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

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.

Recognition of endoscopic diagnosis in differentiated-type early gastric cancer by flexible spectral imaging color enhancement with indigo carmine.

BACKGROUND/AIMS: To evaluate the usefulness of flexible spectral imaging color enhancement with indigo carmine (I-FICE) in early gastric cancer (EGC) demarcation. METHODS: The study participants were 29 patients with differentiated-type EGC. The endoscope was fixed and images of the same area of EGC demarcations in each lesion were obtained using four different methods (WLE, flexible spectral imaging color enhancement (FICE), CE, and I-FICE). FICE mode at R 550 nm (Gain: 2), G 500 nm (Gain: 4), and B 470 nm (Gain: 4) was used. Four endoscopists ranked the images obtained by each method on the basis of the ease of recognition of demarcation using a 4-point system. We calculated the standard deviation of pixel values based on L*, a*, and b* color spaces in the demarcation region (Lab-SD score). RESULTS: The median ranking score for I-FICE images was significantly higher than that obtained from the other methods. Further, the average Lab-SD score was significantly higher for I-FICE images than for images obtained by the other methods. There was a good correlation between the ranking score and Lab-SD score. CONCLUSION: EGC demarcations were most easily recognized both subjectively and objectively using I-FICE image, followed by CE, FICE and WLE images.

Impaired gastric ulcer healing in diabetic mice: role of methylglyoxal.

Methylglyoxal is a reactive dicarbonyl compound produced from cellular glycolytic intermediates that reacts non-enzymatically with proteins to form products such as argpyrimidine at arginine residue. The aim of the present study was to investigate the role of methylglyoxal in the delayed healing of gastric ulcer in diabetes, and to identify the methylglyoxal-modified proteins as a target molecule of this modification. Using male C57BL/6 mice, diabetes was induced by a single i.p. injection of streptozotocin and gastric ulcers were produced by the focal application of 40% of acetic acid to the serosal surface of the stomach. In order to evaluate the effect of OPB-9195, an inhibitor of methylglyoxal modification, on gastric ulcer healing, mice were given orally OPB-9195 (30 mg/kg) twice daily for 14 days, one week before and after the injection of streptozotocin. The area of gastric ulcer on day 7 was significantly increased in diabetic mice compared to non-diabetic mice, indicating delayed ulcer healing. This increase in ulcer area in diabetic mice was significantly reversed by the treatment with OPB-9195 without affecting blood glucose levels. Proteomics analysis showed the methylglyoxal-modification of peroxiredoxin 6 proteins in the diabetic gastric mucosa around gastric ulcer, and this modification was markedly inhibited by the treatment with OPB-9195. In conclusion, the present study suggests a link of increased methylglyoxal modification of proteins including peroxiredoxin 6 to the delayed gastric ulcer healing in diabetes, and also shows the therapeutic potential of the inhibitor of methylglyoxal modification for the treatment of diabetic gastric ulcers.

Inhibition of Bach1 ameliorates indomethacin-induced intestinal injury in mice.

BTB and CNC homolog 1 (Bach1) is a transcriptional repressor of heme oxygenase-1 (HO-1). It plays an important role in the feedback regulation of HO-1 expression, which protects cells from various insults including oxidative stress and inflammatory cytokines. However, the role of Bach1 in intestinal inflammation remains unclear. In this study, the role of Bach1 in intestinal mucosal injury was elucidated using 8-week-old female C57BL/6 (wild-type) and homozygous Bach1-deficient C57BL/6 mice. Intestinal mucosal injuries induced by a single subcutaneous administration of indomethacin were evaluated macroscopically, histologically, and biochemically. Mucosal protein content and chemokine mRNA levels were determined by real-time PCR. Our results showed that the indomethacin-induced intestinal injury was remarkably improved in Bach1-deficient mice. Histological examination showed that the area of injured lesion was decreased in Bach1-deficient mice compared to wild-type mice. Administration of indomethacin induced expression of inflammatory chemokines such as KC, MIP1alpha and MCP1, which was suppressed in Bach1-deficient mice. Myeloperoxidase activity in the intestinal mucosa was also significantly decreased in Bach1-deficient mice. Additionally, Bach1 deficiency enhanced immunopositivity of HO-1 in the intestinal mucosa after indomethacin administration. Disruption of the Bach1 gene thus caused inhibition of mucosal injury, indicating that inhibition of Bach1 may be a novel therapeutic strategy for treating indomethacin-induced intestinal injury.

