Physiological healing of chronic gastric ulcer is not impaired by the hydrogen sulphide (H2S)-releasing derivative of acetylsalicylic acid (ATB-340): functional and proteomic approaches.

Gastric ulcers affect approx. 10% of population. Non-steroidal anti-inflammatory drugs (NSAIDs), including acetylsalicylic acid (ASA) predispose to or impair the physiologically complex healing of pre-existing ulcers. Since H2S is an endogenous cytoprotective molecule, we hypothesized that new H2S-releasing ASA-derivative (ATB-340) could overcome pathological impact of NSAIDs on GI regeneration.Clinically translational gastric ulcers were induced in Wistar rats using state-of-the-art microsurgical model employing serosal application of acetic acid. This was followed by 9 days long i.g. daily treatment with vehicle, ATB-340 (6-24 mg/kg) or equimolar ASA doses (4-14 mg/kg). Ulcer area was assessed macro- and microscopically. Prostaglandin (PG)E2  levels, indicating pharmacological activity of NSAIDs and 8-hydroxyguanozine content, reflecting nucleic acids oxidation in serum/gastric mucosa, were determined by ELISA. Qualitative and/or quantitative pathway-specific alterations at the ulcer margin were evaluated using real-time PCR and mass spectrometry-based proteomics.ASA, unlike ATB-340, dose-dependently delayed/impaired gastric tissue recovery, deregulating 310 proteins at the ulcer margin, including Ras signalling, wound healing or apoptosis regulators. ATB-340 maintained NSAIDs-specific cyclooxygenase-inhibiting capacity on systemic and GI level but in time-dependent manner. High dose of ATB-340 (24 mg/kg daily), but not ASA, decreased nucleic acids oxidation and upregulated anti-oxidative/anti-inflammatory heme oxygenase-1, 24-dehydrocholesterol reductase or suppressor of cytokine signalling (SOCS3) at the ulcer margin.Thus, ASA impairs the physiological healing of pre-existing gastric ulcers, inducing the extensive molecularly functional and proteomic alterations at the wound margin. H2S-releasing ATB-340 maintains the target activity of NSAIDs with limited impact on gastric PGE2 signalling and physiological GI regeneration, enhancing anti-inflammatory and anti-oxidative response, and providing the pharmacological advantage.

Alkaline Phosphatase Relieves Colitis in Obese Mice Subjected to Forced Exercise via Its Anti-Inflammatory and Intestinal Microbiota-Shaping Properties.

Intestinal alkaline phosphatase (IAP) is an enzyme that plays a protective role in the gut. This study investigated the effect of IAP treatment on experimental colitis in mice subjected to forced exercise on a high-fat diet. C57BL/6 mice with TNBS colitis were fed a high-fat diet and subjected to forced treadmill exercise with or without IAP treatment. Disease activity, oxidative stress, inflammatory cytokines, and gut microbiota were assessed. Forced exercise exacerbated colitis in obese mice, as evidenced by increased disease activity index (DAI), oxidative stress markers, and proinflammatory adipokines and cytokines. IAP treatment significantly reduced these effects and promoted the expression of barrier proteins in the colonic mucosa. Additionally, IAP treatment altered the gut microbiota composition, favoring beneficial Verrucomicrobiota and reducing pathogenic Clostridia and Odoribacter. IAP treatment ameliorates the worsening effect of forced exercise on murine colitis by attenuating oxidative stress, downregulating proinflammatory biomarkers, and modulating the gut microbiota. IAP warrants further investigation as a potential therapeutic strategy for ulcerative colitis.

Hydrogen Sulfide-Releasing Indomethacin-Derivative (ATB-344) Prevents the Development of Oxidative Gastric Mucosal Injuries.

Hydrogen sulfide (H2S) emerged recently as an anti-oxidative signaling molecule that contributes to gastrointestinal (GI) mucosal defense and repair. Indomethacin belongs to the class of non-steroidal anti-inflammatory drugs (NSAIDs) and is used as an effective intervention in the treatment of gout- or osteoarthritis-related inflammation. However, its clinical use is strongly limited since indomethacin inhibits gastric mucosal prostaglandin (PG) biosynthesis, predisposing to or even inducing ulcerogenesis. The H2S moiety was shown to decrease the GI toxicity of some NSAIDs. However, the GI safety and anti-oxidative effect of a novel H2S-releasing indomethacin derivative (ATB-344) remain unexplored. Thus, we aimed here to compare the impact of ATB-344 and classic indomethacin on gastric mucosal integrity and their ability to counteract the development of oxidative gastric mucosal injuries. Wistar rats were pretreated intragastrically (i.g.) with vehicle, ATB-344 (7-28 mg/kg i.g.), or indomethacin (5-20 mg/kg i.g.). Next, animals were exposed to microsurgical gastric ischemia-reperfusion (I/R). Gastric damage was assessed micro- and macroscopically. The volatile H2S level was assessed in the gastric mucosa using the modified methylene blue method. Serum and gastric mucosal PGE2 and 8-hydroxyguanozine (8-OHG) concentrations were evaluated by ELISA. Molecular alterations for gastric mucosal barrier-specific targets such as cyclooxygenase-1 (COX)-1, COX-2, heme oxygenase-1 (HMOX)-1, HMOX-2, superoxide dismutase-1 (SOD)-1, SOD-2, hypoxia inducible factor (HIF)-1α, xanthine oxidase (XDH), suppressor of cytokine signaling 3 (SOCS3), CCAAT enhancer binding protein (C/EBP), annexin A1 (ANXA1), interleukin 1 beta (IL-1ß), interleukin 1 receptor type I (IL-1R1), interleukin 1 receptor type II (IL-1R2), inducible nitric oxide synthase (iNOS), tumor necrosis factor receptor 2 (TNFR2), or H2S-producing enzymes, cystathionine γ-lyase (CTH), cystathionine ß-synthase (CBS), or 3-mercaptopyruvate sulfur transferase (MPST), were assessed at the mRNA level by real-time PCR. ATB-344 (7 mg/kg i.g.) reduced the area of gastric I/R injuries in contrast to an equimolar dose of indomethacin. ATB-344 increased gastric H2S production, did not affect gastric mucosal PGE2 content, prevented RNA oxidation, and maintained or enhanced the expression of oxidation-sensitive HMOX-1 and SOD-2 in line with decreased IL-1ß and XDH. We conclude that due to the H2S-releasing ability, i.g., treatment with ATB-344 not only exerts dose-dependent GI safety but even enhances gastric mucosal barrier capacity to counteract acute oxidative injury development when applied at a low dose of 7 mg/kg, in contrast to classic indomethacin. ATB-344 (7 mg/kg) inhibited COX activity on a systemic level but did not affect cytoprotective PGE2 content in the gastric mucosa and, as a result, evoked gastroprotection against oxidative damage.

