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Background and Aim: Efficient intestinal wound healing is essential for good prognoses of ulcerative colitis (UC). Although bile acids and the transmembrane G-protein-coupled receptor (TGR) 5 have been reported to affect wound healing in intestinal epithelial cells, the detailed underlying mechanisms are unclear. Here, we investigated the role of TGR5 in wound healing in the context of colonic epithelial cells in the presence of bile acids., Methods: The expression of TGR5 in the colonic epithelium of both a dextran sulfate sodium (DSS)-induced colitis mouse model (recovery phase), and UC patients in clinical remission, was evaluated. Young adult mouse colonic epithelial (YAMC) cells were then used to evaluate wound healing after treatment with deoxycholic acid (DCA); TGR5 was silenced in YAMC cells via shRNA-transfection, and a wound-healing assay in the presence of DCA was performed. Furthermore, we investigated the role of the activation of AKT in the context of wound healing., Results: The expression of TGR5 was decreased in the colonic epithelium of both mice with DSS-induced colitis and UC patients. Additionally, DCA significantly delayed wound healing in YAMC cells but not in TGR5 silenced ones. Of note, the DCA-induced activation of AKT signaling in YAMC cells was inhibited by TGR5 silencing, and AKT inhibitors prevented the wound healing delay induced by DCA., Conclusions: Overall, we show that DCA delays wound healing in the context of colonic epithelial cells through AKT activation. These results may support the development of new therapeutic approaches for epithelial regeneration in UC., (© 2021 Journal of Gastroenterology and Hepatology Foundation and John Wiley & Sons Australia, Ltd.)

Background and Aim: Mucosal healing is an important clinical goal in patients with inflammatory bowel disease. Recently, short-chain fatty acids (SCFAs) have been reported to have multifaceted effects to host. However, the effects of SCFAs on wound healing in intestinal epithelial cells are unclear. In the present study, we investigated the effects of acetate, one of the major SCFAs, on the wound healing of murine colonic epithelial cells., Methods: Young adult mouse colonic epithelial cells were used to determine the effect of acetate using wound healing assay. Mitogen-activated protein kinase and Rho kinase inhibitor were used to elucidate intracellular signal of wound healing treated with acetate. Meanwhile, Rho activation assays were utilized to measure Rho activation levels. To assess in vivo effects, C57B6 mice with dextran sodium sulfate for 7 days were treated with enema administration of acetate for 7 days. Body weight, disease activity index, colon length, and mucosal break ratio in histology were examined., Results: Acetate enhanced wound healing and fluorescence intensity of actin stress fiber compared with control. These effects were canceled with pretreatment of c-Jun N-terminal kinase (JNK) inhibitor or Rho kinase inhibitor. Furthermore, JNK inhibitor reduced the activation of Rho induced by acetate. In the dextran sodium sulfate-induced colitis model, the mice with enema treatment of acetate significantly exhibited recovery., Conclusions: In this study, we demonstrated that acetate promoted murine colonic epithelial cell wound healing via activation of JNK and Rho signaling pathways. These findings suggested that acetate could have applications as a therapeutic agent for patients with intestinal mucosal damage, such as inflammatory bowel disease., (© 2020 Journal of Gastroenterology and Hepatology Foundation and John Wiley & Sons Australia, Ltd.)

