Exogenous hydrogen sulfide protects against hepatic ischemia/reperfusion injury by inhibiting endoplasmic reticulum stress and cell apoptosis

Abstract

The aim of the present study was to explore the effect of exogenous hydrogen sulphide (H₂S) on endoplasmic reticulum (ER) stress (ERS) in a rat model of hepatic ischemia/reperfusion (I/R) injury. A total of 48 Sprague-Dawley rats were randomly divided into four groups (n=12/group) as follows: Sham, I/R, I/R preceded by NaHS (I/R-NaHS) and I/R preceded by L-C-propargylglycine (PAG), a H₂S inhibitor (I/R-PAG). With the exception of the sham group, the rats in the other groups were subjected to 30 min hepatic warm ischemia followed by reperfusion for 6 or 12 h. Hepatic function was evaluated by serum concentrations of alanine aminotransferase (ALT). Apoptosis of hepatic cells was assessed by TUNEL staining and measurement of caspase-12 expression. The expression levels of ERS-associated proteins and mRNAs of pancreatic ER eukaryotic translation initiation factor-2a kinase (PERK), activating transcription factor-6 (ATF6), glucose-regulated protein (GRP) 78, TNF-receptor-associated factor (TRAF)-2, C/EBP homologous protein (CHOP) and caspase-12 were also measured by western blotting and reverse transcription-quantitative PCR. The serum concentrations of ALT in the I/R and I/R-PAG groups were found to be significantly higher compared with those in the sham and I/R-NaHS groups after 6 h of reperfusion; in addition, the ALT level returned to normal in the I/R group, while it increased further in the I/R-PAG group after 12 h of reperfusion. A higher cell apoptosis rate was observed in the I/R and I/R-PAG groups and the highest cell apoptosis rate was observed in the I/R-PAG group; correspondingly, the expression of caspase-12 was increased in the I/R and I/R-PAG groups. H₂S appeared to significantly attenuate hepatic I/R-induced ERS response, as indicated by the decreased expression of ATF6, PERK, GRP78, TRAF2 and CHOP. Endogenous H₂S may serve a hepatoprotective function after I/R, and inhibition of endogenous H₂S results in aggravation of I/R damage. Exogenous H₂S was shown to inhibit ERS-related gene expression, leading to suppression of inflammatory reaction and improvement of I/R damage. Therefore, exogenous H₂S has therapeutic potential to alleviate hepatic I/R injury.

Keywords: endoplasmic reticulum stress; hepatic ischemia/reperfusion; hydrogen sulfide.

Figure 1

Effects of NaHS and PAG on serum ALT levels after hepatic I/R. Serum ALT level (IU/ml) was measured in sham-operated rats or in rats subjected to 30 min of hepatic ischemia followed by 6 and 12 h of reperfusion treated with saline (I/R group), NaHS (I/R-NaHS group) or PAG (I/R-PAG group). Each bar represents the mean ± SEM of three separate repeated experiments. ^*P<0.05 vs. sham group; ^#P<0.05 vs. 6 h; ^$P<0.05 vs. I/R group. PAG, L-C-propargylglycine; I/R, ischemia/reperfusion; ALT alanine aminotransferase.

Figure 2

Effects of NaHS and PAG on hepatocyte apoptosis following hepatic I/R injury. Livers were harvested at (A-D) 6 h and (E-H) 12 h after reperfusion and TUNEL assay was performed to detect the percentage of apoptotic cells in the (A and E) sham, (B and F) I/R, (C and G) I/R-NaHS and (D and H) I/R-PAG groups. Each bar represents the mean ± SEM of three separate repeated experiments. ^*P<0.05 vs. sham group; ^#P<0.05 vs. 6 h; ^$P<0.05 vs. I/R group. PAG, L-C-propargylglycine; I/R, ischemia/reperfusion.

Figure 3

Western blotting was used to examine the levels of the endoplasmic reticulum stress-associated ATF6, PERK, GRP78, TRAF2, CHOP and caspase-12 proteins. PERK, pancreatic ER eukaryotic translation initiation factor-2a kinase; ATF6, activating transcription factor-6; GRP78, glucose-regulated protein 78; TRAF2, TNF-receptor-associated factor 2; CHOP, C/EBP homologous protein.

Figure 4

Reverse transcription-qPCR analysis was used to examine the mRNA levels of endoplasmic reticulum stress-associated genes. Each bar represents the mean ± SEM of three separate repeated experiments. ^*P<0.05 vs. sham group; ^#P<0.05 vs. 6 h; ^$P<0.05 vs. I/R group. I/R, ischemia/reperfusion; PERK, pancreatic ER eukaryotic translation initiation factor-2a kinase; ATF6, activating transcription factor-6; GRP78, glucose-regulated protein 78; TRAF2, TNF-receptor-associated factor 2; CHOP, C/EBP homologous protein.