Hydrogen Sulfide and Carbon Monoxide Protect Gastric Mucosa Compromised by Mild Stress Against Alendronate Injury

Abstract

BackgroundAlendronate is an inhibitor of osteoclast-mediated bone resorption, but its clinical utility is limited due to gastrointestinal complications including bleeding erosions.AimsWe studied whether potent vasodilators hydrogen sulfide (H2S) and carbon monoxide (CO) can protect against alendronate-induced gastric lesions in rats exposed to mild stress.MethodsThree series (A, B, and C) of Wistar rats received alendronate (150–700 mg/kg i.g., series A) with or without NaHS (5 mg/kg), H2S donor or CORM-2 (5 mg/kg) releasing CO administered i.g. 30 min before alendronate administration (series B) in rats exposed for 3 days before alendronate administration to mild stress (series C). The area of gastric lesions was assessed by planimetry, the gastric blood flow (GBF) was determined by H2-gas clearance technique, and H2S production via CSE/CBS/3-MST activity and the gastric expression of HO-1, HO-2, HIF-1α, NF-κB, iNOS, COX-2, IL-1β, TNF-α, GPx-1 and SOD-2 were analyzed by qPCR or Western blot.ResultsAlendronate dose-dependently produced gastric mucosal lesions and significantly decreased GBF, and these effects were exacerbated by mild stress. NaHS and CORM-2 significantly reduced the alendronate-induced gastric lesions in non-stressed and stressed animals, but only NaHS but not CORM-2 raised H2S production. NaHS and CORM-2 inhibited gastric expression of HIF-1α protein and HO-1, HIF-1α, NF-κB, COX-2, iNOS, IL-1β, TNF-α mRNAs but failed to affect those of HO-2, GPx-1, and SOD-2.ConclusionBoth H2S and CO released from their donors, NaHS and CORM-2, protect gastric mucosa compromised by stress against alendronate-induced gastric damage via mechanism involving downregulation of HIF-1α, NF-κB and proinflammatory factors COX-2, iNOS, IL-1β, and TNF-α.

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].…”

“…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).…”

“…Reverse transcription to complementary DNA was performed with a high-capacity complementary DNA reverse transcription kit (Thermo Fisher Scientific, Life Technologies, MA, USA). Expression of b-actin, heme oxygenase 1 (HO-1), cyclooxygenase (COX)-2, inducible NOS (iNOS), interleukin-1b (IL-1b), GPx-1, and SOD-2 was determined by real-time PCR using specific primers [17,22], SG qPCR master mix (29) including SYBR Green (EURx, Gdansk, Poland), and Quant Studio 12K Flex thermal cycler (Thermo Fisher Scientific, MA, USA). To determine a-CGRP mRNA expression, 5 0 -GCTCACCAGGGAGG-CATCAT-3 0 forward primer and 5 0 -ATGCCTGGTA-CAGGAGCAAGA-3 0 reverse primer were used.…”

“…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.…”