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Poly ADP-ribose polymerase-1 PARP-1 is a nuclear DNA-binding protein that has been shown to play a relevant role in cell necrosis and organ failure in various diseases associated with inflammation. Lung injury was evaluated by quantifying MPO activity and morphological changes. Our results support the potential therapeutic application of PARP inhibitors in the development and severity of acute pancreatitis and associated lung injury.
Acute pancreatitis involves a complex cascade of local and systemic events. A still unknown triggering event converts, within the pancreas, digestive proenzymes into their active forms, leading to membrane disruption, edema, interstitial hemorrhage, necrosis, accompanied by an inflammatory response with infiltrating leukocytes that contributes to the progression of both local pancreatic destruction and systemic manifestations.
In the current study, we have used a well-characterized secretagogue-induced murine model of acute pancreatitis to ascertain the role of PARP-1 and -2 in this pathological process. Interestingly, administration of N - 6-oxo-5,6-dihydro-phenanthridinyl - N , N -dimethylacetamide HCl PJ34 or 3-aminobenzamide 3-AB , potent inhibitors of PARPs activity, 25 , 26 , 27 markedly decreased pancreatitis and pulmonary-associated injury in WT mice in a larger extension than that found in mice that had PARP-1 genetically deleted.
Taken together, our results show the therapeutic potential of PARP inhibitors on the development and severity of acute pancreatitis and pancreatitis-associated lung-injury. Tap water and mouse chow were provided ad libitum. The experimental procedure was performed in accordance with The University of Murcia approved institutional animal care guidelines. Blood, pancreas and lungs were harvested for further studies. The extent of pancreatic edema was evaluated by measuring the wet-to-dry weight ratio.
The difference between the wet and dry tissue weights was calculated, and the degree of edema was expressed as a percentage of the wet tissue weight.
MPO activity was measured photometrically. Analysis of the histopathological changes in pancreas and lung was carried out under light microscopy by two expert pathologists who were blind to the experiment. Pancreatic histological grading of edema, necrosis and infiltration of inflammatory cells was scored by assigning a subjective value as described previously: 30 0, absent; 1, mild; 2, moderate; and 3, severe.
Then, sections were washed three times with PBS with 0. Finally, after rising in distilled water, the slides were counterstained with hematoxylin, rinsed well with tap water and mounted.
Serum concentration of lipase and amylase was measured using commercial kits according to the manufacturer's protocols in a Modular P photometric analyzer Roche, Mannheim, Germany. Statistical analysis was performed by using the Mann—Whitney nonparametric t -test. This treatment has been previously shown to induce acute necrotizing pancreatitis in mice. Interestingly, WT PJ34 and WT 3-AB mice showed the greatest reduction in the serum levels of amylase and lipase as well as a statistically significant reduction in pancreatic water content compared to those of WT mice after cerulein-induced pancreatitis, suggesting a cumulative protective effect after inhibiting PARPs activity.
Effects of genetic and pharmacological inhibition of PARP on cerulein-induced pancreatic injury. The same numbers of control animals from each experimental group received saline white bars.
This figure shows data for 10 to 18 mice per group. Neither of the experimental groups treated with saline exhibited any morphological evidence of acute pancreatitis Figure 2.
To assess these morphological changes in a semiquantitative manner, slides were scored by two pathologists in a blinded manner. More importantly, treatment of WT mice with either PJ34 or 3-AB PARP inhibitors strongly reduced the severity of the cerulein-induced pancreatitis, as noted by a statistically significant decrease in pancreatic edema, inflammation, vacuolization and necrosis, with a decrease in total histologic score from 7.
In contrast, PARP-2 deficiency had no significant protective effect on these histologic parameters Figure 3. Neither of the experimental groups treated with saline exhibited PAR staining. Morphological changes in cerulein-induced pancreatitis. Histological score of cerulein-induced acute pancreatitis. Immunohistochemical localization of PAR in the pancreas. MPO activity in pancreas homogenates was determined as a measure for neutrophil accumulation within the organ Figure 5. WT mice that received repeated cerulein injections showed a profound increase in pancreatic MPO activity.
Same numbers of control animals from each experimental group received saline white bars. This figure shows data for seven to 10 mice per group. A control untreated WT mouse is show. This figure shows data for five mice per group. Several inflammatory mediators have been shown to increase in acute pancreatitis. In addition to measure circulating levels of cytokines, we also determined the local pancreatic level of IL-6 before and after 12 hourly intraperitoneal injections of cerulein.
