| Part 1 Identification and Validation of Hub Genes in Acute Pancreatitis and HypertriglyceridemiaObjective The pathogenesis of acute pancreatitis(AP)and the relationship between acute pancreatitis and hypertriglyceridemia are complex and not fully understood.The purpose of this study was to identify the hub genes along with common differentially expressed genes(DEGs)between acute pancreatitis and hypertriglyceridemia.Methods We downloaded three gene expression profiles of AP and one gene expression profile of hypertriglyceridemia from the Gene Expression Omnibus(GEO)database and filtered the DEGs based on the above four datasets.Next,we identified the hub genes by performing the Gene Ontology(GO)term analysis,Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analysis,and protein-protein interaction(PPI)construction.We also established mouse models with hypertriglyceridemia and AP using a high-fat diet and injection of caerulein(CAE),respectively.Finally,the immunohistochemical analysis was used to verify the differential expressions of hub genes in AP,hypertriglyceridemia,and normal pancreatic tissue.Results A total of 105 DEGs associated with AP and 149 DEGs associated with hypertriglyceridemia were identified.Additionally,we identified six hub genes of AP,all of which were upregulated DEGs and closely related to the cytoskeleton while two DEGs genes were common in both AP and hypertriglyceridemia.We also verified their expression of hub genes in mouse models.Conclusions Overall,we identified six hub genes associated with AP and two common DEGs associated with AP and hypertriglyceridemia.This study could provide new ideas for further elucidation of the pathogenesis of hypertriglyceridemia-induced acute pancreatitis in the future.Part 2 Changes of MCU and SIRT3 Expression In a Mouse Model of Hypertriglyceridemia-induced Acute PancreatitisaObjective To investigate the expression of MCU and SIRT3 in mouse acute pancreatitis(AP)and hypertriglyceridemia-induced pancreatitis(HTGP)models.Methods Twenty 4-week-old C57BL/6J mice were randomly divided into control group,hypertriglyceridemia(HTG)group,AP group and HTGP group.HTG mice were fed with high fat diet for 4 weeks,AP and HTGP models were established by intraperitoneal injection of caerulein.Pancreatic tissues of mice were collected for HE staining,pancreatic histopathological score to assess pancreatic inflammation,venous blood was collected to detect serum amylase and triglyceride levels,western-blot and immunohistochemistry to detect MCU and SIRT3 expression.Results The level of triglyceride in HTG group was significantly higher than that in control group.The triglyceride level in HTGP group was significantly higher than that in AP group.The amylase level in AP group was not significantly higher than that in control group,but the amylase level in HTGP group was significantly higher than that in HTG group.HE staining results showed that the pancreatic tissues of AP group and HTGP group were edema,interlobular space was widened significantly,and a large number of inflammatory cells were infiltrated.Pancreatic histopathological score in AP group and HTGP group was significantly higher than that in control group and HTG group,respectively.The expression level of MCU in AP group and HTGP group was significantly higher than that in Control group and HTG group,and the expression level of MCU in HTGP group was more significantly higher than that in AP group.The expression level of SIRT3 in AP group and HTGP group was significantly lower than that in Control group and HTG group,respectively,and the expression level of SIRT3 in HTGP group was more significantly lower than that in AP group.Conclusions HTG,AP and HTGP models were established successfully.HTGP mice had longer duration of elevated serum amylase,higher pancreatic histopathological scores,and more severe local pancreatic inflammation than AP mice.During the pathogenesis of AP,the expression of MCU was up-regulated,and the expression of MCU was higher in HTGP than in AP.During the pathogenesis of AP,the expression of SIRT3 was down-regulated,and the expression of SIRT3 was down-regulated more severely in HTGP than in AP.Part 3 