Carbon Monoxide (CO) Released from Tricarbonyldichlororuthenium (II) Dimer (CORM-2) in Gastroprotection against Experimental Ethanol-Induced Gastric Damage

Abstract

The physiological gaseous molecule, carbon monoxide (CO) becomes a subject of extensive investigation due to its vasoactive activity throughout the body but its role in gastroprotection has been little investigated. We determined the mechanism of CO released from its donor tricarbonyldichlororuthenium (II) dimer (CORM-2) in protection of gastric mucosa against 75% ethanol-induced injury. Rats were pretreated with CORM-2 30 min prior to 75% ethanol with or without 1) non-selective (indomethacin) or selective cyclooxygenase (COX)-1 (SC-560) and COX-2 (celecoxib) inhibitors, 2) nitric oxide (NO) synthase inhibitor L-NNA, 3) ODQ, a soluble guanylyl cyclase (sGC) inhibitor, hemin, a heme oxygenase (HO)-1 inductor or zinc protoporphyrin IX (ZnPPIX), an inhibitor of HO-1 activity. The CO content in gastric mucosa and carboxyhemoglobin (COHb) level in blood was analyzed by gas chromatography. The gastric mucosal mRNA expression for HO-1, COX-1, COX-2, iNOS, IL-4, IL-1β was analyzed by real-time PCR while HO-1, HO-2 and Nrf2 protein expression was determined by Western Blot. Pretreatment with CORM-2 (0.5–10 mg/kg) dose-dependently attenuated ethanol-induced lesions and raised gastric blood flow (GBF) but large dose of 100 mg/kg was ineffective. CORM-2 (5 mg/kg and 50 mg/kg i.g.) significantly increased gastric mucosal CO content and whole blood COHb level. CORM-2-induced protection was reversed by indomethacin, SC-560 and significantly attenuated by celecoxib, ODQ and L-NNA. Hemin significantly reduced ethanol damage and raised GBF while ZnPPIX which exacerbated ethanol-induced injury inhibited CORM-2- and hemin-induced gastroprotection and the accompanying rise in GBF. CORM-2 significantly increased gastric mucosal HO-1 mRNA expression and decreased mRNA expression for iNOS, IL-1β, COX-1 and COX-2 but failed to affect HO-1 and Nrf2 protein expression decreased by ethanol. We conclude that CORM-2 released CO exerts gastroprotection against ethanol-induced gastric lesions involving an increase in gastric microcirculation mediated by sGC/cGMP, prostaglandins derived from COX-1, NO-NOS system and its anti-inflammatory properties.

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

“…Previous studies have documented that the mechanism of aspirin-induced gastric damage involves an inhibition of cytoprotective prostaglandin E 2 production via COX-1/ COX-2 enzymatic activity and the prominent fall in GBF leading to the formation of severe hemorrhagic lesions of gastric mucosa [5,[29][30][31][32][33]. On the other hand, recent studies reported that the endogenous gaseous mediators H 2 S, CO, and NO contribute to the maintenance of gastric mucosal integrity and prevent the formation of gastric mucosal lesions induced by various noxious stimuli, such as exposure to stress or administration of ethanol, bisphosphonates, and NSAIDs, including aspirin [5,17,19,21,22,26,34].…”

“…Thirty minutes before aspirin application, rats with capsaicin-induced denervation (series A) and rats without capsaicin-induced denervation (series B) were pretreated intragastrically with (1) 0.9% saline (vehicle control) or dimethyl sulfoxide and saline (1:10), (2) CO-releasing CORM-2 [20], applied in a dose of 5 mg/ kg, which has previously been reported by our group to increase CO content in gastric mucosa and to exert gastroprotection against aspirin-and ethanol-induced gastric damage [17,21], or (3) NaHS, the H 2 S-releasing salt applied intragastrically in a dose of 5 mg/kg, which has been demonstrated to protect gastric mucosa against aspirin-, stress-, or alendronate-induced lesions [17,19,22]. In a separate series (C), rats with intact sensory nerves were pretreated with NaHS or CORM-2 in combination with capsazepine (5 mg/kg intragastrically), a TRPV1 antagonist [23], or N G -nitro-L-arginine (L-NNA, 20 mg/kg intraperitoneally), a nonselective nitric oxide synthase (NOS) inhibitor [24].…”

“…The stomach was excised and opened along the greater curvature to determine the area of aspirin-induced gastric lesions by means of computerized planimetry (Morphomat, Carl Zeiss, Berlin, Germany) [26,27]. Next, the gastric mucosal samples from oxyntic mucosa were scraped off on ice, snap-frozen in liquid nitrogen, and stored at -80°C until further analysis [21].…”

“…Determination of mRNA expression of b-actin, a-CGRP, heme oxygenase 1, cyclooxygenase 2, inducible NOS, interleukin-1b, GPx-1, and SOD-2 in rat gastric mucosa by real-time polymerase chain reaction Expression of mRNA for particular genes of interest in gastric mucosa was determined by real-time polymerase chain reaction (PCR) as described previously [21,22]. Briefly, RNA was isolated from gastric mucosal biopsy samples with use of a GeneMATRIX universal RNA purification kit (EURx, Gdansk, Poland).…”

“…Protein expression of b-actin, GPx-1, SOD-1, and iNOS in gastric mucosa was determined by Western blot, as described previously [17,21,22]. Rabbit polyclonal antiiNOS antibody (1:500, ab3523, Abcam, Cambridge, UK), rabbit polyclonal anti-GPx-1 antibody (1:1000, ab22604, Abcam), rabbit polyclonal anti-SOD-1 antibody (1:1000, ab13498, Abcam), and rabbit polyclonal anti-b-actin anitbody (1:1000, 20536-1-AP, Proteintech, Manchester, UK) were used as primary antibodies.…”