Mitochondria-targeted hydrogen sulfide donors versus acute oxidative gastric mucosal injury.

Hydrogen sulfide (H2S) as a gaseous molecule prevents gastrointestinal (GI)-tract against various injuries. This study aimed to evaluate for the first time the detailed molecular mechanism of mitochondria-targeting H2S-prodrugs, AP39 and RT01 in gastroprotection against ischemia/reperfusion (I/R)-induced lesions. Wistar rats exposed to I/R were pretreated i.g. with vehicle, AP39 (0.004-2 mg/kg), RT01 (0.1 mg/kg), or with AP219 (0.1 mg/kg) as structural control without ability to release H2S. AP39 was also administered with mTOR1 inhibitor, rapamycin (1 mg/kg i.g.). Gastric damage area was assessed micro-/macroscopically, gastric blood flow (GBF) by laser flowmetry, mRNA level of HIF-1α, GPx, SOD1, SOD2, annexin-A1, SOCS3, IL-1RA, IL-1ß, IL-1R1, IL-1R2, TNFR2, iNOS by real-time PCR. Gastric mucosal and/or serum content of IL-1ß, IL-4, IL-5, IL-10, G-CSF, M-CSF, VEGFA, GRO, RANTES, MIP-1α, MCP1, TNF-α, TIMP1, FABP3, GST-α, STAT3/5 and phosphorylation of mTOR, NF-κB, ERK, Akt was evaluated by microbeads-fluorescent assay. Mitochondrial complexes activities were measured biochemically. RNA damage was assessed as 8-OHG by ELISA. AP39 and RT01 reduced micro-/macroscopic gastric I/R-injury increasing GBF. AP39-gastroprotection was accompanied by maintained activity of mitochondrial complexes, prevented RNA oxidation and enhanced mRNA/protein expression of SOCS3, IL-1RA, annexin-A1, GST-α, HIF-1α. Rapamycin reversed AP-39-gastroprotection. AP39-gastroprotection was followed by decreased NF-κB, ERK, IL-1ß and enhanced Akt and mTOR proteins phosphorylation. AP39-prevented gastric mucosal damage caused by I/R-injury, partly by mitochondrial complex activity maintenance. AP39-mediated attenuation of gastric mucosal oxidation, hypoxia and inflammation involved mTOR1 and Akt pathways activity and modulation of HIF-1α, GST-α, SOCS3, IL1RA and TIMP1 molecular interplay.

The Combination of Intestinal Alkaline Phosphatase Treatment with Moderate Physical Activity Alleviates the Severity of Experimental Colitis in Obese Mice via Modulation of Gut Microbiota, Attenuation of Proinflammatory Cytokines, Oxidative Stress Biomarkers and DNA Oxidative Damage in Colonic Mucosa.

Inflammatory bowel diseases (IBD) are commonly considered as Crohn's disease and ulcerative colitis, but the possibility that the alterations in gut microbiota and oxidative stress may affect the course of experimental colitis in obese physically exercising mice treated with the intestinal alkaline phosphatase (IAP) has been little elucidated. Mice fed a high-fat-diet (HFD) or normal diet (ND) for 14 weeks were randomly assigned to exercise on spinning wheels (SW) for 7 weeks and treated with IAP followed by intrarectal administration of TNBS. The disease activity index (DAI), grip muscle strength test, oxidative stress biomarkers (MDA, SOD, GSH), DNA damage (8-OHdG), the plasma levels of cytokines IL-2, IL-6, IL-10, IL-12p70, IL-17a, TNF-α, MCP-1 and leptin were assessed, and the stool composition of the intestinal microbiota was determined by next generation sequencing (NGS). The TNBS-induced colitis was worsened in obese sedentary mice as manifested by severe colonic damage, an increase in DAI, oxidative stress biomarkers, DNA damage and decreased muscle strength. The longer running distance and weight loss was observed in mice given IAP or subjected to IAP + SW compared to sedentary ones. Less heterogeneous microbial composition was noticed in sedentary obese colitis mice and this effect disappeared in IAP + SW mice. Absence of Alistipes, lower proportion of Turicibacter, Proteobacteria and Faecalibacterium, an increase in Firmicutes and Clostridium, a decrease in oxidative stress biomarkers, 8-OHdG content and proinflammatory cytokines were observed in IAP + SW mice. IAP supplementation in combination with moderate physical activity attenuates the severity of murine colitis complicated by obesity through a mechanism involving the downregulation of the intestinal cytokine/chemokine network and oxidative stress, the modulation of the gut microbiota and an improvement of muscle strength.

