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.

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.

Administration of Warfarin Inhibits the Development of Cerulein-Induced Edematous Acute Pancreatitis in Rats.

Acute pancreatitis (AP) is a severe disease with high morbidity and mortality in which inflammation and coagulation play crucial roles. The development of inflammation leads to vascular injury, endothelium and leukocytes stimulation, and an increased level of tissue factor, which results in the activation of the coagulation process. For this reason, anticoagulants may be considered as a therapeutic option in AP. Previous studies have shown that pretreatment with heparin, low-molecular-weight heparin (LMWH), or acenocoumarol inhibits the development of AP. The aim of the present study was to check if pretreatment with warfarin affects the development of edematous pancreatitis evoked by cerulein. Warfarin (90, 180, or 270 µg/kg/dose) or saline were administered intragastrically once a day for 7 days consecutively before the induction of AP. AP was evoked by the intraperitoneal administration of cerulein. The pre-administration of warfarin at doses of 90 or 180 µg/kg/dose reduced the histological signs of pancreatic damage in animals with the induction of AP. Additionally, other parameters of AP, such as an increase in the serum activity of lipase and amylase, the plasma concentration of D-dimer, and interleukin-1ß, were decreased. In addition, pretreatment with warfarin administered at doses of 90 or 180 µg/kg/dose reversed the limitation of pancreatic blood flow evoked by AP development. Warfarin administered at a dose of 270 µg/kg/dose did not exhibit a preventive effect in cerulein-induced AP. Conclusion: Pretreatment with low doses of warfarin inhibits the development of AP evoked by the intraperitoneal administration of cerulein.

Protective and Healing Effects of Ghrelin and Risk of Cancer in the Digestive System.

Ghrelin is an endogenous ligand for the ghrelin receptor, previously known as the growth hormone secretagogue receptor. This hormone is mainly produced by endocrine cells present in the gastric mucosa. The ghrelin-producing cells are also present in other organs of the body, mainly in the digestive system, but in much smaller amount. Ghrelin exhibits a broad spectrum of physiological effects, such as stimulation of growth hormone secretion, gastric secretion, gastrointestinal motility, and food intake, as well as regulation of glucose homeostasis and bone formation, and inhibition of inflammatory processes. This review summarizes the recent findings concerning animal and human data showing protective and therapeutic effects of ghrelin in the gut, and also presents the role of growth hormone and insulin-like growth factor-1 in these effects. In addition, the current data on the possible influence of ghrelin on the carcinogenesis, its importance in predicting the risk of developing gastrointestinal malignances, as well as the potential usefulness of ghrelin in the treatment of cancer, have been presented.

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.

Evidence for Cytoprotective Effect of Carbon Monoxide Donor in the Development of Acute Esophagitis Leading to Acute Esophageal Epithelium Lesions.

Exposure to acidic gastric content due to malfunction of lower esophageal sphincter leads to acute reflux esophagitis (RE) leading to disruption of esophageal epithelial cells. Carbon monoxide (CO) produced by heme oxygenase (HMOX) activity or released from its donor, tricarbonyldichlororuthenium (II) dimer (CORM-2) was reported to protect gastric mucosa against acid-dependent non-steroidal anti-inflammatory drug-induced damage. Thus, we aimed to investigate if CO affects RE-induced esophageal epithelium lesions development. RE induced in Wistar rats by the ligation of a junction between pylorus and forestomach were pretreated i.g. with vehicle CORM-2; RuCl3; zinc protoporphyrin IX, or hemin. CORM-2 was combined with NG-nitro-L-arginine (L-NNA), indomethacin, capsazepine, or capsaicin-induced sensory nerve ablation. Esophageal lesion score (ELS), esophageal blood flow (EBF), and mucus production were determined by planimetry, laser flowmetry, histology. Esophageal Nrf-2, HMOXs, COXs, NOSs, TNF-α and its receptor, IL-1 family and IL-1 receptor antagonist (RA), NF-κB, HIF-1α, annexin-A1, suppressor of cytokine signaling (SOCS3), TRPV1, c-Jun, c-Fos mRNA/protein expressions, PGE2, 8-hydroxy-deoxyguanozine (8-OHdG) and serum COHb, TGF-ß1, TGF-ß2, IL-1ß, and IL-6 content were assessed by PCR, immunoblotting, immunohistochemistry, gas chromatography, ELISA or Luminex platform. Hemin or CORM-2 alone or combined with L-NNA or indomethacin decreased ELS. Capsazepine or capsaicin-induced denervation reversed CORM-2 effects. COHb blood content, esophageal HMOX-1, Nrf-2, TRPV1 protein, annexin-A1, HIF-1α, IL-1 family, NF-κB, c-Jun, c-Fos, SOCS3 mRNA expressions, and 8-OHdG levels were elevated while PGE2 concentration was decreased after RE. CO donor-maintained elevated mucosal TRPV1 protein, HIF-1 α, annexin-A1, IL-1RA, SOCS3 mRNA expression, or TGF-ß serum content, decreasing 8-OHdG level, and particular inflammatory markers expression/concentration. CORM-2 and Nrf-2/HMOX-1/CO pathway prevent esophageal mucosa against RE-induced lesions, DNA oxidation, and inflammatory response involving HIF-1α, annexin-A1, SOCS3, IL-1RA, TGF-ß-modulated pathways. Esophagoprotective and hyperemic CO effects are in part mediated by afferent sensory neurons and TRPV1 receptors activity with questionable COX/PGE2 or NO/NOS systems involvement.

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.

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.

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.