The Role Of CDK5RAP3-Regulating Endoplasmic Reticulum Stress Pathway In Pancreatic Acinar Cells Injury Induced By Caerulein And Its Mechanisms

Background:Pancreas is a digestive and endocrine organ consisting of endocrine and exocrine portion.The pancreatic acinar cells(PACs)are the most important cells and functional units in the exocrine portion of the pancreas,which are mainly responsible for the synthesis,storage and secretion of digestive enzymes.Pancreatitis is an inflammatory disease that occurs firstly in PACs and is driven by PACs injury.Therefore,elucidatinge the molecular mechanism of PACs injury is of great scientific significance for the prevention and treatment of pancreatitis.Cyclin-dependent kinase 5 regulatory subunit-associated protein 3(CDK5RAP3),a multifunctional molecule that plays a role in various cell biological processes such as DNA damage response,cell cycle progression,apoptosis,post-translational protein modification,and cell signal transduction.In addition,CDK5RAP3 plays an important role in embryonic development,maintenance of hepatocyte and intestinal epithelial cell function in mammals,and tumorigenesis.However,the relationship between CDK5RAP3 and pancreatitis has not been publicly reported.Interestingly,our previous study found that conditional knockout of CDK5RAP3 in PACs resulted in massive atrophy of PACs and decreased amylase expression of pancreatic tissue in mice,suggesting that CDK5RAP3 plays an important role in maintaining the survival and function of PACs.The purpose of this study is to further explore the role of CDK5RAP3 in pancreatitis induced PACs injury and its potential molecular mechanism.Objective:To investigate the role of CDK5RAP3 in PACs damage induced by Cerulein based on the endoplasmic reticulum stress(ERS)pathway.Methods:1.Grouping and treatment:(1)The mice model of acute pancreatitis(AP)and chronic pancreatitis(CP): model group(intraperitoneal injection of Cerulein)and control group(intraperitoneal injection of PBS);(2)the model of AR42 J injury induced by Cerulein: treated with Cerulein with different concentrations(0,100,400,800 n M groups)for 48 h and treated with 800 n M Cerulein with different timepoints(0,12,24,48 h groups);(3)Exogenous intervention of CDK5RAP3 expression experiment: The Cer group(Cerulein treatment),si NC+ Cer group(transfected with Negative control si RNA + Cerulein treatment),or the vector+Cer group(transfected with vector + Cerulein treatment),the #1+ Cer group(transfected with # 1-CDK5RAP3 si RNA + Cerulein treatment)or over+ Cer group(transfected with CDK5RAP3 over-expressed plasmid + Cerulein treatment);(4)Amylase induction assay: untreated group(normal cultured cells),Cerulein group(Cerulein treatment),dexamethasone group(dexamethasone treatment),Cerulein +dexamethasone group(Cerulein combined with dexamethasone treatment);(5)ERS inhibitor 4-PBA intervention experiment: si NC group(transfected with Negative control si RNA + Cerulein treatment),si RNA group(transfected with #1-CDK5RAP3 si RNA + Cerulein treatment),vehicle group(transfected with Negative control si RNA + PBS + Cerulein treatment),4-PBA group(transfected with Negative control si RNA + 4-PBA+ Cerulein treatment),vehicle + si RNA group(transfected with #1-si RNA + PBS + Cerulein treatment),4-PBA+si RNA(transfection #1-si RNA+ 4-PBA + Cerulein treatment).2.Assessment of AP and CP: AP and CP models induced by Cerulein were established,and hematoxylin-eosin(H&E)staining was performed to observe the pathological morphology of mouse pancreatic tissue.Masson staining was used to evaluate the degree of collagen fiber deposition in pancreatic tissue of CP model mice.3.Protein expression assessment of CDK5RAP3: Western Blot or immunohistochemical methods were used to detect the protein expression level of CDK5RAP3 in tissues or cells.4.AR42 J cell injury assessment:(1)Cell viability was detected by CCK-8assay;(2)LDH leakage rate was detected by cytotoxicity assay.