Organic carbon monoxide prodrug, BW-CO-111, in protection against chemically-induced gastric mucosal damage.

Metal-based carbon monoxide (CO)-releasing molecules have been shown to exert anti-inflammatory and anti-oxidative properties maintaining gastric mucosal integrity. We are interested in further development of metal-free CO-based therapeutics for oral administration. Thus, we examine the protective effect of representative CO prodrug, BW-CO-111, in rat models of gastric damage induced by necrotic ethanol or aspirin, a representative non-steroidal anti-inflammatory drug. Treatment effectiveness was assessed by measuring the microscopic/macroscopic gastric damage area and gastric blood flow by laser flowmetry. Gastric mucosal mRNA and/or protein expressions of HMOX1, HMOX2, nuclear factor erythroid 2-related factor 2, COX1, COX2, iNos, Anxa1 and serum contents of TGFB1, TGFB2, IL1B, IL2, IL4, IL5, IL6, IL10, IL12, tumor necrosis factor α, interferon γ, and GM-CSF were determined. CO content in gastric mucosa was assessed by gas chromatography. Pretreatment with BW-CO-111 (0.1 mg/kg, i.g.) increased gastric mucosal content of CO and reduced gastric lesions area in both models followed by increased GBF. These protective effects of the CO prodrug were supported by changes in expressions of molecular biomarkers. However, because the pathomechanisms of gastric damage differ between topical administration of ethanol and aspirin, the possible protective and anti-inflammatory mechanisms of BW-CO-111 may be somewhat different in these models.

Role of Obesity, Mesenteric Adipose Tissue, and Adipokines in Inflammatory Bowel Diseases.

Inflammatory bowel diseases (IBDs) are a group of disorders which include ulcerative colitis and Crohn's disease. Obesity is becoming increasingly more common among patients with inflammatory bowel disease and plays a role in the development and course of the disease. This is especially true in the case of Crohn's disease. The recent results indicate a special role of visceral adipose tissue and particularly mesenteric adipose tissue, also known as "creeping fat", in pathomechanism, leading to intestinal inflammation. The involvement of altered adipocyte function and the deregulated production of adipokines, such as leptin and adiponectin, has been suggested in pathogenesis of IBD. In this review, we discuss the epidemiology and pathophysiology of obesity in IBD, the influence of a Western diet on the course of Crohn's disease and colitis in IBD patients and animal's models, and the potential role of adipokines in these disorders. Since altered body composition, decrease of skeletal muscle mass, and development of pathologically changed mesenteric white adipose tissue are well-known features of IBD and especially of Crohn's disease, we discuss the possible crosstalk between adipokines and myokines released from skeletal muscle during exercise with moderate or forced intensity. The emerging role of microbiota and the antioxidative and anti-inflammatory enzymes such as intestinal alkaline phosphatase is also discussed, in order to open new avenues for the therapy against intestinal perturbations associated with IBD.

Oxidative gastric mucosal damage induced by ischemia/reperfusion and the mechanisms of its prevention by carbon monoxide-releasing tricarbonyldichlororuthenium (II) dimer.

Endogenous gaseous mediators, such as nitric oxide, hydrogen sulfide or carbon monoxide (CO) are known to exert anti-inflammatory and anti-oxidative activity due to modulation of various molecular pahtways. Therefore, we aimed to investigate if CO released from tricarbonyldichlororuthenium (II) dimer (CORM-2) prevents gastric mucosa against ischemia/reperfusion (I/R)-induced injury in male Wistar rats. Animals were pretreated i.g. With vehicle (DMSO and saline, 1:10), CORM-2 (1, 5 or 10 mg/kg) or zinc protoporphyrin IX (ZnPP, 10 mg/kg i.p.), the HMOXs inhibitor. In separate series, rats were pretreated with CORM-2 (5 mg/kg) applied in combination with glibenclamide (10 mg/kg i.g.), NG-nitro-l-arginine (L-NNA, 20 mg/kg i.p.), 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 10 mg/kg i.p.) or indomethacin (5 mg/kg i.p.). I/R-injuries were induced by clamping celiac artery for 30 min (I) followed by removal of the clamp to obtain R for 3 h. The macroscopic and microscopic area of gastric damage, mucus production and protein expression for HMOX-1/Nrf-2 was determined by planimetry, histology and immunohistochemistry, respectively. Gastric mucosal HMOX-1, HMOX-2, COX-1, COX-2, Kir6.1, Sur2, sGC-α1, sGC-α2, iNOS and eNOS mRNA expression was assessed by real-time PCR. COHb in blood and gastric mucosal CO concentration was analyzed by gas chromatography. Serum content of TGF-ß1, TGF-ß2, TGF-ß3, IL-1α, IL-1ß, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, TNF-α, IFN-γ, GM-CSF was evaluated using Luminex platform. PGE2 concentration and 8-hydroxyguanozine (8-OHG) concentration in gastric mucosa was determined by ELISA. Exposure to I/R induced extensive hemorrhagic erosions in gastric mucosa pretreated with vehicle as compared with intact rats and the area of this gastric damage was reduced by pretreatment with CORM-2 (5 mg/kg i.g.). This effect of CO donor was accompanied by the increased PGE2 content and a significant decrease in 8-OHG and expression of pro- and anti-inflammatory markers mRNA and proteins. Concurrent treatment of CORM-2 with glibenclamide, L-NNA, ODQ but not with indomethacin significantly increased the area of I/R-induced injury and significantly decreased GBF as compared with the group treated with CORM-2 alone. We conclude that CO releasing CORM-2 prevents gastric mucosal oxidative damage induced by I/R improving GBF, decreasing DNA oxidation and inflammatory response on systemic level. This CO-gastroprotection is mediated by the activity of sGC, NOS and K-ATP channels. CO delivered from its donor maintained physiological gastric mucosal PGE2 concentration but the involvement of endogenous COX in beneficial activity of this gaseous mediator at least in this model is questionable.

