The Role Of Adenosine Kinase In Severe Acute Pancreatitis And The Underlying Mechanism

Background:Acute pancreatitis(AP)refers to acute abdomen caused by abnormal activation of pancreatic enzymes to produce digestive effects on the pancreas itself and surrounding organs,which is characterized by local inflammatory response of the pancreas,and can even lead to organ dysfunction.Severe acute pancreatitis(SAP)refers to acute pancreatitis combined with organ dysfunction,or local complications such as necrosis,abscess,or pseudocyst or both.Inflammatory disorder and acinar cell death play an important role in the development of SAP.Various stressors of SAP activate trypsinogen,and active trypsin releases a large amount of calcium from pancreatic acinar cells,leading to pancreatic acinar cell necrosis.Cell debris enters the extracellular space and is recognized by leukocytes,leading to the release of inflammatory factors Interleukin-1β(IL-1β)and Tumor necrosis factor-α(TNF-α)and the development of necroptosis.Endoplasmic reticulum stress(ERS)is one of the core cellular events in the pathogenesis of SAP.Excessive activation of ERS triggers and aggravates pancreatic injury in acute pancreatitis,which is related to multiple mechanisms such as unfolded protein response(UPR)signal transduction,aggravation of inflammatory response,acinar cell apoptosis and necrosis,and pancreatic zymogen-granule autophagy.Adenosine kinase(ADK)is a cytosolic enzyme that catalyzes the conversion of adenosine to adenylate.ADK phosphorylates adenosine to adenosine monophosphate and is the principal enzyme in determining the intracellular and extracellular adenosine levels.Inhibition of ADK increases intracellular and extracellular adenosine levels.Adenosine plays a wide range of physiological functions in living systems as a homeostatic and metabolic regulator.Some studies suggested a potential role of ADK on inflammation and cell death.However,whether ADK regulates inflammation and acinar cell death in SAP is unknown.Objectives:1.To determine the changes of ADK in pancreatic tissue during SAP;2.To clarify the role and molecular mechanism of ADK in SAP;3.To investigate the effect and molecular mechanism of inhibiting ADK on cerulein induced pancreatic acinar cell injury.Methods:1.To establish an experimental SAP model:male C57BL/6 mice were intraperitoneally injected with cerulein(50μg/kg,seven doses at hourly intervals)and LPS(1Omg/kg,at the last cerulein injection).For ADK inhibition,ABT702(1.5mg/kg)was intraperitoneally injected 1 h before cerulein injection;2.To establish a model of cerulein stimulation of pancreatic acinar cells:For cerulein stimulation,AR42J or MPC-83 cells were incubated with cerulein(100nM)for 24h.For ADK inhibition,ABT702(1M)was used 30 min before the first cerulein incubation.For adenosine receptor inhibition,ZM241385(100nM)was used 30 min before ABT702 incubation.For PERK inhibition,GSK2606414(5μM)was used 30 min before cerulein incubation;3.Pancrease weight/body weight measurement:the body weight of mice and pancrease weight were measured at the time of sampling;4.Serum amylase and lipase analysis:Serum of mice were collected,and serumα-amylase and lipase were detected by the respective assay kits;5.S-Adenosylhomocysteine(SAH)analysis:serum were collected and SAH levels were detected by ELISA kit;6.Pancreatic tissue staining and pathological scoring:Samples of the pancreases was stained with hematoxylin-eosin(HE)after paraffin embedding.The histopathological examination and scoring of pancreatic tissue were evaluated by Schmidt score under light microscope;7.Necroptosis assay in pancreatic tissue:Evans blue dye(EBD)(10mg/mL)was injected intraperitoneally for 4h before the mice were euthanized.The pancreatic tissues of mice were taken and frozen sections were used to detect the content of amylase and EBD by immunofluorescence staining;8.Immunohistochemistry and immunofluorescence staining:The pancreatic tissues of mice were collected and paraffin sections were used to detect the expression of neutrophils by immunohistochemistry,and the expression of macrophages by immunofluorescence;9.Western blot was used to detect the protein changes of pancreatic tissues,AR42J cells and MPC-83 cells to detect the changes of inflammation,necroptosis,and endoplasmic reticulum stress;10.All data were analyzed using GraphPad Prism version 6.0.The data are presented as the mean ± SEM.Student’s t-test or one-way analysis of variance followed by Tukey’s post hoc test were used to compare statistical significance.Statistical significance was considered at p<0.05.Results:1.ADK inhibition attenuated the severity of SAP in mice;2.ADK inhibition prevented the infiltration of inflammatory cells and reduced necroptotic response in the pancreas of SAP mice;3.ADK inhibition prevented cerulein-induced inflammation and cell necroptosis in acinar cells;4.ADK inhibition alleviated ERS in SAP mice and cerulein-induced pancreatic acinar cells;5.ERS contributed to cerulein-induced inflammation and cell necroptosis in pancreatic acinar cells;6.Adenosine A2A receptor mediated the effect of ADK inhibition on cerulein-induced inflammation,cell necroptosis and ERS.Conclusion:ADK inhibition reduces SAP inflammation and necroptosis of acinar cells through the adenosine A2A receptor/ERS pathway.ADK is a potential target for the treatment of SAP.