The Effect Of Silencing X-Box Binding Protein 1 With SiRNA On The TNF-?,IL-6 Of Acute Pancreatitis Acinar Cells

Acute pancreatitis(AP)is a common clinical critical illness characterised with acute onset,rapid progression and high mortality.In addition to pancreatic lesions,it can also induce extrapancreatic tissues and organs damage and even cause systemic inflammatory response syndrome(SIRS)and multiple organ dysfunction syndrome(MODS).Although in the past decades,we have made great progress about acute pancreatitis research,the pathogenesis is still complex.In recent years,with the rapid development of molecular biology,cell biology and other life sciences,great progresses have been made about cell biology mechanisms of pancreatic acinar cell damage,especially the endoplasmic reticulum stress(ERS)mechanism.X-box binding protein 1(XBP1)is a protein discovered in recent years and is related to protein folding and the construction of endoplasmic reticulum.XBP1 is essential to modulating ERS events and maintaining viability of adult acinar cells.The expression upregulation of XBP1 is often a sign of ERS.Numerous studies have indicated that XBP1 is closely related to the development of a variety of human diseases,such as diabetes,cancer,inflammatory diseases,viral infections,cardiovascular diseases,central nervous system diseases and so on.However,the research on the relationship between pancreatitis and XBP1 is absent.This study is to explore the role of XBP1 in acute pancreatitis.Objective: By intervening AR42 J cells which were XBP1 gene silenced with caerulein or caerulein combining lipopolysaccharide,we got pancreatitis in vitro model of different levels.Then by 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 silencing X-box binding protein 1 with siRNA on the TNF-?,IL-6 of acute pancreatitis acinar cells.Methods:1 Getting XBP1 gene silencing AR42 J cells: AR42 J cells were transfected by fluorescence labeled siRNA(FAM-siRNA)and we screened out the optimum concentration of siRNA transfection by fluorescence microscopy.In three kinds of siRNA associated with XBP1 silencing,we screened out the siRNA which had the best silencing effect by real-time polymerase chain reaction(RT-PCR).2 Experimental models: The AR42 J cells were randomly divided into transfected group and untransfected group.Transfected group: AR42 J cells were transfected with siRNA which had the best silencing effect.These cells were divided into three groups: caerulein group(CAE group),caerulein combining lipopolysaccharide group(CAE +LPS group)and control group.CAE group was given 10-7 mol/L caerulein.CAE + LPS group was intervened with 10-7 mol/L caerulein and 10 mg/L lipopolysaccharide.Control group was given an equal volume of complete medium.In each group,culture supernatants were collected at the time of 6hs,12 hs and 24 hs respectively.Untransfected group: Untransfected cells were divided into three groups:caerulein group(CAE group),caerulein combining lipopolysaccharide group(CAE + LPS group)and control group.The intervention methods were the same as the former.3 Experimental methods: We used RT-PCR to observe the transfection effect.The levels of AMY in cell culture supernatants were measured by Amylase Assay Kit.ELISA(enzyme linked immunosorbent assay)was used to detect the expression of TNF-? and IL-6.4 Statistical Methods: All statistical analysis was performed by SPSS13.0 for Windows.The significance was assumed at P < 0.05.Results:1 Different kinds of siRNA were used to transfect cells.We screened the effect of silencing by means of RT-PCR which detected XBP1 mRNA expression.Compared with blank group,the XBP1 mRNA expression levelwas lower in XBP1-303 group,XBP1-684 group and XBP1-808 group,especially in XBP1-303 group.There were statistical differences(P<0.05).There was no significant difference between Negative control group,Lipo group and Blank group.2 The levels of AMY in cell culture supernatants: Within two groups: At each time point,the levels of supernatants AMY in Control group were lower and had no significant change;In contrast,the levels of AMY in CAE and CAE + LPS group were significantly higher(P<0.05),gradually increased with time and reached the highest level in 8 h;Compared with the CAE group,the levels of AMY in CAE + LPS group were significantly increased at each time point(P<0.05).Comparison between the two groups: The levels of AMY in Control group had no significant difference at all time points.About the levels of AMY in both CAE and CAE + LPS group,transfected group were significantly increased than untransfected group(P <0.05).3 The levels of TNF-? in cell culture supernatants: Within two groups:The levels of supernatants TNF-? in Control group were lower at all time points;Compared with the Control group,the levels of TNF-? in CAE and CAE + LPS group were significantly increased respectively(P <0.05)and gradually increased with time;The levels of TNF-? in CAE + LPS group were significantly higher than that in CAE group at each time point(P<0.05).Comparison between the two groups: At each time point,no significant difference existed in Control group about TNF-? levels.About the levels of TNF-? in CAE group,transfected group was significantly higher than untransfected group(P<0.05).The result was the same in CAE + LPS group.4 The levels of IL-6 in cell culture supernatants: Within two groups: At each time point,the levels of IL-6 in Control group did not change significantly and were low;The levels of IL-6 in CAE group and CAE + LPS group at all time points were gradually increased with time and were significantly higher than that in Control group(P<0.05);About the levels of IL-6,compared with CAE group at each time point,CAE + LPS group was significantly increased(P<0.05).Comparison between the two groups: Thelevels of IL-6 in Control group at all time points were no significantly different.In CAE group and CAE + LPS group at all time points,the IL-6levels of transfected group compared with untransfected group were significantly higher(P<0.05).Conclusions:1 Application of caerulein and caerulein combining lipopolysaccharide can replicate AP and SAP in vitro model,which can provide the condition to study the role of XBP1 in acute pancreatits.2 In the AP cell model,TNF-? and IL-6 levels were significantly increased and they played an important role in the development of the AP as two key inflammatory factors.3 After the XBP1 silencing,the levels of AMY increased in acute pancreatitis acinar cells and at the same time,TNF-? and IL-6 levels were significantly increased.This indicated that XBP1 involved in acute pancreatitis and played a protective role possibly by reducing the cytokine TNF-? and IL-6 production.