Hyperthermia ameliorates 2,4,6-trinitrobenzene sulphonic acid-induced colitis in rats: the role of heat shock proteins.

PURPOSE: Hyperthermia is known to protect against cellular injury through the expression of heat shock proteins. In this study, the therapeutic effects of hyperthermia on experimental colitis in the rat were evaluated. MATERIALS AND METHODS: Male Wistar rats were given a single intracolonic injection of 2,4,6-trinitrobenzene sulphonic acid (TNBS). Hyperthermia was induced in anesthetized rats by placing them in a temperature-controlled water bath. We started the hyperthermic treatment on the day after the enema. The severity of colitis was evaluated pathologically, and the activities of tissue myeloperoxidase were measured 6 days after the induction of colitis. Furthermore, cytokines, and hyperthermia-induced heat shock proteins in colonic mucosa were detected by enzyme-linked immunosorbent assay and Western blotting. We also investigated the effects of geranylgeranylacetone and zinc protoporphyrin IX on the therapeutic effect of hyperthermia. RESULTS: Hyperthermia significantly improved the macroscopic scores of colitis. The TNBS-induced increases in the activities of myeloperoxidase in the colonic tissue were blunted significantly in hyperthermia-treated animals. Furthermore, hyperthermia attenuated increases in cytokine-induced neutrophil chemoattractants-1 and tumor necrosis factor-alpha in the colon. Furthermore, hyperthermia induced the production of heat shock proteins in rat colonic mucosa, and the combination of geranylgeranylacetone with hyperthermia further induced the heat shock protein HSP70, which resulted in further improvement of TNBS-induced colitis. On the other hand, the combination of zinc protoporphyrin IX with hyperthermia attenuated the therapeutic effect of hyperthermia. CONCLUSIONS: Hyperthermia ameliorates TNBS-induced colitis in rats through the expression of HSP70 and HO-1. It is postulated that hyperthermia may be useful for the treatment of inflammatory bowel diseases.

Interleukin-8 production via protease-activated receptor 2 in human esophageal epithelial cells.

Interaction between proteases and protease-activated receptor (PAR) 2 has been proposed to mediate inflammatory and immune response in the gastrointestinal tract. Recently, increase in interleukin (IL)-8 in the esophageal mucosa has been associated with the pathogenesis of esophagitis induced by reflux of gastric acids, bile acids or trypsin. The aims of the present study were to determine PAR2 expression in normal human esophageal epithelial cells (HEEC) and to evaluate the mediation of IL-8 production by trypsin-PAR2 interaction in HEEC. Reverse transcription polymerase chain reaction (RT-PCR) and Western blot analysis revealed that PAR2 mRNA and protein were constitutively expressed in HEEC without upregulation by the stimulation with tumor necrosis factor alpha or trypsin. IL-8 was produced in a dose-dependent fashion when cells were stimulated with a PAR2 agonist such as trypsin or SLIGKV-amide. Blocking antibody to PAR2, camostat mesilate (a trypsin inhibitor), p-38 mitogen-activated protein kinase (MAPK) inhibitors or ERK1/2 inhibitors reduced IL-8 production from trypsin-stimulated HEEC. Mutation of the NFkappaB-, AP-1- and NF-IL-6-binding site on the IL-8 gene promoter abrogated the induction of luciferase activities stimulated with trypsin by 100, 80 and 50%, respectively. These results indicate that PAR2 activation in HEEC by trypsin induces NFkappaB- and AP-1-dependent IL-8 production in association with activation of p38 MAPK and ERK1/2, suggesting that esophageal inflammation may be induced by PAR2 activation via reflux of trypsin.

Molecular mechanisms involved in anti-inflammatory effects of proton pump inhibitors.