Effect of Forced Physical Activity on the Severity of Experimental Colitis in Normal Weight and Obese Mice. Involvement of Oxidative Stress and Proinflammatory Biomarkers.

Inflammatory bowel diseases are a heterogeneous group of disorders represented by two major phenotypic forms, Crohn's disease and ulcerative colitis. Cross talk between adipokines and myokines, as well as changes in intestinal microcirculation, was proposed in pathogenesis of these disorders. C57BL/6 male mice were fed ad libitum for 12 weeks a standard (SD) or high-fat diet (HFD). After the adaptation period, two groups of animals fed SD or HFD were subjected to 6 weeks of the forced treadmill exercise and the experimental colitis was induced in both groups of sedentary and exercising mice fed SD and HFD by intra-colonic administration of 2,4,6-trinitrobenzenesulfonic acid. The disease activity index (DAI), colonic blood flow (CBF), the weight of animals, caloric intake, the mesenteric fad pad, the colonic oxidative stress markers malondialdehyde (MDA), reduced glutathione (GSH), and superoxide dismutase (SOD) activity and intestinal expression and protein content of proinflammatory markers were evaluated. Macroscopic and microscopic colitis in sedentary SD mice was accompanied by a significant fall in CBF and exacerbated in those fed a HFD. The contents of MDA, GSH, and SOD activity were significantly increased in both SD and HFD fed mice with treadmill exercise as compared with sedentary mice. In sedentary HFD mice a significant increase in the intestinal oxidative stress parameters and mucosal expression of IL-1ß, TNF-α, IL-17, IFNγ, IL-6, and IL-10 protein were observed and these effects were aggravated in mice subjected to forced treadmill exercise. The mucosal expression of mRNA for TNF-α, IL-1ß, iNOS, COX-2, SOD-1, SOD-2, GPx mRNAs, and the hypoxia inducible factor (HIF)-1α protein expression were upregulated in colonic mucosa of treadmill exercising HFD mice with colitis compared with those without exercise. We conclude that forced treadmill running exacerbates the severity of colonic damage in obese mice due to a fall in colonic microcirculation, an increase in oxidative stress, and the rise in expression and activity of proinflammatory biomarkers.

Curcumin: A Potent Protectant against Esophageal and Gastric Disorders.

Turmeric obtained from the rhizomes of Curcuma longa has been used in the prevention and treatment of many diseases since the ancient times. Curcumin is the principal polyphenol isolated from turmeric, which exhibits anti-inflammatory, antioxidant, antiapoptotic, antitumor, and antimetastatic activities. The existing evidence indicates that curcumin can exert a wide range of beneficial pleiotropic properties in the gastrointestinal tract, such as protection against reflux esophagitis, Barrett's esophagus, and gastric mucosal damage induced by nonsteroidal anti-inflammatory drugs (NSAIDs) and necrotizing agents. The role of curcumin as an adjuvant in the treatment of a Helicobacter pylori infection in experimental animals and humans has recently been proposed. The evidence that this turmeric derivative inhibits the invasion and proliferation of gastric cancer cells is encouraging and warrants further experimental and clinical studies with newer formulations to support the inclusion of curcumin in cancer therapy regimens. This review was designed to analyze the existing data from in vitro and in vivo animal and human studies in order to highlight the mechanisms of therapeutic efficacy of curcumin in the protection and ulcer healing of the upper gastrointestinal tract, with a major focus on addressing the protection of the esophagus and stomach by this emerging compound.

Time-dependent course of gastric ulcer healing and molecular markers profile modulated by increased gastric mucosal content of carbon monoxide released from its pharmacological donor.

BACKGROUND AND PURPOSE: Besides hydrogen sulfide (H2S) and nitric oxide (NO), carbon monoxide (CO) contributes to the maintenance of gastric mucosal integrity. We investigated increased CO bioavailability effects on time-dependent dynamics of gastric ulcer healing mediated by particular growth factors, anti-inflammatory and molecular pathways. EXPERIMENTAL APPROACH: Wistar rats with gastric ulcers induced by serosal acetic acid application (day 0) were treated i.g. throughout 3, 6 or 14 days with vehicle or CO-releasing tricarbonyldichlororuthenium (II) dimer (CORM-2, 2.5 mg/kg). Gross and microscopic alterations in gastric ulcer size and gastric blood flow (GBF) at ulcer margin were determined by planimetry, histology and laser flowmetry, respectively. Gastric mRNA/protein expressions of platelet derived growth factors (PDGFA-D), insulin-like growth factor (IGF-1), epidermal growth factor (EGF), hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGFA) and their receptors, heme oxygenases (HMOX), nuclear factor (erythroid-derived 2)-like 2 (Nrf-2), cyclooxygenase (COX-2), hypoxia inducible factor (HIF)-1α, anti-inflammatory annexin-1 and transforming growth factor (TGF-ß1) were assessed by real-time PCR or Western blot. TGF-ß1-3 and IL-10 plasma concentration were measured using Luminex platform. Prostaglandin E2 content at ulcer margin was assessed by ELISA. KEY RESULTS: CORM-2 decreased ulcer area and increased GBF after 6 and 14 days of treatment comparing to vehicle. CO donor upregulated HGF, HGFr, VEGFR1, VEGFR2, TGF-ß1, annexin-1 and maintained increased IGF-1, PDGFC and EGF expression at various time-intervals of ulcer healing. TGF-ß3 and IL-10 plasma concentration were significantly increased after COMR-2 vs. vehicle. CONCLUSIONS: CO time-dependently accelerates gastric ulcer healing and raises GBF at ulcer margin by mechanism involving subsequent upregulation of anti-inflammatory, growth promoting and angiogenic factors response, not observed physiologically.