This figure shows data from six mice per group. Severity of acute pancreatitis is associated with lung injury. Reduced lung inflammation after PARP inhibition has also been reported in other experimental models of acute inflammatory response.
As an additional quantitative assessment of the inflammatory response, lung MPO activity following cerulein-induced pancreatitis was measured. Morphological changes of pancreatitis-associated lung injury. The earliest events in the evolution of acute pancreatitis lead to intra-acinar cell activation of digestive enzymes within the pancreas.
This model is characterized by hypermylasemia and hyperlipasemia, extensive acinar cell vacuolization, pancreatic edema, intrapancreatic activation of digestive zymogens including trypsinogen, neutrophils sequestration within the pancreas and varying degrees of acinar cell necrosis. We found that deletion of PARP-2 significantly reduced the serum level of amylase compared to those found in WT mice after cerulein-induced pancreatitis.
However, our results show that genetic deletion of PARP-2 has only minor effects in the inflammatory response associated to acute pancreatitis. The role of PARP-2 in the inflammatory response is unknown, and to our knowledge, only one report has been previously shown that inhibition of PARP-2, by using antisense oligonucleotide, attenuated inflammation in colitis. The model proposed by this paper suggests that PARP inhibition might control the severity of acute pancreatitis by preventing both the local and systemic inflammatory response, and the mechanisms involved in pancreatic cell necrosis mediated by neutrophils via the enzyme nicotinamide adenine dinucleotide phosphate NADPH oxidase 5 Figure Endothelial cell and acinar expression of adhesion molecules and chemokines during the initial steps of pancreatitis promotes neutrophil adhesion to the endothelial cell surfaces, neutrophil diapedesis and sequestration of neutrophils within the pancreatic parenchyma.
In a second step, once inside the tissue, neutrophil NADPH oxidase initiates the generation of reactive oxygen species ROS including hydrogen peroxide and superoxide. In a third step, severe episodes of acute pancreatitis are associated with a systemic inflammatory response. PARP inhibition might protect against acute pancreatitis at different levels. ROS, reactive oxygen species. In summary, we show that genetic deletion of PARP-1, but not PARP-2, attenuates the severity of acute pancreatitis and pancreatitis-associated lung injury.
Therefore, PARP-1 appears as a new potential therapeutic target molecule in acute pancreatitis. Interestingly, pharmacological inhibition of PARP activity has the strongest effect on controlling the severity of acute pancreatitis and pancreatitis-associated lung injury. PARP inhibition might control acute pancreatitis by preventing both the local and systemic inflammatory response and the mechanisms of pancreatic cell necrosis mediated by ROS released via the neutrophil enzyme NADPH oxidase.
These findings support the potential therapeutic use of PARP inhibitors in the development and severity of acute pancreatitis and associated lung injury. Imrie CW. Acute pancreatitis: overview. Eur J Gastroenterol Hepatol ; 9 — Steer M. Pancreatitis severity: who calls the shots? Gastroenterology ; — Norman J. The role of cytokines in the pathogenesis of acute pancreatitis. Am J Surg ; — Cerulein upregulated ICAM-1 in pancreatic acinar cells, which mediates neutrophils adhesion to these cells.
Am J Physiol ; :G—G Neutrophils and NADPH oxidase mediate intrapancreatic trypsin activation in murine experimental acute pancreatitis. The role of intercellular adhesion molecule 1 and neutrophils in acute pancreatitis and pancreatitis-associated lung injury. Inflammatory mediators in acute pancreatitis. J Pathol ; — Pahl HL. Oncogene ; 18 — Denharn W, Norman J. The potential role of therapeutic cytokine manipulation in acute pancreatitis.
Surg Clin N Am ; 79 — Early NF-kappaB activation is associated with hormone-induced pancreatitis. Inhibition of nuclear factor-kappaB activation improves the survival of rats with taurocholate pancreatitis.
Gut ; 44 — Selective inhibition of NF-kappaB attenuates the severity of cerulein-induced acute pancreatitis. Am J Coll Surg ; — Lab Invest ; 12 — Resistance to endotoxic shock as a consequence of defective NF-kappaB activation in poly ADP-ribose polymerase-1 deficient mice. EMBO J ; 18 — Role of lipopolysaccharide and cecal ligation and puncture on blood coagulation and inflammation in sensitive and resistant mice models.
Am J Pathol ; — Nucleid Acids Res ; 32 — Resistance to acute septic peritonitis in poly ADP-ribose polymerasedeficient mice.
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