OBJECTIVE: Interleukin (IL)-8 has been reported to participate in neutrophil infiltration in Helicobacter pylori (H. pylori)-induced gastritis in humans. In this study, we investigated the anti-inflammatory actions beyond the suppression of acid secretion by proton pump inhibitors (PPI), such as omeprazole and lansoprazole, on IL-8 production by gastric epithelial cells (MKN45) and human umbilical vein endothelial cells (HUVEC) and on the transendothelial migration of polymorphonuclear neutrophils (PMN). MATERIALS AND METHODS: MKN45 and HUVEC were stimulated with H. pylori water extract (HPE) and IL-1beta, respectively, and nuclear factor kappa B (NFkappaB) activation and subsequent IL-8 production was assessed in the absence or presence of PPI. We also assessed the effect of PPI on IL-8-induced PMN transendothelial migration and on the alteration of cytoplasmic calcium concentration in formyl-methionyl-leucyl-phenylalanine (fMLP)-stimulated PMN. RESULTS: HPE and IL-1beta induced a significant increase in IL-8 production by MKN45 and HUVEC, respectively, along with NFkappaB activation, which was significantly inhibited by PPI. PPI also inhibited the IL-8-induced transendothelial migration of PMN and the fMLP-induced cytosolic calcium increase in PMN. CONCLUSIONS: PPI attenuate PMN-dependent gastric mucosal inflammation partly by interfering with NFkappaB activation in vascular endothelial cells and gastric epithelial cells, and partly by modulating the calcium concentration of PMN.

Heme oxygenase-1 (Hsp32) is involved in the protection of small intestine by whole body mild hyperthermia from ischemia/reperfusion injury in rat.

AIM: The aim of the present study was to explore whether heme oxygenase-1 (HO-1) is involved in the hyperthermia-provided protection of the small intestine from ischemia/reperfusion injury in rats. METHODS: Intestinal damage was induced in male Sprague-Dawley rats by clamping both the superior mesenteric artery and the celiac trunk for 30 min, followed by reperfusion. Whole-body hyperthermia was induced in anesthetized rats by placement in a temperature-controlled water bath. Whole-body hyperthermia to a core temperature of 42-43 degrees C for 15 min was followed by passive cooling. We started the hyperthermic treatment 6 h before the vascular clamping. The severity of the mucosal injury was evaluated by several biochemical markers and histological findings. Hyperthermia-induced heat-shock proteins were detected by Western blotting. We also investigated the effect of zinc protoporphyrin IX (an HO-1 inhibitor) on the protective effect of hyperthermia. RESULTS: The rats, which were killed after ischemia/reperfusion, had severe intestinal inflammation. Hyperthermia significantly induced the production of Hsp70 and HO-1 in intestinal mucosa and significantly reduced ischemia/reperfusion-induced mucosal injury. The combination of zinc protoporphyrin IX with hyperthermia extinguished the protective effects of hyperthermia on ischemia/reperfusion injury. CONCLUSION: Hyperthermia protects against ischemia/reperfusion injury in rat small intestine through the expression of heat-shock proteins, especially HO-1.

Ubiquitin-proteasome inhibitor enhances tumour necrosis factor-alpha-induced apoptosis in rat gastric epithelial cells.

BACKGROUND: Tumour necrosis factor (TNF-alpha) is a candidate factor for involvement in inflammation-mediated gastric mucosal injury. However, the effect of this cytokine on gastric epithelial cells has been poorly investigated. In the present study, we examined whether gastric epithelial cells are resistant to TNF-alpha-induced apoptosis, and whether this resistance is related to ubiquitin-proteasome-associated nuclear factor-kappaB (NF-kappaB) activation. METHODS: The rat gastric mucosal cell line RGM-1 was grown in DMEM/F12 medium supplemented with 10% FCS. Confluent monolayers of cells were pretreated or not for 60 min with PSI, a peptide aldehyde known to specifically inhibit the chymotrypsin-like activity of 26S proteasome. Cells were subsequently stimulated with recombinant rat TNF-alpha and their viability was determined by WST-1 assay. Apoptosis was confirmed by fluorescence microscopy after staining with Hoechst 33342 and propidium iodide, and DNA fragmentation was determined by flow cytometry using an APO-BRDU kit. IkappaB-alpha and the p65 binding subunit of NF-kappaB were detected by Western blots. RESULTS: Twenty-four-hour incubation with TNF-alpha alone or PSI alone did not affect the cell viability of RGM-1 cells. Pretreatment with PSI significantly enhanced the level of apoptosis induced by TNF-alpha. In RGM-1 cells treated with TNF-alpha, cytoplasmic IkappaB-alpha decreased and p65 in nuclear extracts increased markedly 30 min after cytokine stimulation. Pretreatment with PSI at 12.5 micromol/L blocked these TNF-alpha-induced changes. CONCLUSION: PSI enhances TNF-alpha-induced apoptosis through inhibition of NF-kappaB activation in RGM-1 cells.

