The Mechanism Of Mitochondrial Calcium Overload In Pancreatic Ductal Epithelial Cell Apoptosis Induced By Caerulin And Palmitic Acid

Objective:The human pancreatic ductal epithelial cell（HPDE6C7）was induced by caerulin（CAE）and palmitic acid（PA）to better mimicked acute pancreatitis（AP）and hypertriglyceridemia pancreatitis（HTGP）in vivo environment.To study apoptosis in the experimental environment,and to observe cytoplasmic and mitochondrial calcium and mitochondrial ultrastructure,as well to detect the expression of mitochondrial calcium uniporter complex（MCUC）and caspase-3.The aim of this study was to investigate the mechanism of mitochondrial calcium overload in HPDE6C7 apoptosis which induced by CAE and PA.Methods:HPDE6C7 were cultured with CAE at 10^‐7mol/L to induce experimental AP,and co-cultured with CAE at 10^‐7mol/L and PA at 2mmol/L to induce experimental HTGP at different times（3h,6h,12h,24h,48h）,respectively.The morphological changes of cells were observed by microscope.The apoptosis was determined by DAPI staining and flow cytometry.Follow by choosing the control group as blank group,and CAE groups and CAE+PA groups at 6h,12h,24h as model groups.Seven groups were selected in a random grouping.We used fluorescence calcium staining techniques to measure calcium level both in cytoplasm and mitochondrion,and observed ultrastructure varies of mitochondrion at ultra-high power by transmission electron microscope（TEM）.The m RNA relative expression of MUC and EMRE were detected by real-time q PCR and the protein relative expression of MUC,EMRE and caspase-3 were detected by western blot.Results:After induced by CAE and CAE+PA,HPDE6C7 cells undergone obvious changes compared with control group,and cell death increased with time of induction.Cells degeneration became more and more apparent with time,as well dead cells increased significantly.Apoptosis in CAE groups and CAE+PA groups increased with time after DAPI staining（P（27）0.05）.Notably,apoptosis rate detected by low cytometry went up steadily with time-dependent character in the CAE groups and CAE+PA groups compared with the control group（P（27）0.05）.Cytoplasm and mitochondrion contained small amount of calcium in the control group.Calcium added with time in the CAE groups and CAE+PA groups versus the control group（P（27）0.05）.And compared with the CAE groups,cytoplasmic and mitochondrial calcium overload in the CAE+PA groups were more obvious at the same time（P（27）0.05）.As for mitochondrial ultrastructure under the TEM,mitochondrion in the CAE groups and the CAE+PA groups appeared distinct and characteristic ultra-damage and morphological changes.The expression of MCU and EMRE,the MCUC gap-associated components,in the CAE groups and the CAE+PA groups were different from those in the control group（P（27）0.05）,and the differences also existed between these two model groups at the same time（P（27）0.05）.Meanwhile,it was found that caspase-3 protein was up-regulated in each model group with time,and that CAE+PA groups differed from CAE groups at 6h and 24h（P（27）0.05）.Conclusions:HPDE6C7 induced by CAE,and CAE and PA increase apoptosis.HTGP induced by CAE and PA,and AP induced by CAE can lead to cytoplasmic calcium overload,which result in mitochondrial calcium overload through MCUC.The apoptosis is related to endogenous apoptosis pathway caused by mitochondrial calcium overload.