Acute ethanol gavage attenuates hemorrhage/resuscitation-induced hepatic oxidative stress in rats

Abstract

Acute ethanol intoxication increases the production of reactive oxygen species (ROS). Hemorrhagic shock with subsequent resuscitation (H/R) also induces ROS resulting in cellular and hepatic damage in vivo. We examined the role of acute ethanol intoxication upon oxidative stress and subsequent hepatic cell death after H/R. 14 h before H/R, rats were gavaged with single dose of ethanol or saline (5 g/kg, EtOH and ctrl; H/R_EtOH or H/R_ctrl, resp.). Then, rats were hemorrhaged to a mean arterial blood pressure of 30 ± 2 mmHg for 60 min and resuscitated. Two control groups underwent surgical procedures without H/R (sham_ctrl and sham_EtOH, resp.). Liver tissues were harvested at 2, 24, and 72 h after resuscitation. EtOH-gavage induced histological picture of acute fatty liver. Hepatic oxidative (4-hydroxynonenal, 4-HNE) and nitrosative (3-nitrotyrosine, 3-NT) stress were significantly reduced in EtOH-gavaged rats compared to controls after H/R. Proapoptotic caspase-8 and Bax expressions were markedly diminished in EtOH-gavaged animals compared with controls 2 h after resuscitation. EtOH-gavage increased antiapoptotic Bcl-2 gene expression compared with controls 2 h after resuscitation. iNOS protein expression increased following H/R but was attenuated in EtOH-gavaged animals after H/R. Taken together, the data suggest that acute EtOH-gavage may attenuate H/R-induced oxidative stress thereby reducing cellular injury in rat liver.

Figure 1

Acute ethanol gavage decreases lipid peroxidation after hemorrhage and resuscitation. Rats were gavaged with saline (ctrl) or ethanol (5 g/kg, 30% EtOH) 12 h before hemorrhagic shock and resuscitation (H/R). Sham-operated animals underwent the same surgical procedures but H/R was not carried out. Immunohistochemical staining for 4-hydroxynonenal (4-HNE) reveals areas of lipid peroxidation. 4-HNE-stained, representative liver sections from sham-operated rats are given in (a) (ctrl) and (e) (EtOH). 4-HNE-stained, representative liver sections from rats at 2 h, 24 h, and 72 h after H/R are given in (c)-(d) (ctrl) and (f)–(h) (EtOH); bar is 50 μm.

Figure 2

Acute ethanol gavage decreases protein nitrosylation after hemorrhage and resuscitation. Immunohistochemical staining for 3-Nitrotyrosine (3-NT) reveals areas of protein nitrosylation. 3-NT-stained, representative liver sections from sham-operated rats are given in (a) (saline control, ctrl) and (e) (acute ethanol gavage, EtOH). 3-NT-stained, representative liver sections from rats at 2 h, 24 h, and 72 h after H/R are given in (c)-(d) (saline control treatment, ctrl) and (f)–(h) (acute ethanol gavage, EtOH); bar is 50 μm.

Figure 3

Semiquantitative analysis of lipid peroxidation and protein nitrosylation after acute ethanol gavage after hemorrhage and resuscitation in a time course. The percentage of 4-HNE (a) and 3-NT (b) positive cells (staining) was semiquantified in liver sections 2 h, 24 h, and 72 h after H/R in a blinded manner. Ctrl: saline gavage, EtOH: ethanol gavage, sham: surgical procedure without the onset of H/R and H/R: rats undergoing hemorrhagic shock with subsequent resuscitation (*P < 0.05 versus both sham groups, ^# P < 0.05 ctrl_H/R_2 h versus EtOH_H/R_2 h group, ^¥ P < 0.05 ctrl_H/R_24 h versus EtOH_H/R_24 h group, n = 3 for 24 h groups, and n = 6 for other groups).

Figure 4

Effects of acute ethanol gavage on hepatic gene expression of BAX, Bcl-2, and caspase 8 at 2 h, 24 h, and 72 h after H/R. Hepatic gene expression of Bax, Bcl-2 (a), and caspase 8 (b) was analyzed. After normalization to GAPDH expression, the ratio of Bcl-2 : Bax gene expression was determined and caspase 8 gene expression was measured as fold change compared to sham-operated control group. Ctrl: saline gavage, EtOH: ethanol gavage, sham: surgical procedure without the onset of H/R and H/R: rats undergoing hemorrhagic shock with subsequent resuscitation (*P < 0.05 versus other groups, n = 3 for 24 h groups, and n = 6 for other groups).

Figure 5

Acute ethanol exposure reduces hepatic iNOS gene expression early after H/R. Saline (Ctrl) or ethanol (EtOH) gavaged rats were subjected to H/R or sham operation. 2 h, 24 h, and 72 h after the end of resuscitation, liver tissue was harvested and western blotting for iNOS and β-actin was performed. (a): lanes 1–6 depict liver protein extracts from rats after sham operation (sham, lanes 1–3: ctrl time course, lane 4–6: EtOH gavage time course) or H/R (lanes 7–12: ctrl time course, lanes 13–18: EtOH gavage time course). In (b), densitometric measurement after normalization to β-actin staining is plotted (*P < 0.05 versus sham and corresponding H/R_EtOH group, n = 3 for 24 h groups, and n = 6 for other groups, representative gel from 3 experiments is shown).