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.

The mitochondria-targeted sulfide delivery molecule attenuates drugs-induced gastropathy. Involvement of heme oxygenase pathway.

Hydrogen sulfide (H2S) signaling and H2S-prodrugs maintain redox balance in gastrointestinal (GI) tract. Predominant effect of any H2S-donor is mitochondrial. Non-targeted H2S-moieties were shown to decrease the non-steroidal anti-inflammatory drugs (NSAIDs)-induced gastrotoxicity but in high doses. However, direct, controlled delivery of H2S to gastric mucosal mitochondria as a molecular target improving NSAIDs-pharmacology remains overlooked. Thus, we treated Wistar rats, i.g. with vehicle, mitochondria-targeted H2S-releasing AP39 (0.004-0.5 mg/kg), AP219 (0.02 mg/kg) as structural control without H2S-releasing ability, or AP39 + SnPP (10 mg/kg) as a heme oxygenase (HMOX) inhibitor. Next, animals were administered i.g. with acetylsalicylic acid (ASA, 125 mg/kg) as NSAIDs representative or comparatively with 75% ethanol to induce translational hemorrhagic or necrotic gastric lesions, that were assessed micro-/macroscopically. Activity of mitochondrial complex IV/V, and DNA oxidation were assessed biochemically. Gastric mucosal/serum content of IL-1ß, IL-10, TNF-α, TGF-ß1/2, ARG1, GST-α, or phosphorylation of mTOR, NF-κB, ERK, Akt, JNK, STAT3/5 were evaluated by microbeads-fluorescent xMAP®-assay; gastric mucosal mRNA level of HMOX-1/2, COX-1/2, SOD-1/2 by real-time PCR. AP39 (but not AP219) dose-dependently (0.02 and 0.1 mg/kg) diminished NSAID- (and ethanol)-induced gastric lesions and DNA oxidation, restoring mitochondrial complexes activity, ARG1, GST-α protein levels and increasing HMOX-1 and SOD-2 expression. AP39 decreased proteins levels or phosphorylation of gastric mucosal inflammation/oxidation-sensitive markers and restored mTOR phosphorylation. Pharmacological inhibition of HMOX-1 attenuated AP39-gastroprotection. We showed that mitochondria-targeted H2S released from very low i.g. doses of AP39 improved gastric mucosal capacity to cope with NSAIDs-induced mitochondrial dysfunction and redox imbalance, mechanistically requiring the activity of HMOX-1.

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.

Carbon monoxide (CO)/heme oxygenase (HO)-1 in gastrointestinal tumors pathophysiology and pharmacology - possible anti- and pro-cancer activities.

Gastrointestinal (GI) tract cancers pose a significant pharmacological challenge for researchers in terms of the discovery of molecular agents and the development of targeted therapies. Although many ongoing clinical trials have brought new perspectives, there is still a lack of successful long-term treatment. Several novel pharmacological and molecular agents are being studied in the prevention and treatment of GI cancers. On the other hand, pharmacological tools designed to release an endogenous gaseous mediator, carbon monoxide (CO), were shown to prevent the gastric mucosa against various types of injuries and exert therapeutic properties in the treatment of GI pathologies. In this review, we summarized the current evidence on the role of CO and heme oxygenase 1 (HO-1) as a CO producing enzyme in the pathophysiology of GI tumors. We focused on a beneficial role of HO-1 and CO in biological systems and common pathological conditions. We further discussed the complex and ambiguous function of the HO-1/CO pathway in cancer cells with a special emphasis on molecular and cellular pro-cancerous and anti-cancer mechanisms. We also focused on the role that HO-1/CO plays in GI cancers, especially within upper parts such as esophagus or stomach.

Impact of Intragastric Balloon Placement on the Stomach Wall: A Prospective Cohort Study.

