Protective effect of endogenous hydrogen sulfide against oxidative stress in gastric ischemia-reperfusion injury

Abstract

Hydrogen sulfide (H(2)S) is a gaseous signaling molecule, which plays a critical role in a number of physiological and pathological progresses. In order to determine the effect of endogenous H(2)S on gastric ischemia-reperfusion (GI-R), we evaluated the gastric mucosal damage in rats intraperitoneally injected with DL-propargylglycine (PAG, 50 mg/kg/day) or L-cysteine (L-cys, 50 mg/kg/day) for 7 days before GI-R. GI-R injury was achieved by clamping the celiac artery for 30 min, followed by reperfusion for 60 min. Gastric mucosal damage was macroscopically assessed in the area of injury and deep damage was assessed by histopathological scoring. PAG increased the area of gastric mucosal injury and deep damage compared with that in untreated GI-R rats (P<0.05). While PAG decreased the H(2)S concentration and cystathionine γ-lyase (CSE) expression in the gastric mucosa, L-cys significantly attenuated the effects of GI-R. Western blot analysis revealed that the increases of malondialdehyde (MDA) and xanthine oxidase (XOD), and decreases of glutathione (GSH), superoxide dismutase (SOD) and the restriction of superoxide (O(2) (-)) production in the PAG group were inhibited by L-cys (P<0.05). Endogenous H(2)S has a protective effect against GI-R in rats by inhibiting oxygen free radical overproduction.

Keywords: DL-propargylglycine; gastric ischemia-reperfusion injury; hydrogen sulfide; oxidative stress.

Figure 1.

Effects of PAG and L-cys on gastric mucosal injury area in rats subjected to gastric ischemia-reperfusion (GI-R). Male Sprague-Dawley rats were intraperitoneally injected with the H₂S synthetase blocker PAG (50 mg/kg) or L-cys (50 mg/kg) for 7 days before the celiac arteries were clamped for 30 min ischemia and then reperfused for 60 min. Sham group, age-matched rats with physiological solute treatment but no GI-R procedure; GI-R group, age-matched rats with physiological solute treatment followed by the GI-R procedure; PAG group, rats intraperitoneally injected with PAG and then subjected to the GI-R procedure; L-cys group, rats intraperitoneally injected with L-cys and then subjected to the GI-R procedure. Data presented as mean ± standard deviation (SD); n=8. ^**P<0.01 vs. the sham group; ^ΔP<0.05 vs. the PAG group. PAG, DL-propargylglycine; L-cys, L-cysteine; H₂S, hydrogen sulfide.

Figure 2.

Effects of PAG and L-cys on gastric mucosal injury scores in rats subjected to gastric ischemia-reperfusion (GI-R) (hematoxylin and eosin stain; magnification, ×100). Male Sprague-Dawley rats were intraperitoneally injected with the H₂S synthetase blocker PAG (50 mg/kg) or L-cys (50 mg/kg) for 7 days before the celiac arteries were clamped for 30 min ischemia and then reperfused for 60 min. Sham group, age-matched rats with physiological solute treatment but no GI-R procedure; GI-R group, age-matched rats with physiological solute treatment followed by the GI-R procedure; PAG group, rats intraperitoneally injected with PAG and then subjected to the GI-R procedure; L-cys group, rats intraperitoneally injected with L-cys and then subjected to the GI-R procedure. Scale bar, 200 μm. Data presented as mean ± standard deviation (SD), n=8. ^**P<0.01 vs. the sham group, ^#P<0.05 vs. the GI-R group, ^ΔP<0.05 vs. the PAG group. PAG, DL-propargylglycine; L-cys, L-cysteine; H₂S, hydrogen sulfide.

Figure 3.

Effects of PAG and L-cys on H₂S concentration in the gastric mucosa (A) and serum (B) from rats subjected to gastric ischemia-reperfusion (GI-R). Male Sprague-Dawley rats were intraperitoneally injected with the H₂S synthetase blocker PAG (50 mg/kg) or L-cys (50 mg/kg) for 7 days before the celiac arteries were clamped for 30 min ischemia and then reperfused for 60 min. Sham group, age-matched rats with physiological solute treatment but no GI-R procedure; GI-R group, age-matched rats with physiological solute treatment followed by the GI-R procedure; PAG group, rats intraperitoneally injected with PAG and then subjected to the GI-R procedure; L-cys group, rats intraperitoneally injected with L-cys and then subjected to the GI-R procedure. Data presented as mean ± standard deviation (SD), n=8. ^*P<0.05 vs. the sham group, ^#P<0.05 vs. the GI-R group, ^ΔP<0.05 vs. the PAG group. PAG, DL-propargylglycine; L-cys, L-cysteine; H₂S, hydrogen sulfide.