Comparison of left side or right side vagotomy in the rat subjected to acute pancreatitis.

PURPOSE: We aimed to evaluate the effects of unilateral vagotomy (right-VR or left-VL) on the severity of caerulein-induced acute pancreatitis (AP). MATERIAL AND METHODS: VR or VL was done in Wistar rats 4 days before AP, except in control, sham operated group. Following 5 h administration of subcutaneous injections of caerulein, the pancreatic blood flow (PBF), serum lipase and IL-10 in caval blood samples were measured. The pancreatic specimens were taken from sacrificed rats for the assessment of MDA-4-HNE and morphology. RESULTS: PBF decreased from 310 ± 20 ml/min/100 g of tissue in control rats to 130 ± 12 units in AP (p < 0.01). VR and VL alleviated this effect to 234 ± 22 and 229 ± 26 units, respectively, (p < 0.01). There was an immense increase of serum lipase in AP, from 100 ± 7 U/L up to 5220 ± 210 U/L (p < 0.01). Only VL limited this increase to 3469 ± 300 U/L (p < 0.01). Serum IL-10 increased uniformly in AP, without any effect of preceding VR or VL. VL performed in rats subjected subsequently to AP resulted in stronger reduction of histological changes, such as pancreatic edema and leukocyte infiltration, than the above parameters in AP rats with VR. MDA+4-HNE increased from 7.5 ± 0.1 pmol/g of tissue in control group to 30.6 ± 3 units in AP group (p < 0.01). Concentration of MDA+4-HNE in pancreatic tissue achieved 16.48 ± 3 pmol/g after VR and 13.84 ± 4 pmol/g following VL. CONCLUSION: Our observation might suggest that protective effect of VL could be stronger than VR in the protection on AP. However changes of PBF seem to be similar in both groups of rats.

Alterations in Gastric Mucosal Expression of Calcitonin Gene-Related Peptides, Vanilloid Receptors, and Heme Oxygenase-1 Mediate Gastroprotective Action of Carbon Monoxide against Ethanol-Induced Gastric Mucosal Lesions.

Carbon monoxide (CO) has been reported to contribute to the maintenance of gastric mucosal integrity, gastroprotection, and ulcer healing. However, involvement of transient receptor potential vanilloid receptor type 1 (TRPV1) located on afferent sensory fibers endings and sensory neuropeptide calcitonin gene-related peptide (CGRP) in CO-mediated gastroprotection against ethanol-induced gastric damage has not been explored. Male Wistar rats with and without denervation of afferent sensory neurons induced by capsaicin (total dose 125 mg/kg within 3 days) were pretreated with vehicle, CO donor tricarbonyldichlororuthenium (II) dimer (CORM-2, 5 mg/kg i.g.), administered alone or with CGRP-α (10 µg/kg i.p.) or TRPV1 antagonist capsazepine (5 mg/kg i.g.), followed 30 min later by intragastric (i.g.) administration of 75% ethanol. The area of gastric damage and gastric blood flow (GBF) were assessed planimetrically and by laser flowmetry, respectively. Microscopic evaluation of ethanol-induced gastric lesions was performed after haematoxylin/eosin (H&E) or alcian blue/periodic acid-Schiff/alcian blue (AB/PAS) staining. Gastric mucosal mRNA fold change for heme oxygenase (HMOX)-1, HMOX-2, CGRP-α, CGRP-ß, inducible nitric oxide synthase (iNOS), endothelial (e)NOS, neuronal (n)NOS, cyclooxygenase (COX)-1, COX-2, and protein expression for HMOX-1 and TRPV1 was determined by real-time PCR or Western blot, respectively. Pretreatment with CORM-2 combined or not with CGRP reduced ethanol-induced gastric lesions and elevated GBF. Capsaicin-denervation or co-treatment with capsazepine or CGRP and CORM-2 in capsaicin-denervated animals failed to affect these beneficial effects of CO donor. In rats with intact sensory nerves, CORM-2 increased gastric mRNA level for HMOX-1 and CGRP-α. In capsaicin-denervated rats, CORM-2 increased eNOS mRNA fold change and TRPV1 protein expression while capsaicin denervation itself decreased HMOX-1 protein expression and eNOS mRNA level. We conclude that CO prevents gastric mucosa from ethanol-induced lesions due to activation of TRPV1/CGRP-α system and accompanying increase in gastric microcirculation but independently on afferent sensory nerve activity despite the stimulation of TRPV1 protein and CGRP-α mRNA expression.

Role of Helicobacter pylori infection in cancer-associated fibroblast-induced epithelial-mesenchymal transition in vitro.