Proliferation and spatial development of colonic epithelial cells are highly regulated along the crypt vertical axis, which, when perturbed, can result in aberrant growth and carcinogenesis. In this study, two key factors were identified that have important and counterbalancing roles regulating these processes: pericrypt myofibroblast-derived Wnt-5a and the microbial metabolite butyrate. Cultured YAMC cell proliferation and heat shock protein induction were analzyed after butryate, conditioned medium with Wnt5a activity, and FrzB containing conditioned medium. In vivo studies to modulate Hsp25 employed intra-colonic wall Hsp25 encoding lentivirus. To silence Wnt-5a in vivo, intra-colonic wall Wnt-5a silencing RNA was used. Wnt-5a, secreted by stromal myofibroblasts of the lower crypt, promotes proliferation through canonical β-catenin activation. Essential to this are two key requirements: (1) proteolytic conversion of the highly insoluble ~40 kD Wnt-5a protein to a soluble 36 mer amino acid peptide that activates epithelial β-catenin and cellular proliferation, and (2) the simultaneous inhibition of butyrate-induced Hsp25 by Wnt-5a which is necessary to arrest the proliferative process in the upper colonic crypt. The interplay and spatial gradients of these factors insures that crypt epithelial cell proliferation and development proceed in an orderly fashion, but with sufficient plasticity to adapt to physiological perturbations including inflammation., Competing Interests: Dr. Yoshito Itoh has an affiliation with a donation-funded department from AstraZeneca Co., Ltd., Eisai Co., Ltd., Otsuka Pharmaceutical Co., Ltd., MSD K.K., Dainippon Sumitomo Pharma Co., Ltd., Chugai Pharmaceutical Co., Ltd., FUJIFILM Medical Co., Ltd. and Merck Serono Co., Ltd. Yuji Naito received scholarship funds from Eisai Co., Ltd., Astellas Pharma Inc., Takeda Pharmaceutical Co., Ltd., and Mitsubishi Tanabe Pharma Co., Ltd. Yoshito Itoh received scholarship funds from MSD K.K. and Bristol-Myers K.K. The other authors have no conflicts of interest to declare.

Background and Aims: Healing of the intestinal mucosal epithelium was found to be a critical factor in the treatment of inflammatory bowel disease (IBD). In this study, we provide further evidence that partially hydrolyzed dietary fiber (PHGG) enhances colonic epithelial cell wound healing, and partially characterize the mechanism that governs this process., Materials and Methods: Young adult mouse colonic (YAMC) epithelial cells were scraped with a 10 μl micro-pipette tip to denude a round of the monolayer and were incubated with PHGG. The area of cell migration was measured using Image J software. Meanwhile, Rho activation assays were utilized to monitor Rho activation levels. To assess in vivo effects, C57B6 mice were treated with DSS for 7 days and then provided food supplemented with PHGG for 8 days., Results: YAMC cells treated with PHGG exhibited significantly enhanced wound healing compared to the control cells; however, this enhancement was inhibited by both Y-27632 (RhoA inhibitor) and U0126 (ERK1/2 inhibitor). Likewise, there was a PHGG-dependent increase in F-actin accumulation and Rho kinase activity that was blocked by U0126. Meanwhile, PHGG-dependent ERK1/2 activity was not inhibited by Y-27632. In the DSS-induced mouse colitis model, animals that received food supplemented with PHGG exhibited significant recovery of the colonic mucosa., Conclusions: In this study, we demonstrate that PHGG promotes colonic epithelial cell wound healing via activation of RhoA, which occurs downstream of ERK1/2 activation. These findings indicate that PHGG could be utilized as a therapeutic agent for patients with intestinal mucosal damage such as those with IBD.

Background/aims: Although the cytotoxicity of aspirin against the intestinal epithelium is a major clinical problem, little is known about its pathogenesis. We assessed the involvement of Multi Drug Resistance (MDR) 1 in intestinal epithelial cell injury caused by aspirin using MDR1 gene-transfected Caco2 cells., Methods: Caco2 cells were treated with various concentrations of aspirin for 24 h. After treatment of Caco2 cells with verapamil, a specific inhibitor of MDR1, we assessed the extent of cell injury using a WST-8 assay at 24 h after aspirin-stimulation. We performed the same procedure in MDR1 gene-transfected Caco2 cells. To determine the function of MDR1 in the metabolism of aspirin, flux study was performed using (14)C-labeled aspirin., Results: The level of aspirin-induced cell injury was higher in verapamil-treated Caco2 cells than in control cells and was less serious in MDR1-transfected Caco2 cells than in control vector-transfected cells. The efflux of (14)C-labeled aspirin was higher in verapamil-treated Caco2 cells than in control cells., Conclusion: These data suggest that aspirin effux occurs through the MDR1 transporter and that the MDR1 transporter is involved in the pathogenesis of aspirin-induced cell injury., (© 2013 S. Karger AG, Basel)