(3)Apoptosis was observed by Hoechst 33342/PI fluorescence double staining.(4)The apoptosisrelated proteins: PARP,Bcl-XL,Bcl2/Bax,Cleaved Caspase 3,Bad were detected by Western Blot.(5)The expression and content of amylase in cells was detected by Western Blot and AMS.Results:1.Cerulein induced PACs injury and down-regulated CDK5RAP3 expression in vivo and in vitro(1)Compared with the control group,pancreatic acinar structure was destroyed,PACs were damaged,inflammatory cells were infiltrated,and pathological scores were higher in the AP and CP model groups(both P<0.001)and more collagen fibers were deposited in pancreatic tissue in CP model group(P<0.01),the expression of CDK5RAP3 protein in pancreas tissues of AP and CP model groups was lower(P<0.001 and P<0.01).(2)Compared with the 0 n M Cerulein group,Cerulein(400 and 800 n M)significantly inhibited AR42 J cell viability(P<0.001),increased the LDH leakage rate of AR42 J cells(P<0.05 and P<0.001)and apoptosis,up-regulated expression of pro-apoptosis-associated proteins Bad,PARP,Cleaved Caspase 3(all P<0.05),and down-regulated the expression of Bcl2/Bax,Bcl-XL and CDK5RAP3(all P<0.001);Compared with the 0 h group,the 48 h Cerulein group showed a significant decrease in AR42 J cell viability(P<0.001),a significant increase in LDH leakage rate(P<0.001)and apoptosis,increase protein expression of Bad,PARP,Cleaved Caspase 3(P<0.05,P<0.01,P<0.05),while the protein expressions of Bcl2/Bax,Bcl-XL and CDK5RAP3 were decreased(P<0.001,P<0.01,P<0.001);Compared with the untreated group,the expression and content of amylase in AR42 J cells in the amylase + dexamethasone group were significantly increased(all P<0.001).2.CDK5RAP3 alleviated AR42 J cell injury induced by Cerulein(1)Compared with the Cer group,the #1+Cer group showed significantly decrease in CDK5RAP3 expression,AR42 J cell viability,increased LDH leakage rate,amylase expression and content(P<0.01,P< 0.05,P< 0.001),increased apoptotic cells,and increased protein expression of Bad,PARP,Cleaved Caspase 3(P<0.05,P<0.01,P<0.01),while the protein expressions of Bcl2/Bax and Bcl-XL decreased(P<0.01,P<0.05).(2)Compared with the Cer group,the over+Cer group showed a significant increase in CDK5RAP3 expression,AR42 J cell viability(P<0.001,P<0.01),a decrease LDH leakage rate,amylase expression and content decreased(P<0.001,P<0.001,P<0.05),apoptotic cells,and a decrease in protein expression of Bad,PARP,Cleaved Caspase 3(P<0.05,P<0.05,P<0.01),while the protein expressions of Bcl2/Bax and Bcl-XL were increased(both P<0.05).3.CDK5RAP3 alleviated AR42 J cell injury induced by Cerulein via regulating ERS(1)Compared with the 0 n M Cerulein group,the 800 n M Cerulein treatment for48 h significantly increased the expression of ERS-related proteins IRE1α,p-e IF2α,ATF6,CHOP and XBP1 s in AR42 J cells(all P<0.01),but the protein expression of GRP78/Bip was decreased(P<0.01);Compared with the 0 h group,the protein expressions of IRE1α,p-e IF2α,ATF6,CHOP,XBP1 s and GRP78/Bip in the 800 n M12 h group were increased(all P<0.01),and the expressions of IRE1α,ATF6,CHOP,XBP1 s,GRP78/Bip were decreased after 24 and 48 h treatment compared with 12 h group(all P<0.05).(2)Compared with the Cer group,the expression of ERS-related proteins IRE1α,p-e IF2α,ATF6,CHOP,XBP1 s,GRP78/Bip was significantly increased in the #1+Cer group(all P<0.001),while the expression of these ERS-related proteins decreased in the over+Cer group(all P<0.05).(3)Compared with the vehicle groups,4-PBA not only alleviated AR42 J cell injury induced by Cerulein and inhibited the activation of ERS pathway,but also partially reversed the CDK5RAP3 si RNA-induced AR42 J cell injury and ERS pathway activation.Conclusion:1.Cerulein induces PACs injury and down-regulate CDKRAP3 protein expression both in vivo and in vitro.2.CDK5RAP3 has a protective effect in Cerlein-induced AR42 J cell injury.3.CDK5RAP3 affects Cerulein-induced AR42 J cell injury through negatively modulating the ERS pathway.