BACKGROUND: Major human gastrointestinal pathogen Helicobacter pylori (H. pylori) colonizes the gastric mucosa causing inflammation and severe complications including cancer, but the involvement of fibroblasts in the pathogenesis of these disorders in H. pylori-infected stomach has been little studied. Normal stroma contains few fibroblasts, especially myofibroblasts. Their number rapidly increases in the reactive stroma surrounding inflammatory region and neoplastic tissue; however, the interaction between H. pylori and fibroblasts remains unknown. We determined the effect of coincubation of normal rat gastric fibroblasts with alive H. pylori (cagA+vacA+) and H. pylori (cagA-vacA-) strains on the differentiation of these fibroblasts into cells possessing characteristics of cancer-associated fibroblasts (CAFs) able to induce epithelial-mesenchymal transition (EMT) of normal rat gastric epithelial cells (RGM-1). MATERIALS AND METHODS: The panel of CAFs markers mRNA was analyzed in H. pylori (cagA+vacA+)-infected fibroblasts by RT-PCR. After insert coculture of differentiated fibroblasts with RGM-1 cells from 24 up to 48, 72, and 96 hours, the mRNA expression for EMT-associated genes was analyzed by RT-PCR. RESULTS: The mRNA expression for CAFs markers was significantly increased after 72 hours of infection with H. pylori (cagA+vacA+) but not H. pylori (cagA-vacA-) strain. Following coculture with CAFs, RGM-1 cells showed significant decrease in E-cadherin mRNA, and the parallel increase in the expression of Twist and Snail transcription factors mRNA was observed along with the overexpression of mRNAs for TGFßR, HGFR, FGFR, N-cadherin, vimentin, α-SMA, VEGF, and integrin-ß1. CONCLUSION: Helicobacter pylori (cagA+vacA+) strain induces differentiation of normal fibroblasts into CAFs, likely to initiate the EMT process in RGM-1 epithelial cell line.

Nitric oxide, afferent sensory nerves, and antioxidative enzymes in the mechanism of protection mediated by tricarbonyldichlororuthenium(II) dimer and sodium hydrosulfide against aspirin-induced gastric damage.

BACKGROUND: Aspirin exerts side effects within the gastrointestinal tract. Hydrogen sulfide (H2S) and carbon monoxide (CO) have been implicated in gastroprotection but the mechanism of beneficial action of these gaseous mediators against aspirin-induced damage has not been fully studied. We determined the involvement of afferent sensory neurons, calcitonin-gene-related peptide (CGRP), lipid peroxidation, and nitric oxide (NO) biosynthesis in gastroprotection of H2S-releasing NaHS and CO-releasing tricarbonyldichlororuthenium(II) dimer (CORM-2) against aspirin-induced injury. METHODS: Wistar rats with or without capsaicin-induced denervation of sensory neurons were pretreated with vehicle, CORM-2 (5 mg/kg intragastrically), or NaHS (5 mg/kg intragastrically) with or without capsazepine (5 mg/kg intragastrically) or N G-nitro-L-arginine (L-NNA, 20 mg/kg intraperitoneally). The areas of aspirin-induced lesions and gastric blood flow (GBF) were assessed by planimetry and laser flowmetry respectively. Gastric mucosal messenger RNA and/or protein expression of CGRP, heme oxygenase 1, inducible nitric oxide synthase, cyclooxygenase 2, interleukin-1ß, glutathione peroxidase 1 (GPx-1), and superoxide dismutase was determined by real-time PCR or Western blot. Malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE) content was determined by colorimetric assay. RESULTS: Aspirin caused gastric lesions, decreased GBF, and raised MDA content, but pretreatment with NaHS and CORM-2 reduced these effects. Capsaicin-induced denervation or co-treatment with capsazepine reversed the gastroprotective and vasodilatory effects of NaHS but not those of CORM-2. L-NNA reversed NaHS-induced gastroprotection and partly reduced CORM-2-induced gastroprotection. NaHS and CORM-2 decreased MDA and 4-HNE content, restoring GPx-1 protein expression. CONCLUSIONS: We conclude that H2S- but not CO-mediated gastroprotection against aspirin-induced injury involves afferent sensory nerves and partly NO activity. NaHS and CORM-2 prevented aspirin-induced gastric mucosal lipid peroxidation via restoration of microcirculation and antioxidative GPx-1 protein expression.

Mechanisms of curcumin-induced gastroprotection against ethanol-induced gastric mucosal lesions.

BACKGROUND: Curcumin, a pleiotropic substance used for centuries in traditional medicine, exhibits antioxidant, anti-inflammatory and antiproliferative efficacy against various tumours, but the role of curcumin in gastroprotection is little studied. We determined the effect of curcumin against gastric haemorrhagic lesions induced by 75% ethanol and alterations in gastric blood flow (GBF) in rats with cyclooxygenase-1 (COX-1) and COX-2 activity inhibited by indomethacin, SC-560 or rofecoxib, inhibited NO-synthase activity, capsaicin denervation and blockade of TRPV1 receptors by capsazepine. METHODS: One hour after ethanol administration, the gastric mucosal lesions were assessed by planimetry, the GBF was examined by H2 gas clearance, plasma gastrin was determined by radioimmunoassay, and the gastric mucosal mRNA expression of Cdx-2, HIF-1α, HO-1 and SOD 2 was analysed by RT-PCR. RESULTS: Curcumin, in a dose-dependent manner, reduced ethanol-induced gastric lesions and significantly increased GBF and plasma gastrin levels. Curcumin-induced protection was completely reversed by indomethacin and SC-560, and significantly attenuated by rofecoxib, L-NNA, capsaicin denervation and capsazepine. Curcumin downregulated Cdx-2 and Hif-1α mRNA expression and upregulated HO-1 and SOD 2, and these effects were reversed by L-NNA and further restored by co-treatment of L-NNA with L-arginine. CONCLUSIONS: Curcumin-induced protection against ethanol damage involves endogenous PG, NO, gastrin and CGRP released from sensory nerves due to activation of the vanilloid TRPV1 receptor. This protective effect can be attributed to the inhibition of HIF-1α and Cdx-2 expression and the activation of HO-1 and SOD 2 expression.

