Role of receptor interacting protein (RIP)1 on apoptosis-inducing factor-mediated necroptosis during acetaminophen-evoked acute liver failure in mice

Highlights

• RIP1 is an early mediator of APAP-induced hepatocyte death.

• Nec-1 improved the survival and prevented from APAP-induced hepatocyte death.

• Nec-1 obviously inhibited APAP-induced hepatic JNK activation.

• Nec-1 attenuates APAP-induced mitochondrial Bax and nuclear AIF translocation.

• Nec-1 is an effective antidote for APAP-induced acute liver failure.

Abstract

Acetaminophen (APAP) overdose induces apoptosis-inducing factor (AIF)-dependent necroptosis, but the mechanism remains obscure. The present study investigated the role of receptor interacting protein (RIP)1, a critical mediator of necroptosis, on AIF-dependent necroptosis during APAP-induced acute liver failure. Mice were intraperitoneally injected with APAP (300 mg/kg). As expected, hepatic RIP1 was activated as early as 1 h after APAP, which is earlier than APAP-induced hepatic RIP3 upregulation. APAP-evoked RIP1 activation is associated with hepatic glutathione (GSH) depletion. Either pretreatment or post-treatment with Nec-1, a selective inhibitor of RIP1, significantly alleviated APAP-induced acute liver failure. Moreover, Nec-1 improved the survival and prevented APAP-induced necroptosis, as determined by TdT-mediated dUTP-biotin nick end labeling (TUNEL) assay. Further analysis showed that Nec-1 significantly inhibited APAP-induced hepatic c-Jun N-terminal kinase (JNK) phosphorylation and mitochondrial Bax translocation. In addition, Nec-1 blocked APAP-induced translocation of AIF from the mitochondria to the nucleus. Of interest, no changes were induced by Nec-1 on hepatic CYP2E1 expression. In addition, Nec-1 had little effect on APAP-induced hepatic GSH depletion at early stage. Taken together, these results suggest that RIP1 is involved in APAP-induced necroptosis. Nec-1 is an effective antidote for APAP-induced acute liver failure.

Introduction

Acetaminophen (APAP) is a widely used analgesic and antipyretic drug. Although safe at therapeutic doses, APAP overdose can cause severe acute liver failure (Jaeschke and Bajt, 2006). APAP-induced liver injury is initiated by the formation of a reactive metabolite, N-acetyl-p-benzoquinone imine (NAPQI), which can be generated by several cytochrome P450 (CYP) isoenzymes, especially CYP2E1 (Zaher et al., 1998). According to several reports, the prolonged activation of c-Jun N-terminal kinase (JNK) plays a key role in APAP-evoked hepatocyte death (Ghosh et al., 2010, Gunawan et al., 2006, Latchoumycandane et al., 2007). Moreover, apoptosis-inducing factor (AIF), which translocates to the nucleus and initiates nuclear DNA fragmentation, may be a critical mediator of hepatocyte death during APAP-induced acute liver failure (Bajt et al., 2006, Bajt et al., 2011, Ramachandran et al., 2011).

There are at least three major kinds of cell death: apoptosis, autophagy and necrosis (Galluzzi et al., 2012). Apoptosis and autophagy are a highly regulated process involving complex signaling networks (Carmona-Gutierrez et al., 2010, Kumar, 2007, Orrenius et al., 2011, Shen and Codogno, 2011). By contrast, necrosis is considered a passive, unregulated form of cell death, morphologically characterized by rounding of the cell, a gain in cell volume, organelle swelling, lack of internucleosomal DNA fragmentation, and plasma membrane rupture (Galluzzi et al., 2007). Increasing evidence demonstrates that some necrosis is evoked by regulated signal transduction pathways such as those mediated by receptor interacting protein (RIP) kinases when caspase cascade cannot be activated efficiently (Hitomi et al., 2008, Vandenabeele et al., 2010, Vanlangenakker et al., 2012). The regulated necrosis and in particular RIP1/RIP3-mediated necrosis, referred to as programmed necrosis or necroptosis, has been becoming an intensively studied form of programmed cell death (Kaiser et al., 2011, Osborn et al., 2010, Trichonas et al., 2010). According to a recent report, RIP3 is a critical mediator of necroptosis during APAP-induced acute liver failure (Ramachandran et al., 2013). Nevertheless, the role of RIP1 in the process of APAP-induced acute liver failure remains obscure.

In the present study, we hypothesize that RIP1 is involved in AIF-dependent necroptosis during APAP-induced acute liver failure. To test this hypothesis, we investigated the effects of necrostatin-1 (Nec-1), a potent and selective inhibitor of RIP1 kinase activity (Degterev et al., 2008, Xie et al., 2013), on APAP-induced necroptosis. We show that pretreatment or post-treatment with Nec-1 alleviated APAP-induced acute liver failure. Moreover, Nec-1 improved the survival and prevented APAP-induced necroptosis. In addition, Nec-1 inhibits APAP-induced hepatic JNK phosphorylation and mitochondrial Bax translocation. We demonstrated that RIP1 is involved in AIF-dependent necroptosis during APAP-induced acute liver failure. Nec-1 is an effective antidote for APAP-induced acute liver failure.