Gender differences in hepatic ischemic reperfusion injury in rats are associated with endothelial cell nitric oxide synthase-derived nitric oxide

Abstract

Aim: This study was designed to examine the hypothesis that gender differences in I/R injury are associated with endothelial cell nitric oxide synthase (eNOS)-derived nitric oxide (NO).

Methods: Wistar rats were randomized into seven experimental groups (12 animals per group). Except for the sham operated groups, all rats were subjected to total liver ischemia for 40 min followed by reperfusion. All experimental groups received different treatments 45 min before the laparotomy. For each group, half of the animals (six) were used to investigate the survival; blood samples and liver tissues were obtained in the remaining six animals after 3 h of reperfusion to assess serum NO, alanine aminotransferase (ALT) and TNF-alpha levels, liver tissue malondialdehyde (MDA) content, and severity of hepatic I/R injury.

Results: Basal serum NO levels in female sham operated (FS) group were nearly 1.5-fold of male sham operated (MS) group (66.7+/-11.0 micromol/L vs 45.3+/-10.1 micromol/L, P<0.01). Although serum NO levels decreased significantly after hepatic I/R (P<0.01, vs sham operated groups), they were still significantly higher in female rat (F) group than in male rat (M) group (47.8+/-8.6 micromol/L vs 23.8+/-4.7 micromol/L, P<0.01). Serum ALT and TNF-alpha levels, and liver tissue MDA content were significantly lower in F group than in M group (370.5+/-46.4 U/L, 0.99+/-0.11 microg/L and 0.57+/-0.10 micromol/g vs 668.7+/-78.7 U/L, 1.71+/-0.18 microg/L and 0.86+/-0.11 micromol/g, respectively, P<0.01). I/R induced significant injury to the liver both in M and F groups (P<0.01 vs sham operated groups). But the degree of hepatocyte injury was significantly milder in F group than in M group (P<0.05 and P<0.01). The median survival time was six days in F group and one day in M group. The overall survival rate was significantly higher in F group than in M group (P<0.05). When compared with male rats pretreated with saline (M group), pretreatment of male rats with 17-beta-estradiol (E2) (M+E2 group) significantly increased serum NO levels and significantly decreased serum ALT and TNF-alpha levels, and liver tissue MDA content after I/R (P<0.01). The degree of hepatocyte injury was significantly decreased and the overall survival rate was significantly improved in M+E2 group than in M group (P<0.01 and P<0.05). The NOS inhibitor N(w) -nitro-L-arginine methyl ester (L-NAME) treatment could completely abolish the protective effects of estrogen in both male and female rats.

Conclusion: The protective effects afforded to female rats subjected to hepatic I/R are associated with eNOS-derived NO.

Figure 1

Serum ALT levels (n = 6, mean±SD). There were no significant differences between MS and FS groups with respect to serum ALT levels. Serum ALT levels increased significantly after hepatic I/R in M or F group vs MS or FS group (^bP<0.01 or ^dP<0.01, respectively), but they were significantly lower in F group vs M group rats (^fP<0.01). When compared with male rats pretreated with saline (M group), pretreatment of male rats with E₂ (M+E₂ group) significantly decreased serum ALT levels after hepatic I/R (^hP<0.01). Pretreatment with L-NAME in female rats or co-pretreatment with E₂ and L-NAME in male rats significantly increased serum ALT levels vs those in F or M+E₂ group rats, respectively, after hepatic I/R (^jP<0.01 or ^lP<0.01).

Figure 2

Serum TNF-α levels (n = 6, mean±SD). No significant differences existed between MS and FS groups with respect to serum TNF-α levels. Serum TNF-α levels increased significantly after hepatic I/R in M or F group vs MS or FS group (^bP<0.01 or ^dP<0.01, respectively), but they were significantly lower in F group vs M group rats (^fP<0.01). When compared with male rats pretreated with saline (M group), pretreatment of male rats with E₂ (M+E₂ group) significantly decreased serum TNF-α levels after hepatic I/R (^hP<0.01). Pretreatment with L-NAME in female rats or co-pretreatment with E₂ and L-NAME in male rats significantly increased serum TNF-α levels vs those in F or M+E₂ group animals, respectively, after hepatic I/R (^jP<0.01 or ^lP<0.01).

Figure 3

Liver tissue MDA content (n = 6, mean±SD). The differences of liver tissue MDA content between MS and FS groups were not significant. Liver tissue MDA content increased significantly after hepatic I/R in M or F group vs MS or FS group (^bP<0.01 or ^dP<0.01, respectively), but they were significantly lower in F group vs M group rats (^fP<0.01). When compared with male rats pretreated with saline (M group), pretreatment of male rats with E₂ (M+E₂ group) significantly decreased liver tissue MDA content after hepatic I/R (^hP<0.01). Pretreatment with L-NAME in female rats or co-pretreatment with E₂ and L-NAME in male rats significantly increased liver tissue MDA content vs that in F or M+E₂ group animals, respectively, after hepatic I/R (^jP<0.01 or ^lP<0.01).

Figure 4

Serum NO₂^-/NO₃^- levels (n = 6, mean±SD). Basal serum NO levels were significantly higher in FS group than those in MS group (^bP<0.01). Although serum NO levels decreased significantly after hepatic I/R in M or F group vs MS or FS group (^dP<0.01 or ^fP<0.01, respectively), they were still significantly higher in F group than those in M group rats (^hP<0.01). When compared with male rats pretreated with saline (M group), pretreatment of male rats with E₂ (M+E₂ group) significantly increased serum NO levels after hepatic I/R (^jP<0.01). Pretreatment with L-NAME in female rats or co-pretreatment with E₂ and L-NAME in male rats significantly decreased serum NO levels vs those in F or M+E₂ group rats, respectively, after hepatic I/R (^lP<0.01 or ⁿP<0.01).