Hydrogen sulfide attenuates hepatic ischemia-reperfusion injury: role of antioxidant and antiapoptotic signaling

Abstract

Hydrogen sulfide (H(2)S) is an endogenously produced gaseous signaling molecule with diverse physiological activity. The potential protective effects of H(2)S have not been evaluated in the liver. The purpose of the current study was to investigate if H(2)S could afford hepatoprotection in a murine model of hepatic ischemia-reperfusion (I/R) injury. Hepatic injury was achieved by subjecting mice to 60 min of ischemia followed by 5 h of reperfusion. H(2)S donor (IK1001) or vehicle were administered 5 min before reperfusion. H(2)S attenuated the elevation in serum alanine aminotransferase (ALT) by 68.6% and aspartate aminotransferase (AST) by 70.8% compared with vehicle group. H(2)S-mediated cytoprotection was associated with an improved balance between reduced glutathione (GSH) vs. oxidized glutathione (GSSG), an attenuated formation of lipid hydroperoxides, and an increased expression of thioredoxin-1 (Trx-1). Furthermore, H(2)S inhibited the progression of apoptosis after I/R injury by increasing the protein expression of heat shock protein (HSP-90) and Bcl-2. These results indicate that H(2)S protects the murine liver against I/R injury through an upregulation of intracellular antioxidant and antiapoptotic signaling pathways.

Fig. 1.

Serum transaminases were measured in mice after 60 min of hepatic ischemia and 5 h of reperfusion. A: aspartate aminotransferase (AST) levels (U/l); B: alanine aminotransferase (ALT levels; U/l). Hydrogen sulfide (H₂S) donor therapy (0.3 and 1.0 mg/kg) significantly reduced hepatic release of AST and ALT. *P < 0.05 vs. vehicle; **P < 0.01 vs. vehicle; ***P < 0.001 vs. vehicle; numbers inside bars represent number of animals investigated in each group.

Fig. 2.

A: hepatic tissue reduced glutathione (GSH)-to-oxidized glutathione (GSSG) ratio at 1, 5, and 24 h after 60 min of ischemia. Data are presented for sham-operated controls, mice receiving vehicle (Veh), and mice treated with the H₂S donor (1.0 mg/kg). H₂S therapy significantly preserved the GSH-to-GSSG ratio compared with the vehicle group. B: representative immunoblot and densitometric analysis (bottom) of hepatic thioredoxin-1 (Trx-1) protein expression. Trx-1 protein levels remained significantly elevated in animals receiving H₂S at 24 h after reperfusion (Rep). **P < 0.01 vs. sham; ***P < 0.001 vs. sham; n = 6 in each group.

Fig. 3.

Lipid hydroperoxide levels (μM) in hepatic tissue lyaste after 1 and 5 h of reperfusion after 60 min of ischemia. Treatment with H₂S (1.0 mg/kg) significantly attenuated hepatic formation of lipid hydroperoxides. **P < 0.01 vs. sham; ***P < 0.001 vs. sham; numbers inside bars represent number of animals investigated in each group.

Fig. 4.

A and B: 90-kDa heat shock protein (HSP-90) protein expression in hepatic tissue, representative immunoblots, and densitometric analysis are shown. H₂S therapy (1.0 mg/kg) significantly increased HSP-90 protein expression at both 5 and 24 h of repefusion compared with vehicle group. C and D: representative immunoblot and densitometric analysis of hepatic Bcl-2 protein expression in the mitochondrial fraction at 5 and 24 h of reperfusion. Bcl-2 protein expression decreased significantly in both vehicle and H₂S groups. However, Bcl-2 levels were relatively preserved in animals treated with H₂S donor therapy. COX-IV, cytochrome c oxidase. **P < 0.01 vs. sham; ***P < 0.001 vs. sham; n = 6 for each group.

Fig. 5.

Representative hepatic tissue immunoblot (A) and densitometric analysis (B) show expression of cleaved caspase-3 at 24 h reperfusion in sham, vehicle, and H₂S donor groups. Cleaved caspase-3 was significantly elevated in vehicle group compared with sham-operated controls. The H₂S donor (1.0 mg/kg) attenuated the increase in hepatic cleaved caspase-3. **P < 0.01 vs. sham; numbers inside bars represent number of animals investigated in each group.