Cross-talk between hydrogen sulfide and carbon monoxide in the mechanism of experimental gastric ulcers healing, regulation of gastric blood flow and accompanying inflammation.

Hydrogen sulfide (H2S) and carbon monoxide (CO) exert gastroprotection against acute gastric lesions. We determined the cross-talk between H2S and CO in gastric ulcer healing process and regulation of gastric blood flow (GBF) at ulcer margin. Male Wistar rats with acetic acid-induced gastric ulcers were treated i.g. throughout 9 days with vehicle (control), NaHS (0.1-10 mg/kg) +/- zinc protoporphyrin (ZnPP, 10 mg/kg), d,l-propargylglycine (PAG, 30 mg/kg), CO-releasing CORM-2 (2.5 mg/kg) +/- PAG. GBF was assessed by laser flowmetry, ulcer area was determined by planimetry/histology. Gastric mucosal H2S production was analysed spectrophotometrically. Protein and/or mRNA expression at ulcer margin for vascular endothelial growth factor (VEGF)A, epidermal growth factor receptor (EGFr), cystathionine-γ-lyase (CSE), cystathionine-ß-synthase (CBS), 3-mercaptopyruvate sulfurtransferase (3-MST), heme oxygenases (HOs), nuclear factor (erythroid-derived 2)-like 2 (Nrf-2), cyclooxygenase (COX)-2, inducible nitric oxide synthase (iNOS), IL-1ß, TNF-α and hypoxia inducible factor (HIF)-1α were determined by real-time PCR or western blot. IL-1α, IL-1ß, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IL-13, IFN-γ, TNF-α, GM-CSF plasma concentration was assessed using Luminex platform. NaHS dose-dependently decreased ulcer area and increased GBF but ZnPP attenuated these effects. PAG decreased H2S production but failed to affect CORM-2-mediated ulcer healing and vasodilation. NaHS increased Nrf-2, EGFr, VEGFA and decreased pro-inflammatory markers expression and IL-1ß, IL-2, IL-13, TNF-α, GM-CSF plasma concentration. CORM-2 decreased IL-1ß and GM-CSF plasma levels. We conclude that NaHS accelerates gastric ulcer healing increasing microcirculation and Nrf-2, EGFr, VEGFA expression. H2S-mediated ulcer healing involves endogenous CO activity while CO does not require H2S. NaHS decreases systemic inflammation more effectively than CORM-2.

Carbon monoxide released from its pharmacological donor, tricarbonyldichlororuthenium (II) dimer, accelerates the healing of pre-existing gastric ulcers.

BACKGROUND AND PURPOSE: Carbon monoxide (CO), a gaseous mediator produced by haem oxygenases (HOs), has been shown to prevent stress-, ethanol-, aspirin- and alendronate-induced gastric damage; however, its role in gastric ulcer healing has not been fully elucidated. We investigated whether CO released from tricarbonyldichlororuthenium (II) dimer (CORM-2) can affect gastric ulcer healing and determined the mechanisms involved in this healing action. EXPERIMENTAL APPROACH: Gastric ulcers were induced in Wistar rats by serosal application of acetic acid. Animals received 9 days of treatment with RuCl3 [2.5 mg·kg-1 intragastrically (i.g.)], haemin (5 mg·kg-1 i.g.), CORM-2 (0.1-10 mg·kg-1 i.g.) administered alone or with zinc protoporphyrin IX (ZnPP, 10 mg·kg-1 i.g.), 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 5 mg·kg-1 i.g.), NG -nitro-l-arginine (l-NNA, 15 mg·kg-1 i.g.), indomethacin (5 mg·kg-1 i.g.) or glibenclamide (10 mg·kg-1 i.g.). Gastric ulcer area and gastric blood flow (GBF) were assessed planimetrically, microscopically and by laser flowmeter respectively. Gastric mRNA/protein expressions of EGF, EGF receptors, VEGFA, HOs, nuclear factor (erythroid-derived 2)-like 2 (Nrf2), COX-2, hypoxia-inducible factor (HIF)-1α and pro-inflammatory iNOS, IL-1ß and TNF-α were determined by real-time PCR or Western blots. KEY RESULTS: CORM-2 and haemin but not RuCl3 or ZnPP decreased ulcer size while increasing GBF. These effects were reduced by ODQ, indomethacin, l-NNA and glibenclamide. CORM-2 significantly decreased the expression of pro-inflammatory markers, Nrf2/HO1 and HIF-1α, and up-regulated EGF. CONCLUSIONS AND IMPLICATIONS: CO released from CORM-2 or endogenously produced by the HO1/Nrf2 pathway accelerates gastric ulcer healing via an increase in GBF, an up-regulation in EGF expression and down-regulation of the inflammatory response.

Exogenous and Endogenous Hydrogen Sulfide Protects Gastric Mucosa against the Formation and Time-Dependent Development of Ischemia/Reperfusion-Induced Acute Lesions Progressing into Deeper Ulcerations.