Role of MCU in Human Pancreatic Duct Epithelial Cell Modelof Hypertriglyceridemia-induced Acute Pancreatitis in VitroObjective HPDE6-C7 cells were treated with triglycerides(TG)and caerulein(CAE)to simulate the development of AP and HTGP in vitro.To investigate the mechanism of MCU in the cytoskeleton of pancreatic ductal epithelial cells,Ruthenium Red(RR)was used as an inhibitor of MCU.Methods MCU gene silencing was used to interfere with MCU gene expression.HPDE6-C7 cells were divided into 8 groups according to different intervention agents:Control group,RR group,TG group,TG+RR group,CAE group,CAE+RR group,TG+CAE group and TG+CAE+RR group.MCU expression,mitochondrial calcium ion,ROS,MDA and GSH concentrations,permeability of monolayer cells and microfilament structure of each group were detected.Results After MCU gene silencing,HPDE6-C7 cells could not grow normally.The expression level of MCU,mitochondrial calcium ion level,ROS,MDA and single-layer cell permeability in CAE and TG+CAE groups were significantly higher than those in Control group and TG group,and the relative expression level of MCU in TG+CAE group was higher than that in CAE group,and the above changes could be reversed by RR intervention.The level of GSH in the CAE group and TG+CAE group was significantly lower than that in the Control group and TG group,respectively,and the decrease degree of GSH in the TG+CAE group was more severe than that in the CAE group,and the above changes could be reversed by RR intervention.In CAE and TG+CAE groups,the disordered structure of microfilaments disappeared,and the microfilaments were loosened and dispersed.After RR intervention,the microfilaments partially recovered and the disordered structure of microfilaments improved.Conclusions In vitro AP model of HPDE6-C7 cells induced by CAE was established successfully.The silencing of MCU gene resulted in a large number of apoptosis of HPDE6-C7 cells.CAE stimulates the high expression of HPDE6-C7 cells through MCU,causing mitochondrial calcium overload and promoting oxidative stress,leading to the destruction of HPDE6-C7 cytoskeleton and the damage of pancreatic duct epithelial mucosal barrier,thus promoting the onset of acute pancreatitis.Part 4 Role of SIRT3 in Human Pancreatic Duct Epithelial Cell Modelof Hypertriglyceridemia-induced Acute Pancreatitis in VitroObjective HPDE6-C7 cells were treated with Triglycerides(TG)and Caerulein(CAE)to simulate the development of AP and HTGP in vitro.The role of SIRT3 in the cytoskeleton of pancreatic ductal epithelial cells was investigated by intervention with 3-TYP,an active inhibitor of SIRT3.Methods HPDE6-C7 cells were divided into 8 groups according to different intervention agents:Control group,3-TYP group,TG group,TG+3-TYP group,CAE group,CAE+3-TYP group,TG+CAE group and TG+CAE+3-TYP group.The expression of SIRT3,the concentration of ROS,MDA and GSH,the permeability of monolayer cells and the structure of microfilaments in each group were detected.Results ROS,MDA and monolayer cell permeability in CAE group and TG+CAE group were significantly higher than those in Control group and TG group,respectively,and these trends could be aggravated by 3-TYP intervention.The level of GSH in the CAE group and TG+CAE group was significantly lower than that in the Control group and TG group,respectively,and the degree of GSH decline in the TG+CAE group was more severe than that in the CAE group,and the above changes could be aggravated by 3-TYP intervention.In CAE and TG+CAE groups,the microfilaments were obviously disordered,and the micro filaments were loosened and dispersed.After 3-TYP intervention,the microfilaments became more disordered.Conclusions CAE stimulation of HPDE6-C7 cells caused enhanced progressive oxidative stress response,leading to acute pancreatitis HPDE6-C7 cytoskeleton destruction,damage to pancreatic ductal epithelial mucosal barrier,thus promoting the onset of acute pancreatitis.The inhibition of SIRT3 activity by 3-TYP further aggravates oxidative stress and changes in microfilament structure during the pathogenesis of acute pancreatitis,and increases the permeability of monolay cells of HPDE6-C7 cells. |
PDF Full Text Request