The Effect And Mechanism Of Tumor Necrosis Factor Receptor-related Factor 6 On Pyroptosis In Pancreatic Ductal Epithelial Cells

Objective: Acute pancreatitis is a multifactorial disease,involving inflammation of the pancreas,production of inflammatory mediators and disorder of digestive enzymes,which eventually leads to multiple organ dysfunctions.The pathogenesis of AP is not completely clear,which may be related to the expansion of inflammatory cascade caused by theactivation of cell pyroptosis and the impairment of pancreatic duct epithelial barrier function.In this study,we investigated whether the human pancreatic ductal epithelial cells(HPDE6C7)can be inducedpyroptosis by the stimulation of caerulin(CAE)and the effect related proteins caspase-1,NLRP3,IL-1β.Then,after up-regulating the TRAF6 gene by lentivirus transfection,we observed whether the TRAF6 gene affects the pyroptosis and the NLRP3 inflammatory pathway signal.Methods: The HPDE6C7 cells were used in this study.First,the cells were divided into four groups according to different time points of stimulated by CAE:control,12 h,24h,and 48 h.Hoechst/PI double staining was used to detect the pyroptosis phenomenon.Extracted the total RNA of each group,the expression level of caspase-1 and NLRP3 mRNA were detected by real-time fluorescence quantitative PCR.The supernatant of each group was collected and detected the concentration of IL-1β by ELISA.Secondly,HPDE6C7 cells were divided into four groups: TRAF6 group,Blank group,TRAF6+CAE group and Blank+CAE group.HPDE6C7 cells were up-regulated TRAF6 gene expression using lentivirus transfection technology in TRAF6 group,and Blank group as its negative control with transecting with blank virus vector.Then both of them were stimulated for 48 h by CAE,as TRAF6+CAE group and Blank+CAE group.The phenomenon of pyroptosis was observed by Hoechst/PI double staining.The expression of TRAF6,caspase-1,caspase-3 and NLRP3 were detected by real-time fluorescence quantitative PCR and western blotting,and the expression of IL-1β in supernatant was detected by ELISA.Results:(1)After CAE intervention,the Hoechst/PI double staining showed that the HPDE6C7 cells in each groups were round,swollen,necrotic and the red fluorescence increased,especially in 48 h.The qPCR showed that the expression levels of NLRP3 and caspase-1 mRNA in HPDE6C7 cells were increased.Among this,the expression level of NLRP3 mRNA increased was time-dependent,and the highest expression level was found at 48h(P < 0.05).While the highestexpression level of caspase-1 mRNA was at 12 h,and then decreased slightly and remained stable,but it was still higher than control group(P < 0.05).Similarly,the results of ELISA showed that the concentrations of IL-1β in the supernatant of cells were 1061.43±6.99pg/ml,1376.17±3.12pg/ml,and 5836.26±3.02pg/ml respectively after CAE intervention for 12 h,24h and48 h,which was significantly higher than control group(P < 0.05).(2)The HPDE6C7 cells was transfected by lentivirus transfection,qPCR and western blotting confirmed that the expression level of TRAF6 gene in TRAF6 group was significantly up-regulated than Blank group(P < 0.05).After up-regulating the TRAF6 gene,the expression levels of caspase-3 and NLRP3 in the TRAF6 group were higher than Blank group(P < 0.05).Hoechst/PI double staining also showed a small amount of cell necrosis was found in TRAF6 group.Then,using CAE stimulated cells for 48 h,Hoechst/PI double staining showed that there were more pyroptosis cells in TRAF6+CAE group and Blank+CAE group.Compared with Blank+CAE group,the pyroptosis cells in TRAF6+CAE group were more obvious.Western blot showed that the expression of caspase-1,caspase-3 and NLRP3 in TRAF6+CAE group was up-regulated compared with Blank+CAE group(P < 0.05).ELISA showed that the concentration of IL-1β in the supernatant of cell culture in TRAF6+CAE group was higher than Blank+CAE group(P < 0.05)Conclusions:(1)The pyroptosis of HPDE6C7 cells are induced by CAE through both the classical pathway of caspase-1 and non-classical pathway of caspase-3.(2)Over-expression of TRAF6 gene in HPDE6C7 cells up-regulatethe expression of caspase-1,NLRP3,caspase-3 and IL-1β,resulting the activation of pyroptosis.