PURPOSE: Endoscopic intragastric balloon (IGB) placement is a minimally invasive treatment for morbid obesity that is sometimes used as a preparatory step before surgical intervention. This study was performed to analyze the changes in the stomach wall induced by IGB placement, with particular emphasis on pathomorphology, inflammatory markers, and tissue growth factors. MATERIAL AND METHODS: In total, 30 patients with morbid obesity were prospectively analyzed. A total of 16 patients with body mass index (BMI) ≥ 53 kg/m2 underwent two-stage treatment comprising IGB placement followed by laparoscopic sleeve gastrectomy (LSG) (IGB group), while 14 patients underwent one-stage LSG (non-IGB group). The gastric specimens removed during LSG were examined. The two groups were compared regarding the surgical results, microscopic structure and inflammatory process exponents of the stomach wall, and receptors for selected tissue growth factors. RESULTS: The IGB group had a longer median hospital stay than that of the non-IGB group. Compared with the non-IGB group, the IGB group had a thicker stomach wall, more submucosal fibrosis, and increased amounts of growth factors and inflammatory markers. CONCLUSION: Patients with IGB placement before LSG showed greater changes in the stomach wall than those of patients who received LSG alone. IGB placement was associated with stomach muscle layer thickening, submucosal fibrosis, and increased levels of inflammatory markers and tissue growth factors.

Role of Obesity, Physical Exercise, Adipose Tissue-Skeletal Muscle Crosstalk and Molecular Advances in Barrett's Esophagus and Esophageal Adenocarcinoma.

Both obesity and esophageal adenocarcinoma (EAC) rates have increased sharply in the United States and Western Europe in recent years. EAC is a classic example of obesity-related cancer where the risk of EAC increases with increasing body mass index. Pathologically altered visceral fat in obesity appears to play a key role in this process. Visceral obesity may promote EAC by directly affecting gastroesophageal reflux disease and Barrett's esophagus (BE), as well as a less reflux-dependent effect, including the release of pro-inflammatory adipokines and insulin resistance. Deregulation of adipokine production, such as the shift to an increased amount of leptin relative to "protective" adiponectin, has been implicated in the pathogenesis of BE and EAC. This review discusses not only the epidemiology and pathophysiology of obesity in BE and EAC, but also molecular alterations at the level of mRNA and proteins associated with these esophageal pathologies and the potential role of adipokines and myokines in these disorders. Particular attention is given to discussing the possible crosstalk of adipokines and myokines during exercise. It is concluded that lifestyle interventions to increase regular physical activity could be helpful as a promising strategy for preventing the development of BE and EAC.

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.

Barrett's Metaplasia Progression towards Esophageal Adenocarcinoma: An Attempt to Select a Panel of Molecular Sensors and to Reflect Clinical Alterations by Experimental Models.

The molecular processes that predispose the development of Barrett's esophagus (BE) towards esophageal adenocarcinoma (EAC) induced by gastrointestinal reflux disease (GERD) are still under investigation. In this study, based on a scientific literature screening and an analysis of clinical datasets, we selected a panel of 20 genes covering BE- and EAC-specific molecular markers (FZD5, IFNGR1, IL1A, IL1B, IL1R1, IL1RN, KRT4, KRT8, KRT15, KRT18, NFKBIL1, PTGS1, PTGS2, SOCS3, SOX4, SOX9, SOX15, TIMP1, TMEM2, TNFRSF10B). Furthermore, we aimed to reflect these alterations within an experimental and translational in vitro model of BE to EAC progression. We performed a comparison between expression profiles in GSE clinical databases with an in vitro model of GERD involving a BE cell line (BAR-T) and EAC cell lines (OE33 and OE19). Molecular responses of cells treated with acidified bile mixture (BM) at concentration of 100 and 250 µM for 30 min per day were evaluated. We also determined a basal mRNA expression within untreated, wild type cell lines on subsequent stages of BE and EAC development. We observed that an appropriately optimized in vitro model based on the combination of BAR-T, OE33 and OE19 cell lines reflects in 65% and more the clinical molecular alterations observed during BE and EAC development. We also confirmed previous observations that exposure to BM (GERD in vitro) activated carcinogenesis in non-dysplastic cells, inducing molecular alternations in the advanced stages of BE. We conclude that it is possible to induce, to a high extent, the molecular profile observed clinically within appropriately and carefully optimized experimental models, triggering EAC development. This experimental scheme and molecular marker panel might be implemented in further research, e.g., aiming to develop and evaluate novel compounds and prodrugs targeting GERD as well as BE and EAC prevention and treatment.

