Dependence of Liver Injury After Hemorrhage/Resuscitation in Mice on NADPH Oxidase-Derived Superoxide

Hemorrhagic shock and resuscitation cause hepatocellular damage by mechanisms involving oxidative stress. However, the sources of free radicals mediating hepatocellular injury remain controversial. Thus, this study tested the hypothesis that NADPH oxidase plays a role in producing hepatocellular injury after hemorrhagic shock and resuscitation. Both wild-type and NADPH oxidase-deficient mice (p47phox knockout mice) were subjected to hemorrhagic shock (3 h at 30 mmHg). The mice were resuscitated over 30 min with the shed blood and additional lactated Ringer's solution (50% of the shed blood volume). Serum alanine aminotransferase (ALT) levels increased at 1 and 6 h postresuscitation in wild-type animals to 4735 ± 1017 IU/L and 1450 ± 275 IU/L (mean ± SE), respectively, whereas in knockout mice, this ALT increase was blunted at both time points (732 ± 241 IU/L and 328 ± 69 IU/L, P < 0.05). Liver necrosis assessed histologically 6 h after the end of reperfusion was also attenuated in the knockout mice (3.5% ± 0.95% of area vs. 0.9% ± 0.26%, P < 0.05). In hemorrhaged wild-type mice, infiltrating neutrophils were twice as numerous compared with hemorrhaged NADPH oxidase-deficient animals 6 h after reperfusion. In knockout animals, hepatic 4-hydroxynonenal content, indicative of lipid peroxidation from reactive oxygen species, was blunted (6.7% ± 0.6% vs. 26.4% ± 2.3% of stained area, P < 0.05), as shown by immunohistochemistry. Immunohistochemical staining for 3-nitrotyrosine, indicative of reactive nitrogen species formation, was also blunted in the livers of knockout mice (11.6% ± 2.8% vs. 37.4% ± 3.4, P < 0.05). In conclusion, hemorrhagic shock and resuscitation cause hepatocellular damage via NADPH oxidase-mediated oxidative stress. The absence of NADPH oxidase substantially attenuates hepatocellular injury after hemorrhagic shock and resuscitation, blunts neutrophil infiltration, and decreases formation of reactive oxygen and reactive nitrogen species.