Inflammatory cascades driven by tumor necrosis factor-alpha play a major role in the progression of acute liver failure and its neurological complications

Abstract

Background/aims: Acute liver failure (ALF) due to ischemic or toxic liver injury is a clinical condition that results from massive loss of hepatocytes and may lead to hepatic encephalopathy (HE), a serious neuropsychiatric complication. Although increased expression of tumor necrosis factor-alpha (TNF-α) in liver, plasma and brain has been observed, conflicting results exist concerning its roles in drug-induced liver injury and on the progression of HE. The present study aimed to investigate the therapeutic value of etanercept, a TNF-α neutralizing molecule, on the progression of liver injury and HE in mice with ALF resulting from azoxymethane (AOM) hepatotoxicity.

Methods/principal findings: Mice were administered saline or etanercept (10 mg/kg; i.p.) 30 minutes prior to, or up to 6 h after AOM. Etanercept-treated ALF mice were sacrificed in parallel with vehicle-treated comatose ALF mice and controls. AOM induced severe hepatic necrosis, leading to HE, and etanercept administered prior or up to 3 h after AOM significantly delayed the onset of coma stages of HE. Etanercept pretreatment attenuated AOM-induced liver injury, as assessed by histological examination, plasma ammonia and transaminase levels, and by hepatic glutathione content. Peripheral inflammation was significantly reduced by etanercept as shown by decreased plasma IL-6 (4.1-fold; p<0.001) and CD40L levels (3.7-fold; p<0.001) compared to saline-treated ALF mice. Etanercept also decreased IL-6 levels in brain (1.2-fold; p<0.05), attenuated microglial activation (assessed by OX-42 immunoreactivity), and increased brain glutathione concentrations.

Conclusions: These results indicate that systemic sequestration of TNF-α attenuates both peripheral and cerebral inflammation leading to delayed progression of liver disease and HE in mice with ALF due to toxic liver injury. These results suggest that etanercept may provide a novel therapeutic approach for the management of ALF patients awaiting liver transplantation.

Figure 1. Etanercept significantly delays the progression of hepatic encephalopathy in mice with AOM-induced ALF.

Time to coma (loss of corneal reflex) in ALF mice treated with etanercept (ETA) 30 min prior to, or 3 h and 6 h after AOM treatment. Data represent mean ± SEM of n = 6 animals in each group. *p<0.001 vs. Saline.

Figure 2. Etanercept attenuates liver damage in mice with AOM-induced ALF.

Etanercept was administered 30 min before AOM. Representative liver sections from (A) saline-treated control mice, (B) AOM-induced ALF comatose mice and (C and D) etanercept-treated ALF mice. ALF mice show areas of confluent hemorrhagic necrosis, microvesicular steatosis as well as sinusoidal dilatation and congestion. Note the presence of minor microvesicular steatosis with little necrosis and sinusoidal congestion following etanercept treatment. Representative pictures from n = 6 mice are shown. Scale bar : 50 µm.

Figure 3. Etanercept treatment significantly attenuates plasma TNF-α, IL-6 and CD40L in mice with AOM-induced ALF.

Etanercept was administered 30 min before AOM. Plasma TNF-α (A), IL-6 (B) and CD40L (C) were quantified by ELISA. Data represent mean ± SEM of n = 12 animals in each group for IL-6, and n = 6 for TNF-α and CD40L. **p<0.001 vs. Saline; †p<0.05 vs. AOM; ††p<0.001 vs. AOM.

Figure 4. Etanercept treatment attenuates IL-6 levels and microglial activation in the brain of mice with ALF.

Etanercept was administered 30 min before AOM. (A) Brain IL-6 levels were measured in cytosolic fractions of cerebral cortex by ELISA. Data represent mean ± SEM of n = 6 animals in each group. *p<0.01 vs. Saline; **p<0.001 vs. Saline; †p<0.05 vs. AOM. (B) Representative micrographs showing the effect of ALF on OX-42 (CD11b) staining in cerebral cortex from saline-treated control, AOM-treated mice at coma stages of encephalopathy, and etanercept-treated ALF mice. Scale bar : 100 µm.

Figure 5. Western blot analysis of human IgG in the cerebral cortex of etanercept-treated AOM mice.

Figure 6. Etanercept treatment attenuates oxidative stress in liver and brain.

Etanercept was administered 30 min before AOM. Total glutathione (GSH_t) and GSH/GSSG ratio were determined in liver (A and B, respectively) and brain (C and D, respectively). Data represent mean ± SEM of n = 6 (liver), n = 12 (brain) and are expressed as µmol/g wet weight (A and C). *p<0.05 vs. Saline; **p<0.001 vs. Saline; †p<0.05 vs. AOM.