Impact of Vagotomy on Postoperative Weight Loss, Alimentary Intake, and Enterohormone Secretion After Bariatric Surgery in Experimental Translational Models.

Obesity may be treated by bariatric procedures and is related to enterohormone release modulation. Nevertheless, a majority of commonly used surgical procedures have a significant impact on vagus nerve function by breaking the connections with its gastric branches. In the case of an intragastric balloon (BAL), this interaction is unclear. However, BAL-induced weight reduction is not long-lasting. Interestingly, this method has not been used in combination with vagotomy (VAG). Thus, we evaluated, for the first time, the short- and long-term effects of combined BAL and VAG using the animal-based translational model and compared these effects with sleeve gastrectomy (SG) and Roux-en-Y gastric bypass (RYGB). Wistar rats were fed a high-calorie diet for 8 weeks to induce obesity before SG, RYGB, BAL + / - VAG. Animals' weight and eating behaviors were monitored weekly. After 90 days, serum samples were collected to evaluate postprandial and fasting GLP-1, GIP, PYY, ghrelin, glucagon, insulin, leptin, and pancreatic polypeptide concentrations by fluorescent assay. VAG, SG, RYGB, and BAL + VAG significantly reduced body weight 30 and 90 days after surgery. BAL alone induced temporal weight reduction observed after 30 days, reversed after 90 days. Calories intake was reduced at the first half of the observation period in all groups. Fluid intake was reduced in all groups except SG and BAL. Enterohormone profile for BAL + VAG was comparable to SG and RYGB but not BAL. VAG and BAL + VAG but not BAL alone maintain weight reduction, alimentary intake changes, and enterohormone release after long-term observation. VAG may improve the effectiveness of bariatric procedures for obesity treatment in clinical practice.

Microbiome Profile and Molecular Pathways Alterations in Gastrointestinal Tract by Hydrogen Sulfide-Releasing Nonsteroidal Anti-Inflammatory Drug (ATB-352): Insight into Possible Safer Polypharmacy.

Aims: Nonsteroidal anti-inflammatory drugs, including ketoprofen, induce adverse effects within the gastrointestinal (GI)-tract. Hydrogen sulfide (H2S) is an antioxidative gaseous mediator contributing to GI-protection. We aimed to evaluate the GI safety of a novel H2S-releasing derivative of ketoprofen (ATB-352) versus classic ketoprofen and the molecular mechanisms of their activity after chronic treatment in experimental animal models. Results: Ketoprofen (10 mg/kg/day) administered intragastrically for 7 days in contrast with ATB-352 (14 mg/kg/day) reduced mucosal H2S content inducing GI damage with significantly increased injury score, altered intestinal microbiome profile, and modulation of more than 50% of 36 investigated molecular sensors (e.g., mammalian target of rapamycin or suppressor of cytokine signaling 3 [SOCS3]). Polypharmacy with aspirin (10 mg/kg/day) enhanced ketoprofen toxicity not affecting GI safety of ATB-352. Omeprazole (20 mg/kg/day) decreased ketoprofen-induced injury to the level of ATB-352 alone. Both compounds combined or not with aspirin or omeprazole maintained the ability to inhibit cyclooxygenase (COX) activity manifested by decreased prostaglandin production. Innovation and Conclusions: Ketoprofen-induced H2S-production decrease and intestinal microbiome profile alterations lead to GI toxicity observed on macro-/microscopic and molecular levels. Ketoprofen but not ATB-352 requires concomitant treatment with omeprazole to eliminate GI adverse effects. ATB-352 applied alone or in a polypharmacy setting with aspirin effectively inhibited COX and maintained GI safety due to H2S-release. Neither compound affected DNA oxidation in the GI mucosa, but ATB-352 had lower impact on molecular oxidative/inflammatory response pathways and intestinal microbiome. The GI safety of ATB-352 could be due to the involvement of heme oxygenase 1 and SOCS3 pathway activation. Antioxid. Redox Signal. 36, 189-210.

