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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.

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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-β1, TGF-β2, IL-1β, 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-β 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-β-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.

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-β, 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.

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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-1β, 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.