Dual role of chloroquine in liver ischemia reperfusion injury: reduction of liver damage in early phase, but aggravation in late phase

Abstract

The anti-malaria drug chloroquine is well known as autophagy inhibitor. Chloroquine has also been used as anti-inflammatory drugs to treat inflammatory diseases. We hypothesized that chloroquine could have a dual effect in liver ischemia/reperfusion (I/R) injury: chloroquine on the one hand could protect the liver against I/R injury via inhibition of inflammatory response, but on the other hand could aggravate liver I/R injury through inhibition of autophagy. Rats (n=6 per group) were pre-treated with chloroquine (60 mg/kg, i.p.) 1 h before warm ischemia, and they were continuously subjected to a daily chloroquine injection for up to 2 days. Rats were killed 0.5, 6, 24 and 48 h after reperfusion. At the early phase (i.e., 0-6 h after reperfusion), chloroquine treatment ameliorated liver I/R injury, as indicated by lower serum aminotransferase levels, lower hepatic inflammatory cytokines and fewer histopathologic changes. In contrast, chloroquine worsened liver injury at the late phase of reperfusion (i.e., 24-48 h after reperfusion). The mechanism of protective action of chloroquine appeared to involve its ability to modulate mitogen-activated protein kinase activation, reduce high-mobility group box 1 release and inflammatory cytokines production, whereas chloroquine worsened liver injury via inhibition of autophagy and induction of hepatic apoptosis at the late phase. In conclusion, chloroquine prevents ischemic liver damage at the early phase, but aggravates liver damage at the late phase in liver I/R injury. This dual role of chloroquine should be considered when using chloroquine as an inhibitor of inflammation or autophagy in I/R injury.

Figure 1

Chloroquine treatment prevented liver I/R injury at early phase, but worsened at late phase following I/R injury. Rats were pre-treated with either chloroquine (60 mg/kg) or vehicle (saline) 1 h before warm ischemia, followed by an additional chloroquine injections at 24 h after reperfusion. Following 60 min (a) or 90 min (b) of warm ischemia, liver injury was assessed at various time points following reperfusion by determination of serum AST and ALT levels. Data are shown as mean±S.D. *P<0.05 compared with vehicle-treated I/R group. (c) Routine histopathology was performed on formalin-fixed liver sections obtained from rats that had undergone 60 min of ischemia followed by 6- and 24-h reperfusion, respectively (original magnification × 400). Representative images from six rats per group were selected

Figure 2

Chloroquine treatment decreased liver expression of inflammatory mediators at early phase following I/R injury. Rats were treated with either chloroquine (60 mg/kg) or vehicle (saline) 1 h before warm ischemia. Rats were killed 0.5 and 6 h after reperfusion. TNF-α (a), IL-6 (b), IL-1β (c) and iNOS (d) mRNA expression levels were measured by quantitative PCR. Results were expressed as relative increase of mRNA expression compared with normal animals. Data are shown as mean±S.D. n=6 per group. *P<0.05 compared with vehicle-treated I/R group

Figure 3

Chloroquine treatment decreased HMGB1 release at early phase following I/R injury. Rats were treated with either chloroquine (60 mg/kg) or vehicle (saline) 1 h before warm ischemia. Liver samples and serum were harvested at 0.5 and 6 h post-reperfusion. (a) HMGB1 cellular location was visualized by immunohistochemical staining (original magnification × 200). Representative images from six rats per group were selected. (b) HMGB1-release into serum was quantified by ELISA. Data are shown as mean±S.D. n=6 per group. *P<0.01 compared with vehicle-treated I/R group

Figure 4

Chloroquine treatment modulated MAP kinase activation. Rats were pre-treated with chloroquine (60 mg/kg) 1 h before warm ischemia, and they were continuously subjected to a daily chloroquine injection for up to 2 days. (a) Western blot analysis for phosphorylation and total ERK, JNK and p38 was performed in the ischemic lobes at various time points following reperfusion. (b) The gray value of bands was calculated by Image J. Data are shown as mean±S.D. n=6 per group. *P<0.05 compared with vehicle-treated I/R group

Figure 5

Chloroquine treatment decreased hepatic autophagy following I/R injury. (a) Rats were treated with either chloroquine (60 mg/kg) or vehicle (saline). Liver samples were harvested at 0.5, 6, 24 and 48 h post-reperfusion. Western blot analysis for LC3 and p62 was performed in the ischemic lobes. (b) The gray value of bands was calculated by Image J. Data are shown as mean±S.D. n=6 per group. *P<0.05 compared with vehicle-treated I/R group

Figure 6

Chloroquine treatment increased hepatic apoptosis following I/R injury. (a) Rats were pre-treated with chloroquine (60 mg/kg) 1 h before warm ischemia, followed by an additional chloroquine injections at 24 h after reperfusion. Caspase-3 and caspase-7 cleavage was observed by western blots in the ischemic lobes at various time points following reperfusion. (b) The gray value of bands was calculated by Image J. Data are shown as mean±S.D. n=6 per group. *P<0.05 compared with vehicle-treated I/R group