HOXA13 in etiology and oncogenic potential of Barrett's esophagus.

Barrett's esophagus in gastrointestinal reflux patients constitutes a columnar epithelium with distal characteristics, prone to progress to esophageal adenocarcinoma. HOX genes are known mediators of position-dependent morphology. Here we show HOX collinearity in the adult gut while Barrett's esophagus shows high HOXA13 expression in stem cells and their progeny. HOXA13 overexpression appears sufficient to explain both the phenotype (through downregulation of the epidermal differentiation complex) and the oncogenic potential of Barrett's esophagus. Intriguingly, employing a mouse model that contains a reporter coupled to the HOXA13 promotor we identify single HOXA13-positive cells distally from the physiological esophagus, which is mirrored in human physiology, but increased in Barrett's esophagus. Additionally, we observe that HOXA13 expression confers a competitive advantage to cells. We thus propose that Barrett's esophagus and associated esophageal adenocarcinoma is the consequence of expansion of this gastro-esophageal HOXA13-expressing compartment following epithelial injury.

Novel Hydrogen Sulfide (H2S)-Releasing BW-HS-101 and Its Non-H2S Releasing Derivative in Modulation of Microscopic and Molecular Parameters of Gastric Mucosal Barrier.

Hydrogen sulfide (H2S) is an endogenously produced molecule with anti-inflammatory and cytoprotective properties. We aimed to investigate for the first time if a novel, esterase-sensitive H2S-prodrug, BW-HS-101 with the ability to release H2S in a controllable manner, prevents gastric mucosa against acetylsalicylic acid-induced gastropathy on microscopic and molecular levels. Wistar rats were pretreated intragastrically with vehicle, BW-HS-101 (0.5-50 µmol/kg) or its analogue without the ability to release H2S, BW-iHS-101 prior to ASA administration (125 mg/kg, intragastrically). BW-HS-101 was administered alone or in combination with nitroarginine (L-NNA, 20 mg/kg, intraperitoneally) or zinc protoporphyrin IX (10 mg/kg, intraperitoneally). Gastroprotective effects of BW-HS-101 were additionally evaluated against necrotic damage induced by intragastrical administration of 75% ethanol. Gastric mucosal damage was assessed microscopically, and gastric blood flow was determined by laser flowmetry. Gastric mucosal DNA oxidation and PGE2 concentration were assessed by ELISA. Serum and/or gastric protein concentrations of IL-1α, IL-1ß, IL-2, IL-4, IL-6, IL-10, IL-13, VEGF, GM-CSF, IFN-γ, TNF-α, and EGF were determined by a microbeads/fluorescent-based multiplex assay. Changes in gastric mucosal iNOS, HMOX-1, SOCS3, IL1-R1, IL1-R2, TNF-R2, COX-1, and COX-2 mRNA were assessed by real-time PCR. BW-HS-101 or BW-iHS-101 applied at a dose of 50 µmol/kg protected gastric mucosa against ASA-induced gastric damage and prevented a decrease in the gastric blood flow level. H2S prodrug decreased DNA oxidation, systemic and gastric mucosal inflammation with accompanied upregulation of SOCS3, and EGF and HMOX-1 expression. Pharmacological inhibition of nitric oxide (NO) synthase but not carbon monoxide (CO)/heme oxygenase (HMOX) activity by L-NNA or ZnPP, respectively, reversed the gastroprotective effect of BW-HS-101. BW-HS-101 also protected against ethanol-induced gastric injury formation. We conclude that BW-HS-101, due to its ability to release H2S in a controllable manner, prevents gastric mucosa against drugs-induced gastropathy, inflammation and DNA oxidation, and upregulate gastric microcirculation. Gastroprotective effects of this H2S prodrug involves endogenous NO but not CO activity and could be mediated by cytoprotective and anti-inflammatory SOCS3 and EGF pathways.

