JNK mediates hepatic ischemia reperfusion injury Tetsuya Uehara, Brydon Bennett, Steve T. Sakata, Yoshitaka Satoh, Graham K. Bilter, John K. Westwick, David A. Brenner  Journal of Hepatology  Volume 42, Issue 6, Pages 850-859 (June 2005) DOI: 10.1016/j.jhep.2005.01.030 Copyright © 2005 European Association for the Study of the Liver Terms and Conditions

Fig. 1 JNK inhibitors increase survival in hepatic I/R. Animal survival is evaluated using a model of total hepatic ischemia. Rats (n=10) are injected intravenously with vehicle, CC0209766 (20mg/kg), or CC0223105 (20mg/kg) 15min before 90min ischemia and 4h after reperfusion (A). In a second series, rats (n=5) are injected with vehicle only or CC-401 at different dosage (20, 10mg/kg) (B). In a third series, rats are injected with CC-401 only 15min before ischemia at several dosages (20, 10, 5, 3mg/kg) (C). Animals surviving for 7 days after surgery are considered survivors. Journal of Hepatology 2005 42, 850-859DOI: (10.1016/j.jhep.2005.01.030) Copyright © 2005 European Association for the Study of the Liver Terms and Conditions

Fig. 2 JNK inhibitors decrease serum ALT levels at 6h after reperfusion. Liver injury is assessed in a rat model of partial hepatic ischemia followed by reperfusion. Rats injected with the inhibitors have significant lower levels of ALT than those receiving vehicle only (n=4, mean±SE; P<0.05, Student's t test). Journal of Hepatology 2005 42, 850-859DOI: (10.1016/j.jhep.2005.01.030) Copyright © 2005 European Association for the Study of the Liver Terms and Conditions

Fig. 3 JNK inhibitors decrease histological liver injury assessed at 6h after reperfusion. (A–F) Hematoxylin and eosin staining is performed on ischemic liver lobes from vehicle (A, B), CC0223105 (C, D), and CC0209766 (E, F) at 6h after reperfusion (×100, ×400). Rat liver treated with vehicle shows pericentral necrosis, increased eosinophilia, and vacuolization (A, B). Some hepatocytes and sinusoidal endothelial cell show apoptotic change (B, arrow). These morphologic changes seen are significantly inhibited by JNK inhibitors (D). Journal of Hepatology 2005 42, 850-859DOI: (10.1016/j.jhep.2005.01.030) Copyright © 2005 European Association for the Study of the Liver Terms and Conditions

Fig. 4 JNK inhibitors decrease necrosis. Rat livers are subjected to partial ischemia followed by 6h of reperfusion. The percentage of necrotic cells are evaluated on HE stained sections by using general morphological criteria from vehicle, CC0223105 and CC0209766 treated I/R livers, and non-ischemic control livers. The percentage of necrotic cells is significantly reduced by JNK inhibitors. JNK inhibitors also decrease the number of neutrophils as compared to treatment with vehicle alone (*P<0.05, **P<0.01). Journal of Hepatology 2005 42, 850-859DOI: (10.1016/j.jhep.2005.01.030) Copyright © 2005 European Association for the Study of the Liver Terms and Conditions

Fig. 5 JNK inhibitors decrease apoptosis. Rat livers are subjected to partial ischemia followed by 6h of reperfusion. The percentage of apoptotic cells is evaluated on HE stained sections by use of general morphological criteria from vehicle, CC0223105 and CC0209766 treated I/R livers, and no treatment liver. The percentage of apoptotic cells is significantly reduced by JNK inhibitors (#P<0.0001). Caspase-3 activity is measured in ischemic liver lobes from vehicle, CC0223105, and CC0209766 at 6h after reperfusion. Non-ischemic rat livers are used as control. Caspase-3 activation is inhibited by these JNK inhibitors (*P<0.05). Journal of Hepatology 2005 42, 850-859DOI: (10.1016/j.jhep.2005.01.030) Copyright © 2005 European Association for the Study of the Liver Terms and Conditions

Fig. 6 JNK inhibitors decrease cytochrome c release into the cytoplasm. Western blotting is performed with cytochrome c antibody (1:1000) on the cytosolic fraction and mitochondrial fraction of non-frozen livers obtained at 180min of reperfusion after 90min of ischemia. Treatment with CC-401 decreases cytochrome c release into the cytoplasm. Journal of Hepatology 2005 42, 850-859DOI: (10.1016/j.jhep.2005.01.030) Copyright © 2005 European Association for the Study of the Liver Terms and Conditions

Fig. 7 Transient inhibition of phosphorylation of c-Jun and AP-1 binding activity by JNK inhibitor. Western blotting is performed with phospho-c-Jun antibody (1:1000) for ischemic liver lobes obtained at 0, 15, 30, and 60min after reperfusion (A). Phosphorylation of c-Jun is seen at 15min after reperfusion, and increased up to 60min after reperfusion. Treatment with CC0209766 blocks this increase at 15min. AP-1 binding activity is measured with 5μg of nuclear extracts from vehicle or CC0209766 treated liver obtained at 0, 15, 30, and 60min after reperfusion (B). Cultured rat hepatocyte treated with TNFα is used as a positive control. Increased AP-1 binding activity is seen in the vehicle treatment, whereas SP9766 decrease this activation. Journal of Hepatology 2005 42, 850-859DOI: (10.1016/j.jhep.2005.01.030) Copyright © 2005 European Association for the Study of the Liver Terms and Conditions

Fig. 8 JNK inhibition blocks Bak induction and Bid degradation. Western blotting is performed with primary antibodies against Akt total (BD Biosciences; San Diego, CA), Akt Ser 473 (Cell Signaling Technology; Beverly, MA), PDK1 Ser 241 (Cell Signaling Technology), FKHRL1 Thr 32 (Cell Signaling Technology), GSK-3β Ser 9 (Cell Signaling Technology), Bak total (Biosource; Camarillo, CA), Bid total (BD Biosciences). Journal of Hepatology 2005 42, 850-859DOI: (10.1016/j.jhep.2005.01.030) Copyright © 2005 European Association for the Study of the Liver Terms and Conditions

Fig. 9 JNK inhibitor decreases hepatic TNFα level. Hepatic TNFα is measured by ELISA in livers from normal rats, or ischemic livers after 60 and 180min of reperfusion treated with vehicle or CC-401. Hepatic TNFα is increased with vehicle treatment at both time points, while JNK inhibitor decreases tissue TNFα (*P<0.05 vs NT, **P<0.01 vs NT, #P<0.05 vs vehicle, n=4). Journal of Hepatology 2005 42, 850-859DOI: (10.1016/j.jhep.2005.01.030) Copyright © 2005 European Association for the Study of the Liver Terms and Conditions

Fig. 10 JNK inhibitors decrease lipid peroxidation. Immunohistochemical staining is performed for 4-hydroxynonenal-modified proteins (brown) in livers from normal rats (A), or ischemic livers after 6h of reperfusion treated with vehicle (B), CC0223105 (C), CC0209766 (D). HNE positive cells are quantified by Image Analyzer (E). Both the cytoplasm and nuclei are positively stained in vehicle treated rats (B), while treatment with JNK inhibitor blocks lipid peroxidation (*P<0.05, **P<0.01). Journal of Hepatology 2005 42, 850-859DOI: (10.1016/j.jhep.2005.01.030) Copyright © 2005 European Association for the Study of the Liver Terms and Conditions