Aim: To investigate the efficacy of rebamipide in a rat model of colitis and restitution of intestinal epithelial cells in vitro., Methods: Acute colitis was induced with trinitrobenzene sulfonic acid (TNBS) in male Wistar rats. Rats received intrarectal rebamipide treatment daily starting on day 7 and were sacrificed on day 14 after TNBS administration. The distal colon was removed to evaluate the various parameters of inflammation. Moreover, wound healing assays were used to determine the enhanced restitution of rat intestinal epithelial (RIE) cells treated with rebamipide., Results: Intracolonic administration of rebamipide accelerated TNBS-induced ulcer healing. Increases in the wet weight of the colon after TNBS administration were significantly inhibited by rebamipide. The wound assay revealed that rebamipide enhanced the migration of RIE cells through phosphorylation of extracellular signal-regulated kinase (ERK) and activation of Rho kinase., Conclusion: Rebamipide enema healed intestinal injury by enhancing restitution of RIE cells, via ERK activation. Rebamipide might be a novel therapeutic approach for inflammatory bowel disease.

Human esophageal epithelium is continuously exposed to physical stimuli or to gastric acid that sometimes causes inflammation of the mucosa. Transient receptor potential vanilloid 1 (TRPV1) is a nociceptive, Ca(2+)-selective ion channel activated by capsaicin, heat, and protons. It has been reported that activation of TRPV1 expressed in esophageal mucosa is involved in gastroesophageal reflux disease (GERD) or in nonerosive GERD symptoms. In this study, we examined the expression and function of TRPV1 in the human esophageal epithelial cell line Het1A, focusing in particular on the role of oxidative stress. Interleukin-8 (IL-8) secreted by Het1A cells upon stimulation by capsaicin or acid with/without 4-hydroxy-2-nonenal (HNE) was measured by ELISA. Following capsaicin stimulation, the intracellular production of reactive oxygen species (ROS) was determined using a redox-sensitive fluorogenic probe, and ROS- and HNE-modified proteins were determined by Western blotting using biotinylated cysteine and anti-HNE antibody, respectively. HNE modification of TRPV1 proteins was further investigated by immunoprecipitation after treatment with synthetic HNE. Capsaicin and acid induced IL-8 production in Het1A cells, and this production was diminished by antagonists of TRPV1. Capsaicin also significantly increased the production of intracellular ROS and ROS- or HNE-modified proteins in Het1A cells. Moreover, IL-8 production in capsaicin-stimulated Het1A cells was enhanced by synthetic HNE treatment. Immunoprecipitation studies revealed that TRPV1 was modified by HNE in synthetic HNE-stimulated Het1A cells. We concluded that TRPV1 functions in chemokine production in esophageal epithelial cells, and this function may be regulated by ROS via posttranslational modification of TRPV1.

Aim: To investigate the expression and function of interleukin-4 receptor alpha (IL-4Ralpha) in human conjunctival epithelial cells (HCjECs)., Methods: The presence of IL-4Ralpha mRNA and protein was examined by reverse transcription (RT) PCR and immunohistology, respectively. Cell surface expression was examined by flow cytometry. The effects of interleukin (IL)-4 or IL-13 on the tyrosine phosphorylation of signal transducer and the activator of transcription 6 (STAT6) were evaluated by immunoblot analyses. The transcripts upregulated upon IL-4 stimulation were examined using GeneChip, and confirmed by quantitative RT-PCR., Results: IL-4Ralpha mRNA and protein were detected in human conjunctival epithelium. IL-4Ralpha protein was expressed on the cell surface of HCjECs. IL-4 and IL-13 induced tyrosine phosphorylation of STAT6. GeneChip analysis showed that nine transcripts were upregulated more than fourfold by IL-4 stimulation in the primary HCjECs from two individuals. Quantitative RT-PCR assay confirmed the upregulation of these transcripts: lecithin retinol acyltransferase (LRAT), calpain (CAPN14), tumour necrosis factor alpha-induced protein 6 (TNFAIP6), RAS guanyl-releasing protein 1 (RASGRP1), endothelin receptor type A (EDNRA), hyaluronan synthase 3 (HAS3), cathepsin C (CTSC), carbonic anhydrase II (CA2) and cytokine-inducible SH2-containing protein (CISH)., Conclusions: HCjECs expressed functioning IL-4Ralpha, and IL-4 stimulation induced the expression of several genes.

