The Effect Of Silencing X-box Binding Protein 1 With SiRNA On The Apoptosis Of Acute Pancreatitis Acinar Cells

Acute pancretitis(AP)is a common acute abdominal pain in clinical.Most patients were clinically self-limited mild acute pancreatitis(MAP).There were still about 20%~30% of patients were suffered severe acute pancreatitis(SAP)at the beginning or in the progression of MAP.SAP ischaracterized assystemic inflammatory response syndrome(SIRS).The injury of pancreas mainly present with apoptosis and necrosis in AP.Necrotic cells could release a lot of cytokines and inflammation mediators.But apoptosis is a programmed cell death,which is realized by the formation of apoptotic bodies.Apoptosis avoids the release of cellular contents.Therefore,a study of how to induce an apoptosis of the inflammatoryacinar cells early,avoiding the course of AP aggravating has become a hotspot in recent years.Endoplasmic reticulum stress(ERS)was a newlydiscovered process which had relations to cell apoptosis recent years after mitochondrial pathway and death receptor pathway.Kinds of physical and chemical factors could lead to the imbalance of the endoplasmic reticulum homeostasis.Then that results in the occurrence of ERS.Unfolded protein response(UPR)is the main mechanismof the response to ERS,which helps ER recover from the stress state to homeostasis through PERK/eIF?,IRE1/XBP1 and ATF6 pathways.Meanwhile,the apoptosis of injured cells may be induced if the ERS were too strong or too long.XBP1 protein plays a key role in UPR,which could up-regulate the expression of genes of ER chaperones and strengthen the capacity of ER processing unfolded and misfolded proteins.Pancreatic acinar cells are very susceptible to ERS.Both ERS and UPR are involved in the pathogenesis and progression of AP.The XBP1knock-out acinar cells may be lack of the ER structure and may come out an extensive apoptosis.In the acinar cells of AP,however,whether there is any treatment to XBP1 that could induce the apoptosis of acinar cells early to avoid the inflammation aggravating to necrosis or not,there is no clear report about that at home or abroad till now.Objective:In this study,RNAi technology was used to knock down the expression of XBP1 gene in AR42 J cells.Caerulein(CAE),caerulein plus lipopolysaccharide(CAE+LPS)were used to treat AR42 J cells respectively to create models of different severity of AP in vitro.We aimed to explore the effect of the down-regulation of the expression of XBP1 on the apoptosis of pancreas acinar cells in AP.Method: Threesmall interference RNA(siRNA)aimed at the XBP1 gene in rat were designed randomly.OnenonsensesiRNA which had no homologywith XBP1 gene was also designed.We named them XBP1-303,XBP1-684,XBP1-808 and negative control.Lipofectamine 2000 was used to transfect these si RNA into AR42 J cells.The siRNA which had the best silence effect of the XBP1 gene would be chosen by real-time RT PCR.The chosen siRNA would be used to transfect AR42 J cells once more.AR42 J cells were treated with caerulein alone,caerulein plus LPS and complete culture mediumrespectivelyat 48 h after transfection.We divided these cells into 3 groups as their treatment: CAE group,CAE+LPS group and negative control group.Cells were collected based on the length of time after treatment.The time point were 4h,8h and 24 h after treatment.CHOP mRNA level of each group was measured by real-time polymerase chain reaction(Real-time PCR).The level of the expression of caspase-12 and GRP78 at 24 h after treatments was measured by western blot.The ratio of cell apoptosis and necrosis of each group of cells was measured by Annexin V-FITC/PI double staining and flow cytometry.Results:1 Thelevel of XBP1 mRNA expression: the expression of XBP1 mRNA which group was transfected by XBP1-303 was the lowest in all groups.2 Thelevel of CHOP mRNA expression: After 4 hours of the treatment of CAE+LPS,the level of CHOP mRNA expression in the transfected group was significantly higher than the untransfected group(P<0.05).However,after 8 hours and 24 hours of the treatment of CAE+LPS,there was no obvious difference between the two groups(P>0.05).The level of CHOP mRNA of transfected cells after treated by CAE alone was significantly higher than the untransfected in each time point(P<0.05).The level of CHOP mRNA of cells after treated by CAE+LPS was significantly higher than the cells treated by CAE alone(P<0.05).The later was significantly higher