The Effect Of CUEDC2 On The TNF-?,IL-6 In Acute Pancreatitis Acinar

Acute pancreatitis(AP)is a common clinical acute abdomen with the characteristic of acute onset,severe and rapid progression.Besides the pancreatic lesions,AP can also causes the injury of other organs or tissues,even induces death because of the systemic inflammatory response syndrome(SIRS)or multiple-organ dysfunction syndrome(MODS).Although in the past decades,we have made great progress about acute pancreatitis research,the pathogenesis is still complex.Whatever the causes of acute pancreatitis,pancreatic injury can cause pancreatic enzymes to digest themselves,as well as inflammatory stimuli stimulate the monocyte-macrophage cells and pancreatic acinar cells release inflammatory mediators and cytokines and inflammatory initiation factor,which trigger inflammatory mediator form waterfall cascade and eventually lead to local or systemic inflammation.CUEDC2 is a multifunctional protein,which contains CUE domain,and widely express in human tissues and organs.A large number of studies have shown that CUEDC2 can be applied to a variety of intracellular signal transduction pathways,to participate in the activities of many cells,such as the regulation of the cell cycle,growth factor signal transduction,development and progression of cancer and inflammation.The present study found that CUEDC2 is not only closely related with the occurrence of tumor development,such as,colitis associated tumor,lung cancer,breast cancer,but also participate in the inflammatory response,such as colitis,et al.However,the research on the relationship between pancreatitis and CUEDC2 has not been reported.This study was to explore the role of CUEDC2 in acute pancreatitis.Objectives:In this study,AR42J cells were induced by caerulein to establish a cell model of acute pancreatitis and using plasmid transfection technology to overexpress CUEDC2 proein.Detecting the levels of amylase(AMY),tumor necrosis factor-alpha(TNF-?)and interleukin-6(IL-6)in cell culture supernatants.We planned to explore the effect of CUEDC2 on the TNF-a,IL-6 of acute pancreatitis acinar cells.Methods:1 Induced AR42J cells cell by caerulein to establish a cell model of acute pancreatitis.2 The CUEDC2 plasmid was transfected into AR42J cells with lipofectamine and examained the transfection efficiency by fluorescence microscopy.3 Experimental models:AR42J cells were divided into 4 groups:Negative plasmid group with caerulein stimulation(Neg-CAE group),Negative plasmid group which was untreated by caerulein(Neg-Control group),CUEDC2 plasmid group with caerulein stimulation(CUED2-CAE group),CUEDC2 plasmid group which was untreated by caerulein(CUEDC2-Control group).Specific grouping methods are as follows:Negative plasmid group:AR42J cells were transfected with negative plasmid.Neg-CAE group was given 10-7 mol/L caerulein.Neg-Control group was given an equal volume of complete medium.The two Groups can be divided into three subgroups:4 h,8 h,24 h.CUEDC2 plasmid group:AR42J cells were transfected with CUEDC2 plasmid.CUEDC2-CAE group was given 10-7 mol/L caerulein.CUEDC2-Control group was given an equal volume of complete medium.The two groups can be divided into three subgroups:4 h,8h,24 h.4 The determination of CUEDC2:The level of CUEDC2 mRNA was determined by PCR.The expression level of CUEDC2 was measured western-blot.5 The measurement of AMY,TNF-?,IL-6:The levels of AMY in cellculture supernatants were measured by Amylase Assay Kit.ELISA(enzyme linked immunosorbent assay)was used to detect the expression of TNF-a and IL-6.6 Statistical Methods:All statistical analysis was performed by SPSS 21.0 for Windows.The significance was assumed at P<0.05.Results:1 acute pancreatitis cell model was successfully established,the levels of supernatants AMY increased with time in CAE group(P<0.05).The levels of supernatants AMY in CAE groups were higher than control group(P<0.05).In CAE group,the levels of CUEDC2mRNA were lower than control group(P<0.05).The levels of CUEDC2mRNA were no significant difference in CAE group at various time(P>0.05).2 After transfected with CUEDC2 plasmid,AR42J cells could visible green fluorescent particles and grow in good condition witch suggests that transfection was successful.The transfection efficiency was about 65%.The levels of CUEDC2 protein in CUEDC2 plasmid group were higher than negative plasmid group(P<0.05).Compared with control group,there had no change in negative plasmid group(P>0.05).3 The levels of AMY in cell culture supernatants:In CUEDC2-Control group and Neg-Control group,the levels of AMY had on significant changes at each time and there had no obvious difference in two groups(P>0.05).In CUEDC2-CAE group and Neg-CAE group,the levels of AMY were increased with time and were higher than respective control group.The levels of AMY in CUEDC2-CAE group were less than Neg-CAE group(P<0.05).4 The levels of TNF-a,IL-6 in cell culture supernatants:The levels of TNF-a,IL-6 in CUEDC2-Control group and NG-Control group were low at each time;Compared with Neg-Control group,the levels of TNF-a,IL-6 in CUEDC2-Control group had no statistical significances(P>0.05).The levels of TNF-a,IL-6 in CUEDC2-CAE group and Neg-CAE group were increased with time and were higher than respective control group(P<0.05).The levels of TNF-a,IL-6 in CUEDC2-CAE group were lower than Neg-CAE group(P<0.05).Conclusions:1 The AP cell model was successfully prepared by caerulein intervention.2 In AP cell model,the level of CUEDC2 mRNA was down-regulated.3 The AP cell model with overexpressing CUEDC2 was successfully constructed,which lay the foundation in study the role of CEUDC2 in acute pancreatitis.4 The levels of AMY,TNF-?,IL-6 can descrease in AR42J cell after tancfection.The study suggests CUEDC2 can weaken the expression of the inflammatory mediators and play the role of protection in AP.