Background and Aims: Ecabet sodium (ES) is a gastric mucosal protective and ulcer-healing agent. Recently enema therapy with ES was found to be effective for the treatment of human ulcerative colitis as well as experimental colitis in an animal model. Whereas ES possesses potential as a novel treatment for ulcerative colitis, its precise mechanism of action remains to be elucidated. In this study, we investigated the therapeutic efficacy of ES in an experimental rat model of colitis, and evaluated the restitution of intestinal epithelial cells treated with ES in vitro., Methods: Acute colitis was induced with trinitrobenzene sulfonic acid (TNBS) in male Wistar rats. Rats received intrarectal treatment with ES daily starting on day 7 and were sacrificed on day 14 after the administration of TNBS. The distal colon was removed to evaluate various parameters of inflammation. Moreover, wound-healing assays were used to determine the enhanced restitution of rat intestinal epithelial (RIE) cells treated with ES., Results: Intracolonic administration of ES accelerated TNBS-induced ulcer healing. Increases in the wet weight of the colon after TNBS administration were significantly inhibited by ES treatment. The wound assay revealed ES enhancement of the migration of RIE cells migration through the phosphorylation of extracellular signal-regulated kinase., Conclusion: Daily administration of an ES enema promoted the healing of intestinal mucosal injury, in part by the enhanced restitution of intestinal epithelial cells via extracellular signal-regulated kinase activation. ES may thus represent a novel therapeutic approach for the treatment of inflammatory bowel disease.

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 microM 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., (Copyright 2009 S. Karger AG, Basel.)

Epidemiological study has shown strong correlation between the Helicobacter pylori (H. pylori) infection and gastric carcinogenesis. However, the mechanism by which H. pylori induces gastric carcinogenesis is not known. In this review, we focused on the product of cytotoxin-associated gene A (CagA), one of the important virulence factors of H. pylori. H. pylori injects CagA protein into the host gastric epithelial cells through its needle-like structure, type IV secretion system. Injected CagA hijacks physiological signal transduction and causes pathological cellular response such as increased cell proliferation, motility, apoptosis and morphological change through different mechanisms. H. pylori has been shown to produce reactive oxygen species (ROS) in infected gastric mucosa. Although the main source of ROS production is possibly host neutrophil, we propose novel source of ROS production in this review; CagA itself can induce ROS production in gastric epithelial cell. Excessive ROS production in gastric epithelial cells can cause DNA damage and thus might involve in gastric carcinogenesis. Understanding the molecular mechanism by which H. pylori-induced carcinogenesis is important for developing new strategies against gastric cancer.

Background/aim: Recently, it has been shown that serine proteases derived from microorganisms stimulate epithelial cells to produce inflammatory mediators through protease-activated receptor (PAR). We investigated the involvement of PAR2 in the interleukin (IL)-8 production by Helicobacter pylori-infected gastric epithelial cells., Methods and Results: Human gastric epithelial cells, MKN45 cells, were used. The expression of PAR2 was assessed by real-time PCR and immunocytochemistry, and IL-8 protein was measured by an enzyme-linked immunosorbent assay. PAR2 mRNA and protein were constitutively expressed on unstimulated MKN45 cells. The treatment of cells with H. pylori resulted in a significant increase in PAR2 expression. In addition, trypsin (a natural PAR2 agonist), SLIGKV amide (a synthetic PAR2 agonist), H. pylori live bacteria or H. pylori culture supernatant significantly induced IL-8 production from MKN45 cells. H. pylori-induced IL-8 production was inhibited by nafamostat mesilate (a serine protease inhibitor), neutralizing antibody to PAR2 and in PAR2-deficient cells treated with siRNA., Conclusions: These results reveal that H. pylori-derived protease activates gastric epithelial cells to produce inflammatory cytokines through PAR2, suggesting an important role for PAR2 in the modulation of gastric inflammation associated with H. pylori.