than the negative control group cells(P<0.05).The level of CHOP mRNA expression in the transfected group treated by CAE+LPS was significantly higher than any other group(P<0.05).3 The expression of GRP78: In the control group without transfection,the expression of GRP78 was very little.The expression of transfected cells in control group was significantly higher than that the non transfected(P<0.05).In the non transfected cells,there was significant difference in the expression between CAE group and CAE+LPS group(P<0.05).Also it was significantly higher than the control group(P<0.05).There was significant difference in the expression between CAE group and CAE+LPS group(P<0.05)in the transfected cells.And it was significantly higher than the other groups(P<0.05).4 The expression of caspase-12:According to whether the XBP1 knock down or not: At each time point,the expression of transfected group was significantly higher than that in the non transfected group(P<0.05).And with the progression,the expression of the caspase-12 level reached the peakat 8 h,and there was no significant difference in the expression between 8h and 24 h(P>0.05).The expression level of transfected cells treated by CAE was also significantly higher than that in the non transfection group at each time point(P<0.05).But there was no significant relationship between the level of their expression and the progression of the treatment in each group.In the control group,the expression levels of transfected cells at each time point were also significantly higher than those of the non transfected cells(P<0.05).There was no obvious change with time progression,either.According to the treatment level,whether transfection or not,in the CAE+LPS treated cells,the expression levels were significantly higher than those in CAE group in addition to the time points at 4 h(P<0.05).Whether the XBP1 gene is knockdown or not,it has an interaction with the treatment levelon AR42 J cells.In our opinion,ERS related apoptosis in the CAE+LPS treatment group in the transfected cells was significantly higher than that in any other group(P<0.05).5 Apoptosis and necrosis ratio were detected with Annexin V/PIdouble staining: The proportion of apoptotic cells in transfected cells treated by CAE was significantly higher than that in the non transfected cells after CAE treatment for 24 h(P<0.05).The proportion of apoptotic cells in transfected cells treated by CAE+LPS was significantly higher than that in the non transfected cells after CAE+LPS treatment for 24 h(P<0.05),the proportion of necrotic cells was on the contrary(P<0.05).In the control group cells,the proportion of apoptotic cells in transfected cells was significantly higher than that in the non transfected group(P<0.05),but there was no obvious difference in the proportion of necrotic cells in each group(P>0.05).The proportion of necrotic cells and apoptotic cells in CAE+LPS group were significantly higher than those in CAE group in the non transfected cells(P<0.05).Also the proportion of apoptotic cells and necrotic cells in CAE group were significantly higher than those in control groupin the non transfected cells(P<0.05).The proportion of necrotic cells and apoptotic cells of CAE+LPS group was significantly higher than that in CAE group in the transfected cells(P<0.05).Also the proportion of apoptotic cells in CAE group was significantly higher than that in control group(P<0.05).But there was no significant difference in the proportion of necrotic cells(P>0.05).Conclusion:1 We built MAP model with caerulein,SAP model with caerulein plus LPS,respectively.Compared with each other,the ERS related apoptotic level of cells in SAP model was significantly higher than cells in MAP model.With the progression of the disease there is an aggravating trend.The necrotic level of cells in SAP model was significantly higher than cells in MAP model.It indicated that these two models were successful.2 ERS was clearly involved in the progression of AP.In SAP and MAP models.3 The apoptosis mediated by ERS after the knockdown of XBP1 may reduce the proportion of necrotic cells,so it may attenuate the severity of AP.This effect is more significant in SAP model.4 XBP1 is one of the important factors in the ERS response mechanism.It can promote the recovery of the function of the endoplasmic reticulum,relieve the intensity of ERS.Silencing of XBP1 may aggravate ERS,and promote the apoptosis of pancrea acinar cells.