Gaseous Mediators as a Key Molecular Targets for the Development of Gastrointestinal-Safe Anti-Inflammatory Pharmacology.

Non-steroidal anti-inflammatory drugs (NSAIDs) represent one of the most widely used classes of drugs and play a pivotal role in the therapy of numerous inflammatory diseases. However, the adverse effects of these drugs, especially when applied chronically, frequently affect gastrointestinal (GI) tract, resulting in ulceration and bleeding, which constitutes a significant limitation in clinical practice. On the other hand, it has been recently discovered that gaseous mediators nitric oxide (NO), hydrogen sulfide (H2S) and carbon monoxide (CO) contribute to many physiological processes in the GI tract, including the maintenance of GI mucosal barrier integrity. Therefore, based on the possible therapeutic properties of NO, H2S and CO, a novel NSAIDs with ability to release one or more of those gaseous messengers have been synthesized. Until now, both preclinical and clinical studies have shown promising effects with respect to the anti-inflammatory potency as well as GI-safety of these novel NSAIDs. This review provides an overview of the gaseous mediators-based NSAIDs along with their mechanisms of action, with special emphasis on possible implications for GI mucosal defense mechanisms.

Activation of transient receptor potential vanilloid channel 4 contributes to the development of ethanol-induced gastric injury in mice.

The transient receptor potential vanilloid channel 4 (TRPV4) is associated with the development of several pathologies, particularly gastric disorders. However, there are no studies associating this receptor with the pathophysiology of gastric erosions. The aim of this study was to investigate the role of TRPV4 in the development of ethanol-induced gastric damage in vivo. Gastric lesions were induced by ethanol in Swiss mice pretreated with TRPV4 antagonists, GSK2193874 (0.1; 0.3 and 0.9 mg/kg) or Ruthenium red (0.03; 0.1 or 0.3 mg/kg) or its agonist, GSK1016790A (0.9 mg/kg). Gastric mucosal samples were taken for histopathology, immunohistochemistry, atomic force microscopy and evaluation of antioxidant parameters. The gastric mucus content and TRPV4 mRNA expression were analyzed. Ethanol exposure induced upregulation of gastric mRNA and protein expression of TRPV4. TRPV4 blockade promoted gastroprotection against ethanol-induced injury on macro- and microscopic levels, leading to reduced hemorrhage, cell loss and edema and enhanced gastric mucosal integrity. Moreover, an increase in superoxide dismutase (SOD) and glutathione (GSH) activity was observed, followed by a decrease in malondialdehyde (MDA) levels. TRPV4 blockade during alcohol challenge reestablished gastric mucus content. The combination of TRPV4 agonist and ethanol revealed macroscopic exacerbation of gastric damage area. Our results confirmed the association of TRPV4 with the development of gastric injury, showing the importance of this receptor for further investigations in the field of gastrointestinal pathophysiology and pharmacology.

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.

Intestinal Alkaline Phosphatase Combined with Voluntary Physical Activity Alleviates Experimental Colitis in Obese Mice. Involvement of Oxidative Stress, Myokines, Adipokines and Proinflammatory Biomarkers.

Intestinal alkaline phosphatase (IAP) is an essential mucosal defense factor involved in the process of maintenance of gut homeostasis. We determined the effect of moderate exercise (voluntary wheel running) with or without treatment with IAP on the course of experimental murine 2,4,6-trinitrobenzenesulfonic acid (TNBS) colitis by assessing disease activity index (DAI), colonic blood flow (CBF), plasma myokine irisin levels and the colonic and adipose tissue expression of proinflammatory cytokines, markers of oxidative stress (SOD2, GPx) and adipokines in mice fed a standard diet (SD) or high-fat diet (HFD). Macroscopic and microscopic colitis in sedentary SD mice was accompanied by a significant decrease in CBF, and a significant increase in the colonic expression of tumor necrosis factor-alpha (TNF-α), IL-6, IL-1ß and leptin mRNAs and decrease in the mRNA expression of adiponectin. These effects were aggravated in sedentary HFD mice but reduced in exercising animals, potentiated by concomitant treatment with IAP, especially in obese mice. Exercising HFD mice demonstrated a substantial increase in the mRNA for adiponectin and a decrease in mRNA leptin expression in intestinal mucosa and mesenteric fat as compared to sedentary animals. The expression of SOD2 and GPx mRNAs was significantly decreased in adipose tissue in HFD mice, but these effects were reversed in exercising mice with IAP administration. Our study shows for the first time that the combination of voluntary exercise and oral IAP treatment synergistically favored healing of intestinal inflammation, strengthened the antioxidant defense and ameliorated the course of experimental colitis; thus, IAP may represent a novel adjuvant therapy to alleviate inflammatory bowel disease (IBD) in humans.

