa. Moreover, Na2S counteracted these effects of PAG.ConclusionsTaken together, the results indicated that the inhibition of endogenous H2S generation caused the deterioration of DSS-induced colitis. We conclude that physiological H2S might act as an anti-inflammatory molecule in colitis.

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.

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.

Carbon monoxide promotes gastric wound healing in mice via the protein kinase C pathway

Abstract Previous studies have shown that carbon monoxide (CO) is involved in a variety of physiological and pathophysiological processes including anti-inflammatory, anti-apoptotic and anti-oxidant responses. However, it remains unclear whether CO promotes gastric ulcer healing. In the present study, we evaluated the efficacy of CO-saturated saline in the treatment of gastric ulcers and its underlying mechanism. Acute gastric ulcers were induced in C57BL/6 male mice using acetic acid. A CO-saturated solution was prepared by bubbling 50% CO gas into saline. To investigate the effect of CO on gastric mucosal healing, CO solution was orally administrated twice a day beginning on day 3 after the induction of gastric ulcer. Mice were sacrificed on day 7 after ulcer induction. The stomach was removed, and the ulcerated lesions were measured. In vitro wound healing assays were used to determine the mechanism of action of CO in the restoration of murine gastric epithelial cells. The oral administration of CO solution accelerated the gastric ulcer healing by promoting re-epithelialization. Furthermore, the wound healing assay performed using the murine gastric epithelial cells revealed that the CO-saturated medium enhanced cell migration through the activation of protein kinase C (PKC). Based on these data, CO may represent a novel therapeutic approach for the treatment of gastric mucosal injuries.