Abstract
Acetaminophen (APAP) hepatotoxicity induces endoplasmic reticulum (ER) stress which triggers the unfolded protein response (UPR) in hepatocytes. However, the mechanisms underlying ER stress remain poorly understood, thus reducing the options for exploring new pharmacological therapies for patients with hyperacute liver injury. Eight-to-twelve-week-old C57BL/6J Xbp1-floxed (Xbp1f/f) and hepatocyte-specific knockout Xbp1 mice (Xbp1∆hepa) were challenged with either high dose APAP [500 mg/kg] and sacrificed at early (1–2 h) and late (24 h) stages of hepatotoxicity. Histopathological examination of livers, immunofluorescence and immunohistochemistry, Western blot, real time (RT)-qPCR studies and transmission electron microscopy (TEM) were performed. Pharmacological inhibition of XBP1 using pre-treatment with STF-083010 [STF, 75 mg/kg] and autophagy induction with Rapamycin [RAPA, 8 mg/kg] or blockade with Chloroquine [CQ, 60 mg/kg] was also undertaken in vivo. Cytoplasmic expression of XBP1 coincided with severity of human and murine hyperacute liver injury. Transcriptional and translational activation of the UPR and sustained activation of JNK1/2 were major events in APAP hepatotoxicity, both in a human hepatocytic cell line and in a preclinical model. Xbp1∆hepa livers showed decreased UPR and JNK1/2 activation but enhanced autophagy in response to high dose APAP. Additionally, blockade of XBP1 splicing by STF, mitigated APAP-induced liver injury and without non-specific off-target effects (e.g., CYP2E1 activity). Furthermore, enhanced autophagy might be responsible for modulating CYP2E1 activity in Xbp1∆hepa animals. Genetic and pharmacological inhibition of Xbp1 specifically in hepatocytes ameliorated APAP-induced liver injury by enhancing autophagy and decreasing CYP2E1 expression. These findings provide the basis for the therapeutic restoration of ER stress and/or induction of autophagy in patients with hyperacute liver injury.
Highlights
Because of its extensive worldwide use and narrow therapeutic window, acetaminophen (APAP) hepatotoxicity is considered a significant public health concern
Increased X-box binding protein 1 (XBP1) expression in human and murine APAP hepatoxicity To investigate whether XBP1 could play a role in the unfolded protein response (UPR) associated with APAP overdose, XBP1 expression was investigated in liver tissue sections obtained from patients undergoing emergency liver transplantation for APAP-induced acute liver failure (ALF)
We found that whilst most hepatocyte nuclei expressed XBP1, cytoplasmic expression of XBP1 became more evident as hepatocyte architecture was altered and serum transaminases increased in patients with APAP overdose
Summary
Because of its extensive worldwide use and narrow therapeutic window, acetaminophen (APAP) hepatotoxicity is considered a significant public health concern. The UPR consists of three primary pathways that sense the accumulation of unfolded or misfolded proteins These sensor protein-transcription factor pairs are: (i) Protein kinase RNA-like endoplasmic reticulum kinase (PERK) and eukaryotic Initiation Factor 2 alpha (eIF2α); (ii) the activating transcription factor 6 (ATF6); and (iii) Inositol requiring kinase 1α (IRE1α) and X-box binding protein 1 (XBP1) [3]. Overnight-fasted 8–12 week-old male Xbp1f/f and Xbp1Δhepa mice were challenged with an i.p. injection of APAP [500 mg/kg] or vehicle. Since it is a high dose and APAP is insoluble in water, APAP was first dissolved in DMSO diluted to 50 mg/ml with PBS (final concentration of DMSO < 1%). Control mice received an equivalent i.p. volume of PBS with DMSO
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