Role of Carbon Monoxide in Host-Gut Microbiome Communication.

Nature is full of examples of symbiotic relationships. The critical symbiotic relation between host and mutualistic bacteria is attracting increasing attention to the degree that the gut microbiome is proposed by some as a new organ system. The microbiome exerts its systemic effect through a diverse range of metabolites, which include gaseous molecules such as H2, CO2, NH3, CH4, NO, H2S, and CO. In turn, the human host can influence the microbiome through these gaseous molecules as well in a reciprocal manner. Among these gaseous molecules, NO, H2S, and CO occupy a special place because of their widely known physiological functions in the host and their overlap and similarity in both targets and functions. The roles that NO and H2S play have been extensively examined by others. Herein, the roles of CO in host-gut microbiome communication are examined through a discussion of (1) host production and function of CO, (2) available CO donors as research tools, (3) CO production from diet and bacterial sources, (4) effect of CO on bacteria including CO sensing, and (5) gut microbiome production of CO. There is a large amount of literature suggesting the "messenger" role of CO in host-gut microbiome communication. However, much more work is needed to begin achieving a systematic understanding of this issue.

Molecular Profile of Barrett's Esophagus and Gastroesophageal Reflux Disease in the Development of Translational Physiological and Pharmacological Studies.

Barrett's esophagus (BE) is a premalignant condition caused by gastroesophageal reflux disease (GERD), where physiological squamous epithelium is replaced by columnar epithelium. Several in vivo and in vitro BE models were developed with questionable translational relevance when implemented separately. Therefore, we aimed to screen Gene Expression Omnibus 2R (GEO2R) databases to establish whether clinical BE molecular profile was comparable with animal and optimized human esophageal squamous cell lines-based in vitro models. The GEO2R tool and selected databases were used to establish human BE molecular profile. BE-specific mRNAs in human esophageal cell lines (Het-1A and EPC2) were determined after one, three and/or six-day treatment with acidified medium (pH 5.0) and/or 50 and 100 µM bile mixture (BM). Wistar rats underwent microsurgical procedures to generate esophagogastroduodenal anastomosis (EGDA) leading to BE. BE-specific genes (keratin (KRT)1, KRT4, KRT5, KRT6A, KRT13, KRT14, KRT15, KRT16, KRT23, KRT24, KRT7, KRT8, KRT18, KRT20, trefoil factor (TFF)1, TFF2, TFF3, villin (VIL)1, mucin (MUC)2, MUC3A/B, MUC5B, MUC6 and MUC13) mRNA expression was assessed by real-time PCR. Pro/anti-inflammatory factors (interleukin (IL)-1ß, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IL-13, tumor necrosis factor α, interferon γ, granulocyte-macrophage colony-stimulating factor) serum concentration was assessed by a Luminex assay. Expression profile in vivo reflected about 45% of clinical BE with accompanied inflammatory response. Six-day treatment with 100 µM BM (pH 5.0) altered gene expression in vitro reflecting in 73% human BE profile and making this the most reliable in vitro tool taking into account two tested cell lines. Our optimized and established combined in vitro and in vivo BE models can improve further physiological and pharmacological studies testing pathomechanisms and novel therapeutic targets of this disorder.