Previous investigations have demonstrated that the cellular signaling induced by hypoxia-reoxygenation is a major pathway contributing to gastric mucosal injury induced by stress, non-steroidal anti-inflammatory drugs, and Helicobacter pylori. The aim of the present study was to perform a gene expression analysis of the gastric mucosal cellular response and to define the protective molecules in hypoxia and reoxygenation using a high-density DNA microarray analysis. Normal rat gastric mucosal (RGM-1) cells were subjected to hypoxia for 2 h, and reoxygenation was initiated by placing the cells in an environment of normoxia for 2, 4, or 8 h. Total RNA was extracted, and differences in the gene expression profiles between the normoxia and hypoxia groups or among the different durations of reoxygenation were investigated using a high-density DNA microarray. HIF-1- and apoptosis-related genes were modulated by hypoxia. Moreover, inflammation-, stress-, and wound- healing-related genes were regulated by reoxygenation following hypoxia. In particular, the expression of heat shock protein-70, amphiregulin and cyclooxygenase-2 were upregulated during reoxygenation following hypoxia, suggesting that these upregulations may play an important role in maintaining cell survival and supporting cell function., (Copyright 2006 S. Karger AG, Basel.)

Recent investigations have demonstrated that the signaling of hypoxia-re-oxygenation is a major contributing pathway leading to gastric mucosal injury induced by stress, non-steroidal anti-inflammatory drugs, and Helicobacter pylori. The aim of the present study was to perform a gene expression analysis on the gastric mucosal cellular response to hypoxia-reoxygenation using a high-density oligonucleotide array. Cells were subjected to hypoxia with 95% N(2) and 5% CO(2) at 37 degrees C for 2 h. Reoxygenation was initiated by placing the cells in an environment of normoxia for 2 h. Total RNA was extracted, and differences in gene expression profiles between the normoxia and hypoxia-reoxygenation groups were investigated using a GeneChip of Rat Toxicology U34 array (Affymetrix). Hypoxia-reoxygenation up-regulated the stress-related genes (heat shock protein-70 [HSP-70], catalase). The enhanced expression of HSP-70 was confirmed by Western blot analysis. In conclusion, these results suggest that up-regulation of the HSP-70 gene after reoxygenation may play a role in maintaining cell survival and supporting cell function as a molecular chaperone.

Immunoglobulin A (IgA)-mediated mucosal immunity is important for the host because it contributes to reducing infection risk and to establishing host–microbe symbiosis. BTB and CNC homology 1 (Bach1) is a transcriptional repressor with physiological and pathophysiological functions that are of particular interest for their relation to gastrointestinal diseases. However, Bach1 effects on IgA-mediated mucosal immunity remain unknown. For this study using Bach1-deficient (Bach1−/−) mice, we investigated the function of Bach1 in IgA-mediated mucosal immunity. Intestinal mucosa, feces, and plasma IgA were examined using immunosorbent assay. After cell suspensions were prepared from Peyer's patches and colonic lamina propria, they were examined using flow cytometry. The expression level of polymeric immunoglobulin receptor (pIgR), which plays an important role in the transepithelial transport of IgA, was evaluated using Western blotting, quantitative real-time PCR, and immunohistochemistry. Although no changes in the proportions of IgA-producing cells were observed, the amounts of IgA in the intestinal mucosa were increased in Bach1−/− mice. Furthermore, plasma IgA was increased in Bach1−/− mice, but fecal IgA was decreased, indicating that Bach1−/− mice have abnormal secretion of IgA into the intestinal lumen. In fact, Bach1 deficiency reduced pIgR expression in colonic mucosa at both the protein and mRNA levels. In the human intestinal epithelial cell line LS174T, suppression of Bach1 reduced pIgR mRNA stability. In contrast, the overexpression of Bach1 increased pIgR mRNA stability. These results demonstrate that Bach1 deficiency causes abnormal secretion of IgA into the intestinal lumen via suppression of pIgR expression. NEW & NOTEWORTHY: The transcriptional repressor Bach1 has been implicated in diverse intestinal functions, but the effects of Bach1 on IgA-mediated mucosal immunity remain unclear. We demonstrate here that Bach1 deficiency causes abnormal secretion of IgA into the intestinal lumen, although the proportions of IgA-producing cells were not altered. Furthermore, Bach1 regulates the expression of pIgR, which plays an important role in the transepithelial transport of IgA, at the posttranscriptional level. [ABSTRACT FROM AUTHOR]

