Volume 135, Issue 4, Pages 1311-1321 (October 2008) Deletion of Apoptosis Signal-Regulating Kinase 1 Attenuates Acetaminophen-Induced Liver Injury by Inhibiting c-Jun N-Terminal Kinase Activation  Hayato Nakagawa, Shin Maeda, Yohko Hikiba, Tomoya Ohmae, Wataru Shibata, Ayako Yanai, Kei Sakamoto, Keiji Ogura, Takuya Noguchi, Michael Karin, Hidenori Ichijo, Masao Omata  Gastroenterology  Volume 135, Issue 4, Pages 1311-1321 (October 2008) DOI: 10.1053/j.gastro.2008.07.006 Copyright © 2008 AGA Institute Terms and Conditions

Figure 1 Analysis of APAP-induced acute liver injury in WT, ASK1−/−, JNK1−/−, JNK2−/−, and JNK inhibitor (15 mg/kg)–pretreated mice. (A) Serum ALT levels at 6 and 24 hours after 300 mg/kg APAP treatment (WT and ASK1−/− mice, n = 8; JNK1−/−, JNK2−/−, and JNK inhibitor–pretreated mice, n = 5). (B) Liver histology with H&E staining at 24 hours post-treatment. (C) Histopathology scores at 6 and 24 hours post-treatment. (A and C) *P < .05 compared with WT mice at 6 hours; P < .05 compared with WT mice at 24 hours. (D) TUNEL-stained liver sections at 6 hours post-treatment. (E) Average number of TUNEL-positive hepatocytes per low-power field (lpf) at 6 hours post-treatment (n = 3 for each group). *P < .05 compared with WT mice. (F) Total hepatic GSH levels at baseline and at 1.5 and 6 hours post-treatment (baseline GSH, n = 3; GSH after APAP administration, n = 5). Baseline GSH was measured in mice killed 1.5 hours after vehicle only administration. *P < .05 compared with WT mice at 6 hours. Gastroenterology 2008 135, 1311-1321DOI: (10.1053/j.gastro.2008.07.006) Copyright © 2008 AGA Institute Terms and Conditions

Figure 2 Gene expression profiles influenced by APAP overdose in the liver. (A) Among the genes that showed similar expression in WT and ASK1−/− untreated liver (<1.3-fold increased and <1.3-fold decreased), 959 genes showed a marked change in expression after APAP administration in WT mouse liver (>3-fold up-regulated or >3-fold down-regulated). (B) ASK1-dependent genes in APAP-induced liver injury; 140 genes were induced in WT mouse liver to a greater extent than in ASK1−/− mouse liver (>2.5-fold up-regulated). The gene list is available in supplementary Table 1 (see supplementary material online at www.gastrojournal.org). (C) Representative data for ASK1-dependent genes are given. Gastroenterology 2008 135, 1311-1321DOI: (10.1053/j.gastro.2008.07.006) Copyright © 2008 AGA Institute Terms and Conditions

Figure 3 Analysis of ASK1, MKK4, and JNK activation in mouse liver after APAP administration. (A) ASK1 activation after 300 mg/kg APAP treatment in WT mouse liver. (B) Analysis of Trx−ASK1 complex dissociation. Total liver lysates were immunoprecipitated with ASK1 antibody and analyzed for the dissociation of Trx from ASK1 by immunoblotting with ASK1 and Trx antibodies. Representative data are shown from 5 experiments. (C) Comparison of MKK4 and JNK activation in WT, ASK1−/−, JNK1−/−, JNK2−/−, and JNK inhibitor–pretreated mouse liver at 6 hours post-treatment. Control mice were killed at 6 hours after vehicle control administration. β-actin was used as a loading control. (D) Time course of JNK activation in WT and ASK1−/− mouse liver after 300 mg/kg APAP treatment. Representative data are shown from 3 experiments. Numbers below the immunoblots represent the fold-increase in JNK phosphorylation (1.0 activity in untreated WT mice). Gastroenterology 2008 135, 1311-1321DOI: (10.1053/j.gastro.2008.07.006) Copyright © 2008 AGA Institute Terms and Conditions

Figure 4 Effect of ASK1 deletion on APAP-induced p38 activation and involvement of the p38 pathway in APAP-induced liver injury. (A) Western blot analysis showing the time course of p38 activation in WT and ASK1−/− mice livers after 300 mg/kg APAP treatment. Representative data are shown from 3 experiments. (B) Western blot analysis of total and phosphorylated p38 protein levels in WT and p38α+/– mice livers at 6 hours post-treatment. (C) Serum ALT levels at 6 hours post-treatment in WT and p38α+/– mice (n = 4 for each group). (D) Serum ALT levels at 6 and 24 hours posttreatment in mice pretreated with 15 mg/kg p38 inhibitor or vehicle only (n = 4 for each group). Gastroenterology 2008 135, 1311-1321DOI: (10.1053/j.gastro.2008.07.006) Copyright © 2008 AGA Institute Terms and Conditions

Figure 5 Analysis of signal transduction and hepatocellular damage by APAP in mouse primary hepatocytes. (A) ASK1 activation by APAP in primary hepatocytes. Primary hepatocytes isolated from WT mice were incubated with 5 mmol/L APAP, and ASK1 activation was determined by Western blotting. (B) Effect of ASK1 deletion on JNK and p38 activation in primary hepatocytes. Primary hepatocytes isolated from WT or ASK1−/− mice were incubated in 5 mmol/L APAP. Activation of JNK and p38 was examined by Western blotting. (C) Effect of ASK1 deletion on hepatocellular damage by APAP. Primary WT or ASK1−/− hepatocytes were incubated with 10 mmol/L APAP for 24 hours and cytotoxicity was assessed using a modified 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. *P < .05 compared with WT hepatocytes. (D) Effects of p38 inhibitor and JNK inhibitor on APAP-induced hepatocellular damage. Primary WT hepatocytes were pretreated with 15 μmol/L SB203580, 15 μmol/L SP600125, or dimethyl sulfoxide (dmso) only 1 hour before 10 mmol/L APAP administration. Cytotoxicity was assessed using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay at 24 hours after APAP administration. *P < .05 compared with the DMSO control. Gastroenterology 2008 135, 1311-1321DOI: (10.1053/j.gastro.2008.07.006) Copyright © 2008 AGA Institute Terms and Conditions

Figure 6 Involvement of ASK1 in the inflammatory response during APAP-induced liver injury. Relative mRNA levels of (A) IL-1α, (B) IL-1β, and (C) IL-6 at 6 hours after 300 mg/kg APAP treatment in WT or ASK1−/− mouse liver was analyzed by real-time PCR (n = 5 for each group). Data are expressed as fold increases. *P < .05 compared with WT mice. (D) Primary splenocytes isolated from WT and ASK1−/− mice were stimulated with necrotic hepatocytes for 24 hours, and enzyme-linked immunosorbent assay was used to measure IL-6 concentration in the supernatant. (E) Western blot analysis of necrotic hepatocyte-induced JNK and p38 activation in primary splenocytes. Gastroenterology 2008 135, 1311-1321DOI: (10.1053/j.gastro.2008.07.006) Copyright © 2008 AGA Institute Terms and Conditions