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. 1999 Feb;44(2):253–258. doi: 10.1136/gut.44.2.253

Inhibition of nuclear factor-κB activation improves the survival of rats with taurocholate pancreatitis

A Satoh 1, T Shimosegawa 1, M Fujita 1, K Kimura 1, A Masamune 1, M Koizumi 1, T Toyota 1
PMCID: PMC1727365  PMID: 9895386

Abstract

Background—Death in the early stages of severe acute pancreatitis is frequently the result of multiple organ dysfunction, but its mechanism is not clear. 
Aims—To investigate the state of nuclear factor-κB (NF-κB) in macrophages of rats with lethal pancreatitis, and to assess the effectiveness of pyrrolidine dithiocarbamate, an inhibitor of NF-κB, on the pathology and mortality. 
Methods—Taurocholate pancreatitis was produced in rats, and the severity of the disease, the mortality, and activation of NF-κB in peritoneal and alveolar macrophages were compared in rats receiving pyrrolidine dithiocarbamate (PDTC) treatment and those that were not. 
Results—Taurocholate pancreatitis produced massive necrosis, haemorrhage, and severe leucocyte infiltration in the pancreas as well as alveolar septal thickening in the lung. NF-κB was activated in peritoneal and alveolar macrophages six hours after pancreatitis induction. Pretreatment with PDTC dose-dependently attenuated the NF-κB activation and improved the survival of the rats, although it did not affect the early increase in serum amylase and histological findings. 
Conclusions—Early blockage of NF-κB activation may be effective in reducing fatal outcome in severe acute pancreatitis. 



Keywords: pancreatitis; multiple organ dysfunction; nuclear factor-κB; pyrrolidine dithiocarbamate; macrophages; rat

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Figure 1 .

Figure 1

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Histological findings for the pancreas (A), lung (B), and liver (C) 24 hours after the induction of taurocholate (TCA) pancreatitis. Original magnification × 40. 


Figure 2 .

Figure 2

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Electrophoretic mobility shift assay of NF-κB using the 32P labelled probe. Peritoneal macrophages (A) and alveolar macrophages (B) were collected from rats with taurocholate pancreatitis at the indicated time after the induction of pancreatitis. 


Figure 3 .

Figure 3

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Competition assays of the activated NF-κB. Peritoneal macrophages were collected 12 hours after the induction of taurocholate pancreatitis. Nuclear extracts were preincubated with no reagents (lane 1), a 10-fold (lane 2) or a 100-fold (lane 3) molar excess of unlabelled κB oligonucleotide, or a 100-fold molar excess of mutant κB oligonucleotide (lane 4). In the competition assay, an excess of the oligonucleotide competitor was preincubated with the nuclear extract for 60 minutes at room temperature. 


Figure 4 .

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Supershift assays of activated NF-κB. Peritoneal macrophages (lanes 1-5) and alveolar macrophages (lanes 6-10) were collected 12 hours after the induction of taurocholate pancreatitis. Nuclear extracts were preincubated with no reagents (lanes 1 and 6), or with 2 µg each of the rabbit polyclonal antibody against p50 (lanes 2 and 7), p65 (lanes 3 and 8), c-Rel (lanes 4 and 9), or p52 (lanes 5 and 10). In the supershift assays, incubation with the respective antibody was carried out at 4°C for 60 minutes. 


Figure 5 .

Figure 5

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(A) Effects of pyrrolidine dithiocarbamate (PDTC) on NF-κB activation. Peritoneal macrophages (lanes 1-4) and alveolar macrophages (lanes 5-8) were obtained 12 hours after the induction of taurocholate pancreatitis. Lanes 2-4 and 6-8 show NF-κB activation in the respective type of macrophages obtained from rats pretreated with the indicated doses of PDTC (1, 10, and 100 mg/kg) one hour before the induction of pancreatitis, whereas lanes 1 and 5 show NF-κB activation in the respective type of macrophages obtained from rats not pretreated with PDTC. (B) Specificity studies of activated NF-κB in the peritoneal macrophages of pancreatitis rats pretreated with 1 mg/kg PDTC. Nuclear extracts from macrophages were preincubated with no reagents (lane 1), with a 100-fold molar excess of unlabelled κB oligonucleotide (lane 2), with 2 µg/ml of the rabbit polyclonal antibody against either p50 (lane 3) or p65 (lane 4). Then the mixtures were subjected to electrophoretic mobility shift assay. 


Figure 6 .

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Effects of pyrrolidine dithiocarbamate (PDTC) on the histological findings in the pancreas. PDTC at a dose of 10 mg/kg was given intraperitoneally one hour before the induction of pancreatitis and the tissues were obtained 24 hours after the induction of pancreatitis. Original magnification × 40. 


Figure 7 .

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Effects of pyrrolidine dithiocarbamate (PDTC) on the mortality of the rats with taurocholate pancreatitis. PDTC was given intraperitoneally one hour before the induction of pancreatitis, and mortality was examined for seven days after the induction of pancreatitis. *p<0.05 compared with the rats not given treatment. 


Figure 8 .

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Effect of timing of pyrrolidine dithiocarbamate (PDTC) (10 mg/kg) administration on mortality. *p<0.05 compared with the rats not given treatment. 


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