Tatsushi Omatsu

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.

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.

The impact of non-steroidal anti-inflammatory drugs on the small intestinal epithelium

The small intestine has been called as a dark continent of digestive tract and it had been very difficult to diagnose or treat the disease of small intestine. However recent technological development including video capsule endoscopy or balloon-assisted endoscopy has made us to aware the various diseases of small intestine. By using capsule endoscopy, many researchers reported that more than 70% of patients treated continuously with non-steroidal anti-inflammatory drugs (NSAID) exhibit the mucosal damage of small intestine. In some cases, NSAID not only causes mucosal damage but also results in life threatening bleeding from small intestine, which had not been prevented or cured by gastro-protective drug or anti-gastric acid secretion drug administration. Therefore to investigate and identify the effective drug that protects small intestine from mucosal damage is urgently expected. In spite of extensive investigation in clinical field, only a few drugs such as misoprostol, a synthetic prostaglandin E1 analogue, has been reported as an effective one but is not satisfactory enough to fulfill the requirement of patients who suffer from NSAID-induced mucosal damage of small intestine. And now, extensive study is being performed using several gastro-mucoprotective drugs by many researchers. In this review, we introduce the current clinical situation in small intestinal injury of patients under NSAID treatment, and to summarize the molecular mechanism by which NSAID, including acetyl salicylic acid, cause small intestinal damage. In addition, we present results of clinical trials performed so far, and refer the possible preventive method or treatment in the near future.

Identification of inflammation‐related proteins in a murine colitis model by 2D fluorescence difference gel electrophoresis and mass spectrometry

Background and Aims:  The aim of this study was to identify new intestinal proteins potentially associated with acute inflammation using proteomic profiling of an in vivo mice model of ulcerative colitis.

The role of mitochondria-derived reactive oxygen species in the pathogenesis of non-steroidal anti-inflammatory drug-induced small intestinal injury

Abstract Non-steroidal anti-inflammatory drugs (NSAIDs) have been implemented in clinical settings for a long time for their anti-inflammatory effects. With the number of NSAID users increasing, gastroenterological physicians and researchers have worked hard to prevent and treat NSAID-induced gastric mucosal injury, an effort that has for the large part being successful. However, the struggle against NSAID-induced mucosal damage has taken on a new urgency due to the discovery of NSAID-induced small intestinal mucosal injury. Although the main mechanism by which NSAIDs induce small intestinal mucosal injury has been thought to depend on the inhibitory effect of NSAIDs on cyclooxygenase (COX) activity, recent studies have revealed the importance of mitochondria-derived reactive oxygen species (ROS) production, which occurs independently of COX-inhibition. ROS production is an especially important factor in the increase of small intestinal epithelial cell permeability, an early stage in the process of small intestinal mucosal injury. By clarifying the precise mechanism, together with its clinical features using novel endoscopy, effective strategies for preventing NSAID-induced small intestinal damage, especially targeting mitochondria-derived ROS production, may be developed.

Protective effect of the Japanese traditional medicine juzentaihoto on myelosuppression induced by the anticancer drug TS-1 and identification of a potential biomarker of this effect

BackgroundTS-1 is an oral anticancer drug containing a 5-fluorouracil derivative (Tegafur) that is widely used in Japan for the treatment of cancer, especially gastrointestinal tumors. Frequently, however, TS-1 therapy has to be discontinued because of leukopenia. If it were possible to predict the development of bone marrow suppression before the white blood cell (WBC) count had actually decreased, treatment could be improved by strict dosage control and/or the prophylactic administration of hematopoietic drugs. Juzentaihoto (JTT), a traditional Japanese medicine (Kampo), has been reported to activate hematopoiesis and reduce the side effects associated with chemotherapy and radiotherapy. Here, we 1) evaluate the efficacy of JTT in alleviating myelosuppression induced by TS-1 therapy in mice, and 2) explore biomarkers that reflect both induction by TS-1 and alleviation by JTT of bone marrow suppression using a proteomics approach.MethodsTen mg/kg of TS-1 was administered to Balb/c mice with or without 1 g/kg of oral JTT for 3, 5 and 7 days. WBC count and ratio of CD34+ bone marrow cells (BMCs) were estimated by flow cytometry. Plasma samples were analyzed using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI TOF-MS). A biomarker candidate from SELDI profiling was identified using a combination of cation exchange spin column purification, SDS-PAGE, enzymatic digestion and LC-MS/MS.ResultsAfter administration of TS-1, a significant decrease in WBC count and CD34+ BMC ratio were observed at days 5 and 3, respectively. JTT treatment improved WBC count on day 7 and CD34+ BMC ratio on days 5 and 7. SELDI analysis highlighted three protein peaks that had increased on day 3 after treatment with TS-1 but remained unchanged in mice co-treated with JTT. One of the three peaks, m/z 4223.1, was further investigated and identified as a specific C-terminal fragment of albumin.ConclusionThis study indicates that bone marrow suppression by treatment with TS-1 in mice might be improved by coadministration of JTT. A C-terminal fragment of albumin was identified as a candidate biomarker for predicting TS-1-induced myelosuppression. However, the sensitivity and specificity of the biomarker candidate must be validated in future clinical studies.