Toll-like receptor 3 (TLR3) and interferon-beta promoter stimulator-1 (IPS-1) are associated with antiviral responses to double-stranded RNA viruses and contribute to innate immunity. We previously reported that conjunctival epithelial cell (CEC) TLR3 and IPS-1 pathways respond to the common ligand polyinosinic:polycytidylic acid (polyI:C) to regulate different gene expression patterns as well as CD11c + cell migration in murine-model corneas. However, the differences in the functions and the roles of TLR3 and IPS-1 remain unclear. In this study, we investigated the differences of TLR3 or IPS-1-induced gene expression in corneal epithelial cells (CECs) in response to polyI:C stimulation using cultured murine primary CECs (mPCECs) derived from TLR3 and IPS-1 knockout mice via comprehensive analysis. The genes associated with viral responses were upregulated in the wild-type mice mPCECs after polyI:C stimulation. Among these genes, Neurl3, Irg1, and LIPG were dominantly regulated by TLR3, while interleukin (IL)-6 and IL-15 were dominantly regulated by IPS-1. CCL5, CXCL10, OAS2, Slfn4, TRIM30α, and Gbp9 were complementarily regulated by both TLR3 and IPS-1. Our findings suggest that CECs may contribute to immune responses and that TLR3 and IPS-1 possibly have different functions in the corneal innate immune response. [ABSTRACT FROM AUTHOR]

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 vitrowound 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. [ABSTRACT FROM AUTHOR]

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. Copyright © 2011 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Background: Reactive oxygen species (ROS) and reactive nitrogen species (RNS) have been reported to impact gastric inflammation and carcinogenesis. However, the precise mechanism by which Helicobacter pylori induces gastric carcinogenesis is presently unclear. Aim: This review focuses on H. pylori-induced ROS/RNS production in the host stomach, and its relationship with gastric carcinogenesis. Results: Activated neutrophils are the main source of ROS/RNS production in the H. pylori-infected stomach, but H. pylori itself also produces ROS. In addition, extensive recent studies have revealed that H. pylori-induced ROS production in gastric epithelial cells might affect gastric epithelial cell signal transduction, resulting in gastric carcinogenesis. Excessive ROS/RNS production in the stomach can damage DNA in gastric epithelial cells, implying its involvement in gastric carcinogenesis. Conclusion: Understanding the molecular mechanism behind H. pylori-induced ROS, and its involvement in gastric carcinogenesis, is important for developing new strategies for gastric cancer chemoprevention. [ABSTRACT FROM AUTHOR]

To 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. Cell 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. We 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. The protective effect of PZ on indomethacin-induced small intestinal injury may be dependent on its ROS-quenching effect. [ABSTRACT FROM AUTHOR]