The inducible nitric oxide synthase inhibitor ONO-1714 blunts dextran sulfate sodium colitis in mice.

In mice with acute dextran sulfate sodium colitis, we examined the effect of inducible nitric oxide synthase inhibition by (1S,5S,6R,7R)-7chloro-3-amino-5methyl-2-azabicyclo[4.1.0]heptane hydrochloride (ONO-1714) on colonic biochemistry, injury, and inflammation. Colonic luminal nitrate and nitrite were measured by the Griess reaction; inducible nitric oxide synthase messenger RNA expression by reverse transcription-polymerase chain reaction; and nitrotyrosine by immunohistochemistry. Mice with colitis showed increases in nitrate and nitrite, inducible nitric oxide synthase messenger RNA, and numbers of cells staining for nitrotyrosine. Colonic inflammation was severe. ONO-1714 inhibited increases in nitrate and nitrite and numbers of nitrotyrosine-positive cells; injury and inflammation also were reduced. Dextran sulfate sodium-induced increases in thiobarbituric acid-reactive substances, a lipid peroxidation marker, were blunted by ONO-1714, which also inhibited increases in mucosal inflammatory cytokines. Nitric oxide produced by inducible nitric oxide synthase may contribute to colonic inflammation by nitrosation, oxidative damage, and enhanced inflammatory cytokines.

Telomerase activity and expression of telomerase RNA component and catalytic subunits in precancerous and cancerous colorectal lesions.

To investigate the role of telomerase activity in colorectal adenoma-carcinomas, telomerase activity, human telomerase RNA component (hTERC) and human telomerase reverse transcriptase (hTERT) mRNA were quantitatively analyzed in human cancerous and precancerous colorectal tissues. Sixty-six colorectal tumor specimens, including 10 invasive carcinomas, 6 mucosal carcinomas and 50 adenomas were evaluated. Ten specimens of normal tissue were also included in the study. Telomerase activity was assayed by semiquantitative fluorescence using the TRAP-eze(TM) telomerase detection kit. Analysis of the expression of each telomerase subunit gene was performed by real-time PCR amplification. There was a positive correlation between histological atypia and telomerase activity (rho = 0.700, p < 0.0001), hTERT mRNA expression (rho = 0.603, p < 0.0001), and hTERC expression (rho = 0.290, p < 0.05). There was also a positive correlation between the levels of hTERT mRNA and telomerase activity (r = 0.455, p < 0.01). Significant differences in the levels of hTERT mRNA were shown between normal tissues and the adenomas (p < 0.05) and between the mucosal carcinomas and invasive carcinomas (p < 0.05). The values of hTERC expression in neoplastic tissues were significantly higher than in the normal tissues; however, there were no significant differences between the adenomas and the carcinomas. In summary, although upregulation of hTERC expression is an early event in adenoma development, hTERT mRNA expression is gradually upregulated during the adenoma-carcinoma sequence and may be a rate-limiting determinant of telomerase activity.

Postlaminectomy ossified extradural pseudocyst. Case report.

A large ossified spurious meningocele accompanied by recurrent lumbar disc herniation occurred 7 years after posterior intervention for laminectomy and discectomy in a 53-year-old man. The cyst wall, histologically composed of mature bone tissue, was sparsely covered with connective tissue and lined with fibrocyte- or fibroblast-like cells on the inside. The ossified pseudocyst was presumed to have originated from a minute defect in the dura mater which occurred at the time of the first operation.