High-mobility group box chromosomal protein 1 (HMGB1), originally discovered 30 years ago as a nuclear DNA-binding protein, 1 was recently identified as a late-acting mediator of sepsis (endotoxin lethality). 2 Injection of HMGB1 itself was lethal, and serum levels of HMGB1 increased 8 to 32 hours after the administration of endotoxin in mice, when the tumor necrosis factor peak had already occurred. 2 The HMGB1 was also found to have the capacity to induce cytokines and activate inflammatory cells when it applied extracellularly. 2-4 This implicates HMGB1 as a proinflammatory mediator. It has been demonstrated that HMGB1 is secreted actively by living inflammatory cells, such as stimulated macrophages/monocytes, and is released passively from necrotic or damaged cells. 5-7 In a recent study, we have first demonstrated that serum HMGB1 levels were significantly elevated in patients with severe acute pancreatitis (SAP) within 72 hours after the onset and were correlated with disease severity. 8 The HMGB1 levels were higher in patients with organ dysfunction and infection during the clinical course. Moreover, the HMGB1 levels in nonsurvivors were higher than those in survivors. These results suggest that HMGB1 may play a pivotal role in the pathogenesis of SAP, and that HMGB1 may act as a key mediator for inflammation and organ failure in this disease. The aim of this study was to investigate the expression of HMGB1 protein in rat acute pancreatitis.

Male Wistar rats weighing 250 to 300 g were used. Sham-operated rats (laparotomy only) were set as the control. Mild acute pancreatitis was induced by intravenous administration of caerulein (5 μg/kg/h for 4 hours)(CN pancreatitis). The SAP was induced by retrograde injection of 3% or 20%(wt/vol) sodium deoxycholate (DCA)(0.1 mL) to biliopancreatic ducts with the temporary clamp of common bile duct at the hilus of the liver (DCA pancreatitis). The 24-hour mortality rates of 3% and 20% DCA pancreatitis were approximately 60% and 100%, respectively. The HMGB1 levels in serum and ascitic fluid were determined with an established available enzyme-linked immunosorbent assay kit (Shino-Test Corporation, Sagamihara, Japan). 9 The detection limit with this enzyme-linked immunosorbent assay kit is 1.0 ng/mL. To evaluate the expressions of HMGB1 protein in major organs during SAP, tissues of pancreas, liver, kidney, lung, and small intestine were homogenized with 3 mL of buffer (20 mM Tris-hydrochloride, pH 7.5, 10 μM [4-amidino-phenyl]-methane-sulfonyl fluoride, 1 mM EDTA, 1 mM dithiothreitol). The homogenates were centrifuged at 15,000 g for 30 minutes at 4 C, and the supernatants were collected. Each sample (100 μg of protein) was analyzed by Western blotting. Anti-HMGB1 antibodies were from Abcam Ltd (Cambridge, UK) and used at a 1: 2000 dilution. The results were expressed as mean±SEM. Statistical analyses were performed using the Mann-Whitney U test. Statistical significance was set at P< 0.05.