Goblet cells, which contribute to mucosal defense and repair in the intestinal epithelium, are depleted in human and rodent colitis. The Notch signal pathway regulates the differentiation of intestinal stem cells into epithelial cells and inhibits the differentiation of secretory lineages, including goblet cells. The aim of our study was to clarify whether the blocking of the Notch pathway at an early stage of colitis would preserve goblet cells and facilitate the healing process in dextran sulfate sodium (DSS)-induced colitis in mice. DSS was orally administered to C57/BL6 mice for 7 days, and dibenzazepine (DBZ), a Notch pathway blocker, was administered for 5 consecutive days, beginning on the first day of DSS treatment. Colonic mucosal inflammation was evaluated clinically, biochemically, and histologically. The expression of the goblet cell-associated genes Math1 and MUC2 and proinflammatory cytokines was evaluated by real-time reverse-transcriptase-PCR, with the expression of Math1 and MUC2 also visualized by immunohistochemical examination. The administration of DBZ at 4 μmol/kg significantly reduced the severity of the colitis. Compared with the DSS only-treated intestine, the number of goblet cells was relatively sustained, and the expression of Math1 and MUC2 was also elevated in the DSS/DBZ-treated intestine. DBZ treatment suppressed the mRNA levels for interleukin-1β and -6, and matrix metalloproteinases-3 and -9 in the DSS-treated intestine. Early-stage blocking of Notch signaling may ameliorate acute DSS colitis by preventing reduction in the number of goblet cells. [ABSTRACT FROM AUTHOR]

Abstract: 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-β. 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. [Copyright &y& Elsevier]

The 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. The 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. Indomethacin 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. Taken together, reactive oxygen species production is one of the mechanisms by which indomethacin induced small intestinal injury. [ABSTRACT FROM AUTHOR]

Background/Aim: Recently, it has been shown that serine proteases derived from microorganisms stimulate epithelial cells to produce inflammatory mediators through protease-activated receptor (PAR). We investigated the involvement of PAR2 in the interleukin (IL)-8 production by Helicobacter pylori-infected gastric epithelial cells. Methods and Results: Human gastric epithelial cells, MKN45 cells, were used. The expression of PAR2 was assessed by real-time PCR and immunocytochemistry, and IL-8 protein was measured by an enzyme-linked immunosorbent assay. PAR2 mRNA and protein were constitutively expressed on unstimulated MKN45 cells. The treatment of cells with H. pylori resulted in a significant increase in PAR2 expression. In addition, trypsin (a natural PAR2 agonist), SLIGKV amide (a synthetic PAR2 agonist), H. pylori live bacteria or H. pylori culture supernatant significantly induced IL-8 production from MKN45 cells. H. pylori-induced IL-8 production was inhibited by nafamostat mesilate (a serine protease inhibitor), neutralizing antibody to PAR2 and in PAR2-deficient cells treated with siRNA. Conclusions: These results reveal that H. pylori-derived protease activates gastric epithelial cells to produce inflammatory cytokines through PAR2, suggesting an important role for PAR2 in the modulation of gastric inflammation associated with H. pylori. Copyright © 2008 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

ASPP2 regulates cell polarity and cell-cell adhesion by binding to, and co-localizing with PAR3 at tight junctions. Here we show a novel role of ASPP2 in suppressing gastric cancer (GC) invasiveness. Immunoprecipitation and immunofluorescence analyses showed that ASPP2 promoted the recruitment of PAR3 to cell-cell junctions in GC cells. Diminished expression of ASPP2 and loss of junctional PAR3 localization were significantly associated with diffuse-type histology, deeper invasion depth, positive peritoneal dissemination and worse prognosis in primary GC. ASPP2 suppressed migration and invasion of GC cells in vitro and peritoneal dissemination of GC cells in vivo in a mouse model. ASPP2 suppressed epithelial-mesenchymal transition (EMT) induced by TGF-β1-Smad2/3 signaling in GC cells through suppression of the degradation of Smad7, a negative regulator of TGF-β1-Smad2/3 signaling, by interacting with the E3 ubiquitin ligase ITCH. In conclusion, ASPP2 suppresses invasion, peritoneal dissemination and TGF-β1-induced EMT by inhibiting Smad7 degradation mediated by ITCH. [ABSTRACT FROM AUTHOR]