Evaluation Of Severity And Pathogenesis Of Severe Acute Pancreatitis In Mice Based On Bile Acid Targeted Metabolomics

Objective: A C57BL/6 mouse model of severe acute pancreatitis(SAP)was established by injecting sodium taurocholate under the pancreatic capsule.Ultra-Performance Liquid Chromatography Mass Spectroscopy(UPLC-MS)was used to target and analyze the plasma cholic acid metabolism profiles of mice at different stages of onset,screen differential metabolites,and identify potential pathways,Analyze its significance in evaluating the severity and pathogenesis of SAP,in order to provide a theoretical basis and diagnosis and treatment ideas for clinical practice.Methods:1.Establish SAP mouse model and collect specimens: Sixty male C57BL/6 mice were randomly divided into SAP 2h group,SAP 6h group,SAP 12 h group,SAP 16 h group(before death),and control group.SAP group modeling method: After ether inhalation anesthesia,open the abdomen and inject 5% sodium taurocholate evenly under the pancreatic capsule to establish an SAP mouse model;The control group was injected with an equal amount of physiological saline.SAP group mice were subjected to enucleation of mouse eyeballs at 2,6,12,and 16 hours after modeling,and plasma was obtained by centrifugation.Pancreas and lungs were collected for pathological analysis,while specimens were collected using the same method in the control group.The SAP disease progression model was successfully established based on Schmidt score validation.2.Using ultra high performance liquid chromatography tandem mass spectrometry(UPLC-MS)technology to detect bile acid targeted metabolites in each group of plasma:Multiple statistical analysis was used to screen for differential bile acid in the model.PLS-DA first principal variable weight value(VIP),p-value,and fold change(FC)tests were used to screen for differential bile acid,with p<0.05,VIP>1,FC>2,or<0.5 being statistically significant.The identification of differential cholic acid is first preliminarily confirmed based on the precise molecular weight,and its possible molecular formula is speculated.Then,differential cholic acid is confirmed by comparing MS/MS fragment information with public databases(Metlin,HMDB,massbank,Lipid Maps,Mzcloud).3.Conduct pathway research through KEGG and related literature to preliminarily elucidate the pathogenesis and progression pathway of SAP.Results:1.After three months of exploration,we successfully constructed a male C57BL/6mouse SAP disease progression model.2.Bile acid detection results: Based on the existing bile acid profile(a total of 44 bile acids)targeted bile acid detection using UPLC-MS technique,20 bile acids were determined:3A-H-7-O-5B bile acid,bile acid impurity(12-Ketodeoxycholic acid,12-KDCA),allocholic acid(ACA),β-Murine cholic acid(β-MCA),Cholic acid(CA),Chenodeoxycholic acid(CDCA),Deoxycholic acid(DCA),Glycocholic acid(GCA),Hyodeoxycholic acid(HDCA),Murine deoxycholic acid(MDCA),Taurocholic acid(TCA),Taurochenodeoxycholic acid(TCDCA),Taurochenodeoxycholic acid(TCDCA),Taurocholic acid(THCA),Taurine hyodeoxycholic acid(THDCA),Taurolithocholic acid(TLCA),Tauroursodeoxycholic acid(THDCA),Tauroursodeoxycholic acid(TLCA),and Tauroursodeoxycholic acid(TLCA).Tauroursodeoxycholic Acid(TUDCA),Ursodeoxycholic acid(UDCA),ω-Murine cholic acid/α-Murine cholic acid(ω-MCA/α-MCA).3.Screening of target bile acids: A total of 7 differentially expressed BAs were screened(ω-MCA/α-MCA,β-MCA,CA,ACA,MDCA,UDCA,TCA),including 4 types of BAs(ω-MCA/α-MCA,β-MCA,CA,ACA)was statistically significant.The comparison between the model group and the control group shows that compared to the control groupω-MCA/α-MCA significantly increased in the SAP 6-hour group;Compared to the control group β-MCA was significantly increased in the SAP 2h group,6h group,and 12 h group;Compared with the control group,the expression of CA was significantly increased in the SAP 2h and 6h groups;Compared to the control group,the expression of ACA was significantly increased in the SAP 6-hour group..4.Potential metabolic pathway: Preliminary exploration revealed that cholesterol metabolism disorder-bile acid metabolism-mitochondrial damage-pancreatic apoptotic necrosis may be the metabolic pathway for the development of SAP.Conclusion: Based on UPLC-MS technology,the differential cholic acid and its metabolic characteristics in different stages of SAP can be identified,and the progression path of SAP can be effectively analyzed by combining the changes in cholic acid and cholic acid metabolism spectrum.At different stages of SAP onset,the differential combination of bile acids is also different,which can be used as a marker to evaluate the severity or stage of SAP disease.The bile acid metabolism spectrum can effectively analyze the progression path of SAP,and differential bile acid combinations can serve as effective biomarkers to determine different stages of disease,preliminarily elucidating the pathogenesis of SAP,providing a basis and treatment ideas for clinical research.