Elsevier

Journal of Hepatology

Volume 42, Issue 6, June 2005, Pages 850-859
Journal of Hepatology

JNK mediates hepatic ischemia reperfusion injury

https://doi.org/10.1016/j.jhep.2005.01.030Get rights and content

Background/Aims

Hepatic ischemia followed by reperfusion (I/R) is a major clinical problem during transplantation, liver resection for tumor, and circulatory shock, producing apoptosis and necrosis. Although several intracellular signal molecules are induced following I/R including NF-κB and c-Jun N terminal kinase (JNK), their roles in I/R injury are largely unknown. The aim of this study is to assess the role of JNK during warm I/R injury using novel selective JNK inhibitors.

Methods

Male Wistar rats (200±25 g) are pretreated with vehicle or with one of three compounds (CC0209766, CC0223105, and CC-401), which are reversible, highly selective, ATP-competitive inhibitors of JNK. In the first study, rats are assessed for survival using a model of ischemia to 70% of the liver for 90 min followed by 30% hepatectomy of the non-ischemic lobes and then reperfusion. In the second study, rats are assessed for liver injury resulting from 60 or 90 min of ischemia followed by reperfusion with analysis over time of hepatic histology, serum ALT, hepatic caspase-3 activation, cytochrome c release, and lipid peroxidation.

Results

In the I/R survival model, vehicle-treated rats have a 7-day survival of 20–40%, while rats treated with the three different JNK inhibitors have survival rates of 60–100% (P<0.05). The decrease in mortality correlates with improved hepatic histology and serum ALT levels. Vehicle treated rats have pericentral necrosis, neutrophil infiltration, and some apoptosis in both hepatocytes and sinusoidal endothelial cells, while JNK inhibitors significantly decrease both types of cell death. JNK inhibitors decrease caspase-3 activation, cytochrome c release from mitochondria, and lipid peroxidation. JNK inhibition transiently blocks phosphorylation of c-Jun at an early time point after reperfusion, and AP-1 activation is also substantially blocked. JNK inhibition blocks the upregulation of the pro-apoptotic Bak protein and the degradation of Bid.

Conclusions

Thus, JNK inhibitors decrease both necrosis and apoptosis, suggesting that JNK activity induces cell death by both pathways.

Introduction

Ischemia reperfusion (I/R) injury is an important clinical problem for several organs including brain, heart, kidney, and liver. Hepatic I/R injuries occur during transplantation, liver resection for tumor, and circulatory shock [1]. Possible consequences of hepatic I/R injury include liver failure and/or multi-organ system failures, resulting in morbidity and mortality [2]. Hepatic I/R injury produces two types of cell death, apoptosis and necrosis in hepatocytes and non-parenchymal cells [3], [4], [5]. Several intracellular signaling molecules are activated by I/R including NFκB and c-Jun N terminal kinase (JNK) [6]. However, their role in the molecular pathogenesis of hepatic I/R injury is largely unknown.

JNK is phosphorylated and activated by several types of stresses, including stimulation by cytokines, such as TNFα [7] and IL-1 [8], and environmental stresses such as radiation and oxidant stress [9], [10]. Substrates for JNK include the transcription factors c-Jun and ATF-2. Recent studies have proposed that activated JNK may also directly affect mitochondria through undefined substrates leading to apoptosis [11], [12], [13], [14]. JNK is strongly induced during warm hepatic I/R injury [8] and during the cold ischemia/warm repetition injury of liver transplantation [6], [15].

Using selective JNK inhibitors, we now report that JNK blockade suppresses liver injury in a rat model of hepatic warm I/R. These are the first data from animal studies directly demonstrating that JNK plays a harmful role during liver I/R injury.

Section snippets

Reagents

Specific JNK inhibitors [16], [17], [18], [19], CC0209766, CC0223105, and CC-401 were synthesized by Signal Pharmaceuticals, Inc. Each JNK inhibitor is dissolved in vehicle before injection (5% 1-methyl-2-pyrrolidone, 30% PEG-400, 25% PEG-200, 20% propylene glycol, USP, 20% 0.9% sodium chloride for injection, USP). JNK inhibitors (3–20 mg/kg rat, dissolved in 0.6–8.0 mg/ml vehicle) or equivalent volume of vehicle only are administrated intravenously at 15 min before starting ischemia. Some rats

JNK inhibitors improve survival in model of hepatic I/R injury

The effect of JNK inhibitors on animal survival was evaluated using a model of total hepatic I/R in which survival depends on recovery of the ischemic liver after reperfusion. Rats (n=10 per group) were injected intravenously with vehicle, CC0209766 (20 mg/kg rat), or CC0223105 (20 mg/kg rat) 15 min prior to 90 min ischemia and 4 h after reperfusion (Fig. 1A). Vehicle treated rats have a 20% survival rate, whereas CC0209766 or CC0223105 treated rats have a 60 or 70% survival rate, respectively. In

Discussion

JNK is rapidly activated during the reperfusion phase of liver transplantation and following warm ischemia [6], [8], [26], although the functional consequences of JNK activation were unknown. Specific JNK inhibitors have been developed that inhibit the JNK1, JNK2, and JNK3 isoforms through specific competition for their ATP binding sites [16]. These inhibitors block c-Jun phosphorylation, expression of inflammatory genes in cultured cells, proliferation of cultured cells, and TNFα production by

Acknowledgements

Grant support: National Institutes of Health.

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