Protective effects of SP600125 a new inhibitor of c-jun N-terminal kinase (JNK) and extracellular-regulated kinase (ERK1/2) in an experimental model of cerulein-induced pancreatitis

Abstract

Extracellular regulated kinases (ERK1/2) and c-Jun N-terminal Kinases (JNK), are generally considered to play a key role in signal transduction pathways activated by a wide range of stimuli. We studied the effects of SP600125, a novel inhibitor of both JNK and ERK1/2, in male C57/BL6 mice given with an hyper-stimulating dose of cerulein (50 μg/kg for each of four injections at hourly intervals) to elicit secretagogue-induced pancreatitis. A control group received four intra-peritoneal injections of 0.9% saline at hourly intervals. Animals were randomized to receive either SP600125 (15 mg/kg i.p. administered 2h before and 30 min after the first injection of cerulein) or its vehicle (1 ml/kg of a 10% DMSO/NaCl solution). A group of animals was killed 30 minutes after the last cerulein injection to evaluate pancreatic JNK and ERK1/2 activation by Western Blot analysis. Another group was sacrificed 2 hours after the last cerulein injection to evaluate serum lipase and amylase levels, pancreas oedema, pancreatic content of Tumor Necrosis Factor-α (TNF-α) and Intercellular adhesion molecule-1 (ICAM-1) and the histological alterations. SP600125 inhibited almost totally JNK activation (90%) and partially ERK1/2 activation (45%), reduced the serum lipase and amylase levels and the degree of oedema, blunted the increased pancreatic content of TNF-α  and ICAM-1 and protected against the histological damage. Our data confirm that both JNK and ERK1/2 activation plays a key role in acute pancreatitis and that SP600125 may represent a potential therapeutic approach to the treatment of patients at high risk of developing this life-threatening condition.

Introduction

Mitogen-activated protein kinases (MAPKs) belong to the extracellular signal-regulated kinases family and are seronine/threonine protein kinases activated by a variety of cell surface receptors (Cano and Mahadevan, 1995, Davis, 1994, Seger and Krebs, 1995).

These proteins are of vital importance for signal transduction pathways and especially two of these were investigated for their role: extracellular signal-regulated kinase (ERK1/2) and c-jun N-terminal kinase (JNK).

ERK1/2 and JNK activation requires phosphorylation of both tyrosine and threonine residues by upstream dual specific kinases. Active MAPKs are responsible for the phosphorylation of a variety of effector proteins including several transcription factors (Widmann et al., 1999).

It has already been demonstrated that pancreas hyperstimulation with cerulein induces acute pancreatitis in rats and can activate MAPKs cascade (Wagner et al., 2000, Grady et al., 1996).

Actually JNK and ERK1/2 were proposed as important early mediators during cerulein-induced pancreatitis due to their pattern and activation time course (Grendell, 1997).

Acute pancreatitis is an autodigestive process resulting in acute inflammation of the pancreas characterized by oedema, leukocyte infiltration, hemorrhage and cellular necrosis. The severity of the disease ranges from mild oedema to severe fulminant multi-systemic organ failure (Steer, 1998, Bhatia et al., 2000).

A large body of evidence suggests that during pancreatic damage inflammatory mediators including cytokines, chemokines and adhesion molecules such as Intercellular adhesion molecule 1 (ICAM-1) are involved (Norman, 1998, Schmid and Adler, 1999).

This disorder remains to be fully understood in its pathophysiology and the treatment is still confined to general supportive measures with no causal approach.

In light of these findings the possibility to interact with drugs that blunt MAP-kinases system in transduction signal could represent a new strategic alternative.

Experimental evidence suggested that selective JNK (Wagner et al., 2000, Fleischer et al., 2001) inhibition could ameliorate the severity of pancreatitis cerulein-induced in the rat model. Furthermore PD98059 and UO126, both inhibitors of ERK1/2, afford significant protection against inflammatory sequelae following experimental acute pancreatitis (Clemons et al., 2002).

However the effects of the concomitant and combined blockade of both JNK and ERK1/2 in the development of acute pancreatitis have not been yet investigated. We therefore studied the effects of SP600125 (anthra[1,9cd]pyrazol-6(2H)-one 1,9 pyrazoloanthrone), a new JNK and ERK1/2 inhibitor, on the severity of pancreatic damage. Previous in vitro experiments demonstrated that SP600125 exhibits over 300-fold greater selectivity for JNK as compared to ERK1/2 MAP kinase and blocks the expression of TNF-α (Bennett et al., 2001, Shin et al., 2002).