Effects Of CUEDC2 On Apoptosis Of Acute Pancreatitis Acinar Cells

Acute pancreatitis(AP)is an acute inflammatory response caused by multiple factors.AP is one of the common clinical acute abdomen with abrupt onset,rapid development,severe illness and complications.Most cases with mild acute pancreatitis(MAP)are clinical self-limitation.As for severe acute pancreatitis(SAP)associated with pancreatic surrounding tissue necrosis,so that the clinical condition of SAP is dangerous and high mortality.The pathogenesis of AP is complicated and apoptosis play an important role in the development of AP.Apoptosis and necrosis are two main forms of pancreatic acinar cell injury in AP.The main difference is that apoptotic cells format apoptotic bodies and are swiftly engulfed by macrophages,so that apoptotic cells rarely cause inflammatory reaction.The severity of AP is positively correlated with degree of necrosis,and negatively correlated with the degree of apoptosis.Inducing apoptosis of acinar cell in early stage is the key to treatment AP.CUE domain containing protein 2(CUEDC2)is a newly discovered protein which can inhibit the nuclear factor-?B(NF-?B)and Janus kinase/ signal transducer and activator of tran-scriptions(JAK/STAT)signaling pathway,but also can inhibit macrophage activation and the chaperone activity of heat shock protein 70.CUEDC2 is also involved in the regulation of cell cycle.Literature study found that the expression level and function of CUEDC2 are different in various tumors.Moreover,CUEDC2 is involved in the regulation of inflammatory responses,such as unilateral ureteral obstruction and colitis-associated cancer.In addition,CUEDC2 is involved in the regulation of cell apoptosis,such as myocardial ischemia-reperfusion injury and chronic myeloid leukemic.At present,there is no report on the role of CUEDC2 in acute pancreatitis,so that this study analyzed the effect of CUEDC2 on apoptosis of the acute pancreatitis acinar cells.Objective: Caerulein(CAE)was used to treat AR42 J cells to create AP model in vitro.Plasmid transfection technology was used to overexpress the expression of CUEDC2 protein in AR42 J cells.The purpose of our study is to explore the effect of CUEDC2 on apoptosis of the acute pancreatitis acinar cells.Methods:1 Caerulein was used to treat AR42 J cells to create AP model in vitro.Plasmid transfection technology was used to overexpress the expression of CUEDC2 protein in AR42 J cells.2 The experimental group:(1)negative plasmid control group(Neg-CON): The pEGFP-N1 plasmid was transfected into AR42 J cells and the cells was cultured in complete medium.(2)CUEDC2 plasmid control group(CUEDC2-CON): The p EGFP-N1-CUEDC2 plasmid was transfected into AR42 J cells and the cells was cultured in complete medium.(3)negative plasmid caerulein group(Neg-CAE): The pEGFP-N1 plasmid was transfected into AR42 J cells and the cells was cultured in 10-7mmol/L caerulein medium.(4)CUEDC2 plasmid caerulein group(CUEDC2-CAE): The p EGFP-N1-CUEDC2 plasmid was transfected into AR42 J cells and the cells was cultured in 10-7 mmol/L caerulein medium.The cells were divided into three subgroups: 4h,8h and 24 h.3 The expression of CUEDC2 mRNA was detected by using RT-PCR.The expression of CUEDC2,Bax and Bcl-2 were detected by using Western blot.While,the ratio of cell early apoptosis and late apoptosis and necrosis were detected by Annexin V-PE/7-AAD double staining combined with flow cytometry.Results:1 The level of CUEDC2 mRNA expression: the expression level of CUEDC2 mRNA was lower in caerulein group than that in control group(P<0.05).There was no significant difference in the expression between caerulein groups at different point(P>0.05).2 The building of AR42 J cell model of overexpression CUEDC2: the expression of CUEDC2 which group was transfected by pEGFP-N1-CUEDC2 plasmid was the highest in all group(P<0.05).There was no significant difference between the control group and negative plasmid group(P>0.05).It suggests that the cell model was built successfully.3 The expression of Bax: the expression level of Bax was higher in caerulein group than that in control group(P<0.05).In control groups,the expression level of Bax was higher in CUEDC2 plasmid control group than that in the negative plasmid control group(P<0.05),and the expression of Bax in control group was no obvious change with time progression.In caerulein groups,the expression level of Bax was higher in CUEDC2 plasmid caerulein group than that in the negative plasmid caerulein group at 4h and 8h(P<0.05),but there was no statistical difference at 24h(P>0.05).And with the time progression,the expression of the Bax level in negative plasmid caerulein group reached the peak at 8h,and there was no significant difference in the expression between 8h and 24 h.But,the expression of Bax in CUEDC2 plasmid caerulein group had no correlation with the time progression.4 The expression of Bcl-2: the expression level of Bcl-2 was lower in caerulein group than that in control group(P<0.05).In control groups,the expression level of Bcl-2 was lower in CUEDC2 plasmid control group than that in the negative plasmid control group(P<0.05),and the expression of Bcl-2 in control group was no obvious change with time progression.In caerulein groups,the expression level of Bcl-2 was lower in CUEDC2 plasmid caerulein group than that in the negative plasmid caerulein group at 4h and 8h(P<0.05),but there was no statistical difference at 24h(P>0.05).And with the time progression,the expression of the Bcl-2 level in negative plasmid caerulein group was lowest at 8h,and there was no significant difference in the expression between 8h and 24 h.But,the expression of Bcl-2 in CUEDC2 plasmid caerulein group had no correlation with the time progression.5 The Bax/Bcl-2 ratio: the Bax/Bcl-2 ratio was higher in caerulein group than that in control group(P<0.05).In control groups,the Bax/Bcl-2 ratio was higher in CUEDC2 plasmid control group than that in the negative plasmid control group(P<0.05),and the Bax/Bcl-2 ratio in control group was no obvious change with time progression.In caerulein groups,the Bax/Bcl-2 ratio was higher in CUEDC2 plasmid caerulein group than that in the negative plasmid caerulein group at 4h and 8h(P<0.05),but there was no statistical difference at 24h(P>0.05).And with the time progression,the Bax/Bcl-2 ratio level in negative plasmid caerulein group reached the peak at 8h,and there was no significant difference in the expression between 8h and 24 h.But,the Bax/Bcl-2 ratio in CUEDC2 plasmid caerulein group had no correlation with the time progression.6 Early apoptosis rate and late apoptosis and necrosis rate: the early apoptosis rate was higher in caerulein group than that in control group(P<0.05),and the late apoptosis and necrosis rate was also higher in caerulein group than that in control group(P<0.05).In control groups,the early apoptosis rate was higher and the late apoptosis and necrosis rate was lower in CUEDC2 plasmid control group than that in the negative plasmid control group(P<0.05).In caerulein groups,the early apoptosis rate was higher and the late apoptosis and necrosis rate was lower in CUEDC2 plasmid caerulein group than that in the negative plasmid caerulein group at 8h(P<0.05),but there was no statistical difference at 24h(P>0.05).Conclusions:1 We built AP model in vitro with caerulein.And we also built the AR42 J cell model of overexpression CUEDC2 successfully by using plasmid transfection technology.2 The expression level of CUEDC2 mRNA is down-regulated in AP compared with normal cell.3 Early apoptosis rate and late apoptosis and necrosis rate in AP was higher than that in normal cells.Overexpressing CUEDC2 could increase the apoptosis rate and reduce the necrosis rate in the early period of AP.It suggests that CUEDC2 promote the apoptosis of pancreas acinar cells and CUEDC2 is a protective protein in AP.