The Functions And Mechanism Study On Kinase PKR In Acute Pancreatitis

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PKR is associated with pancreatic tissue inflammation and injury in rat acute pancreatitis modelPurposes:To initial identify the relationship of PKR,pancreatic inflammation and injury in rat acute pancreatitis model.Methods:The rat AP model was constructed by up to four hourly intraperitoneal(i.p.)injections of 50 ?g/kg/h cerulein.The rats in the experimental group were divided into 4 h,8 h,12 h and 24 h groups(12 rats in each group).In the control group(12 rats),50/kg saline was injected intraperitoneally once.Pancreatic protein of control and AP groups was collected,PKR expression was detected by Western blot,PKR expression in control and AP 8 h pancreas was detected by immunohistochemistry,TNF-a,IL-6 and LDH level in control and AP pancreatic tissues was measured by enzyme-linked immunosorbent assay(ELISA).The pathologic changes of pancreatic tissue following cerulean i.p.injections were detected by HE(haematoxylin-eosin)stain.Spearman correlation analysis was used to identify the relationship between KR expression and TNF-a,IL-6 and LDH level,respectively.Results:Pancreatic tissue PKR protein level in AP each time point groups increased compared to control group(P<0.05).Immunohistochemistry showed AP 8 h group had more PKR expression than that of control group.Compared to control group,pancreatic tissue in AP 8 h group showed hemorrhage,edema,and infiltration of inflammatory cells.level in pancreatic tissue of control group was low,increased at AP 4 h,reached the peak at AP 8 h,then slightly decreased at AP 12 h and 24 h.TNF-?,IL-6 and LDH level in pancreatic tissue of AP group increased compared to control group(P<0.05).PKR expression showed positive correlation with TNF-a,IL-6 and LDH level,respectively.Conclusions:Pancreatic tissue PKR,TNF-?,IL-6 and LDH level in AP group increased,suggesting PKR was associated with AP pancreatic tissue inflammation and injury.Part ? The mechanic study on PKR promotes pancreatic cell inflammation and injuryPurposes:Initially investigate the expression,functions and mechanisms of PKR in pancreatic acinar cells under cerulein stimulation conditions.Methods:10 nmol/L Cerulein was used to stimulate rat pancreatic acinar AR42J cells for 4 h,8 h,12 h,24 h,PKR protein level in AR42J cells was detected by Western blot,TNF-a,IL-6 level in AR42J cells and LDH level in AR42J cell culture supernatant was detected by ELISA.The scramble siRNA and PKR siRNA were transfected into AR42J cells,PKR protein level in AR42J cells was detected by Western blot.TNF-a,IL-6 and LDH level in AR42J cell culture supernatant was detected by ELISA.inhibitor of kappa B kinase a(IKK?),P65(NF-?B subunit),phosphorylated P65 expression in AR42J cells of control and cerulein groups was detected by Western blot.The interaction between PKR and IKKa was identified by Co-immunoprecipitation.The co-localization of PKR and IKKa was detected by cell immunofluorescence.Nuclear and cytoplasmic fraction and Western blot were used to detect the nuclear and cytoplasmic localization of P65 in AR42J cells.The PKR antibody,PKR antibody and Hoechst 33342(DNA dye)were used for cell immunofluorescence to observe the subcellular localization in AR42J cells.Results:PKR,TNF-a,IL-6 and LDH level in AP each time point was higher than that of control group(P<0.05).PKR,TNF-a,IL-6 and LDH level in AR42J cells of control group was low,increased at cerulein stimulation for 4 h,reached the peak at 8 h,slightly decreased at 12 and 24 h.PKR siRNA inhibited TNF-a,IL-6 and LDH level in AR42J cells,suggesting PKR promoted cerulein-induced pancreatic acinar cell inflammation and injury.IKK?,p-P65 protein level in AR42J cells after cerulein stimulation(P<0.05).P65 protein level didn't change.Co-immunoprecipitation showed the interaction between PKR and IKKa.Additionally,the interaction enhanced after cerulein stimulation for 8 h.AR42J cell immunofluorescent double stain showed PKR and IKKa mainly localized in cytoplasm and co-localized primarily in cytoplasm.After cerulein stimulation,PKR and IKKa translocated to nucleus and co-localized in nucleus.Using cerulein stimulation 8 h plus PKR siRNA transfection,Western blot showed P65translocated to nucleus compared to control group.Compared to cerulein stimulation group,P65 nuclear translocation was inhibited in cerulein stimulation 8 h plus PKR siRNA transfection group.P65 nuclear translocation was not affected in PKR siRNA transfection group,compared to control group.AR42J cell immunofluorescent double stain showed PKR and P65 mianly localized in cytoplasm in control group.PKR and P65 translocated to nucleus in ceulein stimulation 8 h plus scramble siRNA transfection group,compared to control group.PKR and P65 nuclear translocation was inhibited in ceulein stimulation plus PKR siRNA transfection group,compared to ceulein stimulation plus scramble siRNA transfection group.The data suggested PKR promoted cerulein-induced phosphorylation and nuclear translocation of P65 via interacting with IKK?.Conclusions:Cerulein stimulation up-regulated IKKa,p-P65 expression in AR42J cells.PKR promoted cerulein-induced AR42J cell inflammation and injury.PKR promoted cerulein-induced phosphorylation and nuclear translocation of P65 via interacting with IKK?.Part ? PKR expression in peripheral blood mononuclear cells of acute pancreatitis patientsPurposes:To detect PKR level in peripheral blood mononuclear cells(PBMC)of AP patients and explore the relativity of PKR level and severity of AP.Methods:109 patients diagnosed as AP which were divided into mild acute pancreatitis(MAP)group(33 cases),moderate severe acute pancreatitis(MSAP)group(38 cases),severe acute pancreatitis(SAP)group(38 cases)and healthy population(56 cases)group were screened.The peripheral blood was collected and PBMC was separated.PKR level in PBMC was detected by PKR ELISA kit.Results:PKR level in PBMC of AP patients obviously increased,compared to healthy controls(P<0.05).PKR level increased along with the severity of AP.P<0.05 when PKR level in PBMC of moderately severe acute pancreatitis(MSAP)and severe acute pancreatitis(SAP)compared to that in mild acute pancreatitis(MAP).P<0.05 when PKR level in PBMC of SAP compared to that of MSAP.Conclusions:PKR level in PBMC of AP patients was higher that of healthy controls,showing the positive correlation to the severity of AP.