Chloroquine ameliorates carbon tetrachloride-induced acute liver injury in mice via the concomitant inhibition of inflammation and induction of apoptosis

Abstract

This is the first study to investigate the hepatoprotective effect of CQ on acute liver injury caused by carbon tetrachloride (CCl₄) in a murine model and the underlying molecular mechanisms. Ninety-six mice were randomly divided into the control (n = 8), CQ (n = 8), CCl₄ (n = 40), and CCl₄ + CQ (n = 40) treatment groups. In the CCl₄ group, mice were intraperitoneally (i.p) injected with 0.3% CCl₄ (10 mL/kg, dissolved in olive oil); in the CCl₄ + CQ group, mice were i.p injected with CQ at 50 mg/kg at 2, 24, and 48 h before CCl₄ administration. The mice in the control and CQ groups were administered with an equal vehicle or CQ (50 mg/kg). Mice were killed at 2, 6, 12, 24, 48 h post CCl₄ treatment and their livers were harvested for analysis. The results showed that CQ pre-treatment markedly inhibited CCl₄-induced acute liver injury, which was evidenced by decreased serum transaminase, aspartate transaminase and lower histological scores of liver injury. CQ pretreatment downregulated the CCl₄-induced hepatic tissue expression of high-mobility group box 1 (HMGB1) and the levels of serum HMGB1 as well as IL-6 and TNF-α. Furthermore, CQ pre-treatment inhibited autophagy, downregulated NF-kB expression, upregulated p53 expression, increased the ratio of Bax/Bcl-2, and increased the activation of caspase-3 in hepatic tissue. This is the first study to demonstrate that CQ ameliorates CCl₄-induced acute liver injury via the inhibition of HMGB1-mediated inflammatory responses and the stimulation of pro-apoptotic pathways to modulate the apoptotic and inflammatory responses associated with progress of liver damage.

Fig. 1. Chloroquine pretreatment attenuates CCl₄-induced acute liver injury in mice.

a Time course of ALT serum levels (n = 8). b Time course of AST serum levels (n = 8). c Representative histopathological images of H&E-stained liver sections from CCl₄-treated mice pretreated with vehicle or CQ. Bar = 100 μm. d The histological scores for liver sections from CCl₄-treated mice pretreated with vehicle or CQ (n = 4). Data are presented as mean ± SD. *P < 0.05 and **P < 0.01, compared with the control group; ^#P < 0.05 and ^##P < 0.01, compared with the CCl₄ + vehicle group

Fig. 2

The impact of chloroquine pretreatment on the changes of body weight and liver weight/body weight ratio following CCl4- induced acute liver injury in mice. a The changes of body weight. b The changes of liver weight/body weight ratio (%). Data are presented as mean ± SD (n = 8). **P < 0.01, compared with the CCl₄ alone group

Fig. 3. Chloroquine pretreatment attenuates CCl₄-induced lethal death in mice.

In the CCl₄ group, mice were ip injected with 2.5% CCl₄ with or withoutpre-treatment of CQ. The death ratios were recorded during 24 h. (n = 10 per group)

Fig. 4. Chloroquine pretreatment downregulates CCl₄-induced hepatic HMGB1 expression and serum levels.

a Western blotting analysis of HMGB1 protein expression in the liver tissue of mice from the vehicle and chloroquine (CQ) pretreated CCl₄ groups (n = 4). b Serum HMGB1 levels measured by ELISA in mice from the vehicle and CQ pre-treatment groups (n = 8) at 24 h. Data are presented as mean ± SD. *P < 0.05 and **P < 0.01, compared with the control group; ^#P < 0.05 and ^##P < 0.01, compared with the CCl₄ + vehicle group. c Representative immunohistochemical results of hepatocytic HMGB1 protein expression at 24 h (n = 4). Bar = 50 μm

Fig. 5. Effect of different dose of chloroquine on the levels of serum HMGB1 in mice exposed with CCl₄.

Serum HMGB1 levels measured by ELISA in mice from the vehicle and CQ pre-treatment groups at 24 h (n = 6). Data are presented as mean ± SD. *P < 0.05 and **P < 0.01, compared with the CCl₄ alone group

Fig. 6. Chloroquine pretreatment attenuates CCl₄-induced inflammatory responses.

a Serum levels of TNF-α. b Serum levels of IL-6. c Hepatic tissue expression levels of TNF-α mRNA. d Hepatic tissue expression levels of IL-6 mRNA. Data are presented as mean ± SD (n = 8). *P < 0.05 and **P < 0.01, compared with the control group; ^#P < 0.05 and ^##P < 0.01, compared with the CCl₄ + vehicle group

Fig. 7. The impact of chloroquine pretreatment on biomarkers of the MAPK family, NF-κB pathway, autophagy, and apoptosis following CCl₄- induced acute liver injury in mice.

a Western blot analysis for LC3, Beclin1, phosphorylation (p)-Erk, p- JNK, p-p38, p53, Bax, Bcl-2, NF-κB, IκBa, and cleaved caspase-3 were performed on the liver tissue of mice at 2, 6, 12, 24, and 48 h from the CCl₄ + vehicle and CCl₄ + chloroquine (CQ) groups (n = 3). b The densitometric analysis of the bands was conducted using Image J. Data are shown as mean ± SD (n = 3). *P < 0.05 and **P < 0.01, compared with the control group; ^#P < 0.05 and ^##P < 0.01, compared with the CCl₄ + vehicle group

Fig. 8. Effect of chloroquine pre-treatment on CCl₄-induced cell apoptosis in liver tissues.

a Representative TUNEL-stained sections showing apoptosis in the liver tissue of mice from the vehicle and chloroquine (CQ) pretreated CCl₄ groups (n = 4). Bar = 100 μm. b TUNEL-positive cells were counted and statistical analyses presented as mean ± SD. **P < 0.01, compared with the control group; ^#P < 0.05, compared with the CCl₄ + vehicle group

Fig. 9. CCl₄ blocks autophagy flux.

a p62 protein expression was examined in the liver tissue of mice at 2, 6, 12, 24, and 48 h following CCl₄ exposure. b HMGB1, p62, and LC3II protein expression was examined using western blotting (left panel) and the corresponding analysis (right panel). Data are shown as mean ± SD (n = 3). *P < 0.05 and **P < 0.01, compared between different group. c CCl₄ causes accumulation of autophagosomes by inhibiting the later stage of autophagy. Live-cell imaging of HepG2 cells transfected with a tandem mRFP-GFP-LC3 construct for 12 h or treated with 20 μM CQ for 12 h. Bar = 25 μm