Hydrogen sulfide (H2S) is an endogenous mediator, synthesized from l-cysteine by cystathionine γ-lyase (CSE), cystathionine ß-synthase (CBS) or 3-mercaptopyruvate sulfurtransferase (3-MST). The mechanism(s) involved in H2S-gastroprotection against ischemia/reperfusion (I/R) lesions and their time-dependent progression into deeper gastric ulcerations have been little studied. We determined the effect of l-cysteine, H2S-releasing NaHS or slow H2S releasing compound GYY4137 on gastric blood flow (GBF) and gastric lesions induced by 30 min of I followed by 3, 6, 24 and 48 h of R. Role of endogenous prostaglandins (PGs), afferent sensory nerves releasing calcitonin gene-related peptide (CGRP), the gastric expression of hypoxia inducible factor (HIF)-1α and anti-oxidative enzymes were examined. Rats with or without capsaicin deactivation of sensory nerves were pretreated i.g. with vehicle, NaHS (18-180 µmol/kg) GYY4137 (90 µmol/kg) or l-cysteine (0.8-80 µmol/kg) alone or in combination with (1) indomethacin (14 µmol/kg i.p.), SC-560 (14 µmol/kg), celecoxib (26 µmol/kg); (2) capsazepine (13 µmol/kg i.p.); and (3) CGRP (2.5 nmol/kg i.p.). The area of I/R-induced gastric lesions and GBF were measured by planimetry and H2-gas clearance, respectively. Expression of mRNA for CSE, CBS, 3-MST, HIF-1α, glutathione peroxidase (GPx)-1, superoxide dismutase (SOD)-2 and sulfide production in gastric mucosa compromised by I/R were determined by real-time PCR and methylene blue method, respectively. NaHS and l-cysteine dose-dependently attenuated I/R-induced lesions while increasing the GBF, similarly to GYY4137 (90 µmol/kg). Capsaicin denervation and capsazepine but not COX-1 and COX-2 inhibitors reduced NaHS- and l-cysteine-induced protection and hyperemia. NaHS increased mRNA expression for SOD-2 and GPx-1 but not that for HIF-1α. NaHS which increased gastric mucosal sulfide release, prevented further progression of acute I/R injury into deeper gastric ulcers at 6, 24 and 48 h of R. We conclude that H2S-induced gastroprotection against I/R-injury is due to increase in gastric microcirculation, anti-oxidative properties and afferent sensory nerves activity but independent on endogenous prostaglandins.

Interaction between endogenous carbon monoxide and hydrogen sulfide in the mechanism of gastroprotection against acute aspirin-induced gastric damage.

Acetylsalicylic acid (ASA) is mainly recognized as painkiller or anti-inflammatory drug. However, ASA causes serious side effects towards gastrointestinal (GI) tract which limits its usefulness. Carbon monoxide (CO) and hydrogen sulfide (H2S) have been described to act as important endogenous messengers and mediators of gastroprotection but whether they can interact in gastroprotection against acute ASA-induced gastric damage remains unknown. In this study male Wistar rats were pretreated with 1) vehicle (saline, i.g.), 2) tricarbonyldichlororuthenium (II) dimer (CORM-2, 5mg/kg i.g.), 3) sodium hydrosulfide (NaHS, 5mg/kg i.g.), 4) zinc protoporphyrin (ZnPP, 10mg/kg i.p.), 5) D,L-propargylglycine (PAG, 30mg/kg i.g.), 6) ZnPP combined with NaHS, 7) PAG combined with CORM-2 or 8) 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 10mg/kg i.p.) combined with CORM-2 or NaHS and 30min later ASA was administered i.g. in a single dose of 125mg/kg. After 1h, gastric blood flow (GBF) was determined by H2 gas clearance technique and gastric lesions were assessed by planimetry and histology. CO content in gastric mucosa and COHb concentration in blood were determined by gas chromatography and H2S production was assessed in gastric mucosa using methylene blue method. Protein and/or mRNA expression for cystathionine-γ-lyase (CSE), cystathionine-ß-synthase (CBS), 3-mercaptopyruvate sulfurtransferase (3-MST), heme oxygenase (HO)-1, HO-2, hypoxia inducible factor-alpha (HIF)-1α, nuclear factor (erythroid-derived 2)-like 2 (Nrf-2), cyclooxygenase (COX)-1 and COX-2, inducible nitric oxide synthase (iNOS) and interleukin (IL)-1ß were determined by Western blot or real-time PCR, respectively. ASA caused hemorrhagic gastric mucosal damage and significantly decreased GBF, H2S production, CO content, mRNA or protein expression for CSE, 3-MST, HO-2 and increased mRNA and/or protein expression for CBS, HO-1, Nrf-2, HIF-1α, iNOS, IL-1ß, COX-2 in gastric mucosa and COHb concentration in blood. Pretreatment with CORM-2 or NaHS but not with PAG decreased ASA-damage and increased GBF. ZnPP reversed protective and hyperemic effect of NaHS but PAG failed to affect CORM-2-induced gastroprotection. CORM-2 elevated CO content, mRNA or protein expression for HO-1, Nrf-2, and decreased expression of CBS, HIF-1α, COX-2, IL-1ß, iNOS, the H2S production in gastric mucosa and COHb concentration in blood. NaHS raised mRNA or protein expression for CSE, COX-1 and decreased mRNA expression for IL-1ß and COHb level in blood. We conclude that CO is involved in gastroprotection induced by H2S while beneficial protective action of CO released from CORM-2 in gastric mucosa seems to be H2S-independent. In contrast to H2S, CO ameliorates hypoxia, regulates Nrf-2 expression but similarly to H2S acts on sGC-dependent manner to restore gastric microcirculation and exhibit anti-inflammatory activity in gastric mucosa compromised by ASA.