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

Abstract

Hydrogen sulfide (H₂S) is an endogenous mediator, synthesized from l-cysteine by cystathionine γ-lyase (CSE), cystathionine β-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.

Keywords: afferent sensory neurons; gastric blood flow; hydrogen sulfide; ischemia/reperfusion; prostaglandins.

Figure 1

Gross macroscopic appearance of gastric mucosa pretreated 30 min prior to gastric ischemia-reperfusion (I/R) with vehicle (A); NaHS (90 μmol/kg i.g.); (B), l-cysteine (80 μmol/kg i.g.); (C) or GYY4137 (90 μmol/kg i.g.); (D) and exposed to 30 min of I of celiac artery followed by 3 h of R (I/R).

Figure 2

Mean lesion area and the GBF in gastric mucosa of rats pretreated with vehicle, NaHS (18–180 μmol/kg i.g.), l-cysteine (0.8–80 μmol/kg i.g.) or GYY4137 (90 μmol/kg i.g.) and exposed to I followed by 3 h of R. Results are mean ± S.E.M of 4–5 rats per each experimental group. Significant changes as compared with the respective values in the vehicle-control group is indicated by an asterisk (p < 0.05).

Figure 3

Mean lesion area and GBF in gastric mucosa of rats pretreated with vehicle, NaHS (A) or l-cysteine (B) alone or in combination with indomethacin (INDO, 14 μmol/kg i.g.), SC-560 (14 μmol/kg i.p.) or celecoxib (CELE, 26 μmol/kg i.g.) and exposed to I followed by 3 h of R. Results are mean ± S.E.M of 4–5 rats per each experimental group. A significant change as compared with the respective values in the vehicle-control is indicated by asterisks (p < 0.05).

Figure 4

Mean lesion area and the GBF in rats pretreated with vehicle, NaHS (90 μmol/kg i.g.) or l-cysteine (80 μmol/kg i.g.) alone or in combination with capsazepine (13 μmol/kg i.p.) and exposed to I followed by 3 h of R. Results are mean ± S.E.M of 4–5 per each experimental group. Significant changes as compared with the respective values in the vehicle-control group are indicated by an asterisk (p < 0.05). A cross indicates a significant change compared to the values obtained in the group treated with NaHS and l-cysteine alone (p < 0.05).

Figure 5

Effect of capsaicin denervation on the mean lesion area and GBF in rats exposed to 30 min of I followed by 3 h of R and pretreated with vehicle, NaHS (90 μmol/kg i.g.) or l-cysteine (80 μmol/kg i.g.) alone or in combination with CGRP (2.5 nmol/kg i.p.). Results are mean ± S.E.M of 4–5 rats per each experimental group. Significant change as compared with the respective values in vehicle-control group was indicated by asterisk (p < 0.05). Asterisks and crosses indicate significant changes compared to the values obtained in the group treated with saline, NaHS or l-cysteine without denervation (p < 0.05). A cross indicates a significant change as compared with the respective capsaicin-denervated animals without co-treatment with CGRP (p < 0.05).

Figure 6

Expression of mRNA for CSE (A), CBS (B) and 3-MST (C) in gastric mucosa of rats pretreated with vehicle (saline) or NaHS (90 μmol/kg i.g.) with or without exposure to I followed by 3 h of R. Results are mean ± S.E.M of 4–5 rats per each experimental group. An asterisk indicates a significant change as compared with intact rats (p < 0.05). A cross indicates a significant difference as compared with vehicle pretreated group (p < 0.05).

Figure 7

Expression of mRNA for HIF-1α (A), GPx-1 (B) and SOD-2 (C) in gastric mucosa of rats exposed to I followed by 3 h of R and pretreated with vehicle (saline) or NaHS (90 μmol/kg i.g.). Results are mean ± S.E.M of 4–5 rats per each experimental group. An asterisk indicates a significant change as compared with intact rats (p < 0.05). A cross indicates a significant difference as compared with the vehicle pretreated group (p < 0.05).

Figure 8

Mean lesion area and the GBF in gastric mucosa of rats pretreated with single dose of vehicle or NaHS (90 μmol/kg i.g.) and exposed to 30 min of I followed by 3, 6, 24 and 48 h of R. Results are mean ± S.E.M. of 4–5 rats per each group. An asterisk indicates a significant change as compared with the value obtained in the vehicle-control group exposed to I and 3 h of R (p < 0.05). Double asterisks denote a significant difference as compared with the values obtained in vehicle pretreated groups at respective times of R (p < 0.05). Crosses indicate a significant difference as compared with the values obtained in NaHS-pretreated groups at 3 h and 6 h of R, respectively (p < 0.05).

Figure 9

Total sulfide pool released by CSE/CBS activity pathway in gastric mucosa of intact rats and those pretreated with vehicle (saline) or NaHS (90 μmol/kg i.g.) and exposed to I followed by 3, 6, 24 or 48 h of R. Results are mean ± S.E.M of 4–5 rats per each experimental group. An asterisk indicates a significant change as compared with the value obtained in intact rats (p < 0.05).

Figure 10

Total sulfide pool released by activity of CSE/CBS (A) or 3-MST (B) pathway from gastric mucosa of intact rats and those pretreated with vehicle (saline), l-cysteine (80 μmol/kg i.g.) or NaHS (90 μmol/kg i.g.) and exposed to I followed by 3 h of R. Results are mean ± S.E.M of 4–5 rats per each experimental group. Asterisk indicates a significant change as compared with the value obtained in intact rats (p < 0.05).