Figure 4.

Effects of PAG and L-cys on CSE expression in gastric mucosa from rats subjected to gastric ischemia-reperfusion (GI-R). Male Sprague-Dawley rats were intraperitoneally injected with the H₂S synthetase blocker PAG (50 mg/kg) or L-cys (50 mg/kg) for 7 days before the celiac arteries were clamped for 30 min ischemia and then reperfused for 60 min. Sham group, age-matched rats with physiological solute treatment but no GI-R procedure; GI-R group, age-matched rats with physiological solute treatment followed by the GI-R procedure; PAG group, rats intraperitoneally injected with PAG and then subjected to the GI-R procedure; L-cys group, rats intraperitoneally injected with L-cys and then subjected to the GI-R procedure. Data presented as mean ± standard deviation (SD), n=3. ^**P<0.01 vs. the sham group, ^##P<0.01 vs. the GI-R group, ^ΔΔP<0.01 vs. the PAG group. PAG, DL-propargylglycine; L-cys, L-cysteine; H₂S, hydrogen sulfide; CSE, cystathionine γ-lyase.

Figure 5.

Effects of PAG and L-cys on (A) MDA and (B) GSH content in gastric mucosa from rats subjected to gastric ischemia-reperfusion (GIR). Male Sprague-Dawley rats were intraperitoneally injected with the H₂S synthetase blocker PAG (50 mg/kg) or L-cys (50 mg/kg) for 7 days before the celiac arteries were clamped for 30 min ischemia and then reperfused for 60 min. Sham group, age-matched rats with physiological solute treatment but no GI-R procedure; GI-R group, age-matched rats with physiological solute treatment followed by the GI-R procedure; PAG group, rats intraperitoneally injected with PAG and then subjected to the GI-R procedure; L-cys group, rats intraperitoneally injected with L-cys and then subjected to the GI-R procedure. Data presented as mean ± standard deviation (SD), n=8. ^*P<0.05, ^**P<0.01 vs. the sham group; ^#P<0.05 vs. the GI-R group; ^ΔP<0.05, ^ΔΔP<0.01 vs. the PAG group. PAG, DL-propargylglycine; L-cys, L-cysteine; H₂S, hydrogen sulfide; MDA, malondialdehyde; GSH, glutathione.

Figure 6.

Alteration of oxidative stress in gastric mucosal tissue from rats subjected to 30 min gastric ischemia and 60 min reperfusion (GI-R). (A) Effects of PAG and L-cys on XOD expression in gastric mucosal tissue induced by GI-R (n=3). (B) Effects of PAG and L-cys on SOD expression in gastric mucosal tissue induced by GI-R (n=3). β-actin was used to normalize for loading variations. (C) Effects of PAG and L-cys on SOD activity in gastric mucosal tissue induced by GI-R (n=8). (D) Effects of PAG and L-cys on the alteration of O₂⁻ production in gastric mucosal tissue induced by GI-R. Male Sprague-Dawley rats (n=8) were intraperitoneally injected with the H₂S synthetase blocker PAG (50 mg/kg) or L-cys (50 mg/kg) for 7 days before the celiac arteries were clamped for 30 min ischemia and then reperfused for 60 min. Sham group, age-matched rats with physiological solute treatment but no GI-R procedure; GI-R group, age-matched rats with physiological solute treatment followed by the GI-R procedure; PAG group, rats intraperitoneally injected with PAG and then subjected to the GI-R procedure; L-cys group, rats intraperitoneally injected with L-cys and then subjected to the GI-R procedure. Data presented as mean ± standard deviation (SD). ^*P<0.05, ^**P<0.01 vs. the sham group, ^#P<0.05, ^##P<0.01 vs. the GI-R group, ^ΔΔP<0.01 vs. the PAG group. PAG, DL-propargylglycine; L-cys, L-cysteine; H₂S, hydrogen sulfide; XOD, xanthine oxidase; SOD, superoxide dismutase.