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

Fang, H; Liu, A; Dahmen, U; Dirsch, O

To elucidate the molecular basis of chloroquine-mediated injury in the late phase in liver I/R injury, we investigated its effect on the activation of autophagy. Autophagy is a highly conserved regulated cellular process by which cytoplasmic constituents, including organelles are broken down and are recycled through the lysosomal degradation pathway. 23, 24 The execution of autophagy involves a set of evolutionarily conserved autophagy-related gene (Atg) proteins, which are required for the induction of autpophagy, and the generation, maturation, and recycling of autophagosomes. 35 LC3, a homologue of yeast Atg8, exists in two forms, LC3-I and its proteolytic derivative LC3-II. After synthesis, pro-LC3 is cleaved by Atg4B to expose a C-terminal glycine residue, which represents the cytosolic LC3-I form. LC3-1 is subsequently conjugated with phosphatidylethanolamine to become LC3-II, which localizes on the isolation membrane and the autophagic membranes. As the amount of LC3-II correlates with the number of autophagosomes, LC3-II thus can be used to estimate autophagic activity. 36 p62 is selectively incorporated into autophagosomes via direct binding to LC3 and is identified as one of the specific substrates that degrade through the autophagy-lysosomal pathway. Thus, the total cellular expression levels of p62 inversely correlate with autophagic activity. 36 Chloroquine has been recently used to inhibit autophagy in vivo without noticeable side effects. Chloroquine raises the lysosomal pH, which leads to inhibition of lysosome-autophagosome fusion and lysosomal protein degradation. In this study, chloroquine blocked autophagic flux as shown by LC3-II and P62 accumulation and aggravated liver I/R injury after reperfusion. Impaired autophagy leads to the development of MPT in hypoxic hepatocytes. 37 Onset of the MPT leads to a pH-dependent necrotic cell death as well as apoptotic cell death by releasing proapoptotic proteins factors, which are normally sequestered in the mitochondrial intermembrane space. [38] [39] [40] Indeed, induction of autophagy is associated with attenuation of I/R injury in liver, as demonstrated recently. 21, 41, 42 As treatment with chloroquine significantly decreased autophagy and worsened liver injury, we concluded that the decreased autophagy accounted, in part, for the hepatic harmful effects of chloroquine in the late phase after reperfusion.