Characteristics Of Lipid Metabolism In Hyperlipidemic Acute Pancreatitis And Effect Of Ceramide On Inflammatory Response Of Pancreatic Acinar Cell

Objective:Based on the untargeted lipidomics,the metabolic characteristics and abnormal metabolism associated with HLAP were comprehensively elucidated.The lipid molecule with the most significant differences was screened,and its effects on the inflammatory response of pancreatic acinar cells were further examined to verify the pathophysiological effects and mechanisms.Methods:1.From January to October 2020,HLAP patients who were admitted to the Department of Gastroenterology and Department of General Surgery of affiliated hospital of north sichuan medical college were enrolled,and also healthy controls who underwent routine physical examination in the Health Examination Center of affiliated hospital of north sichuan medical college were enrolled as control group.Blood samples and clinical data of the two groups were collected at admission.To analyze lipid metabolism characteristics of HLAP based on untargeted metabolomics,the blood samples of the subjects were detected by ultra-performance Liquid chromatography-mass spectrometry(LC-MS),and the results were identified as various lipids through the signal characteristics of mass spectrometry.A model of Orthogonal partial least squares discriminant analysis(OPLS-DA)was constructed to detect the different metabolites.Univariate analysis was used to determine the difference in metabolites between groups.The affected metabolic pathways were searched in Kyoto Encyclopedia of Genes and Genomes(KEGG)database and Biopan database.2.Murine pancreatic acinar cell line AR42J was used as the cell line,and the cells were divided into Control group,Model group and Cer group(Model+Cer).Cells in the model group were treated with STC solution with a final concentration of 0.5 mmol/L for 2 hours,while cells in the control group were replaced with PBS solution without STC at the same concentration.Cer(d18:1/18:0)solution with a final concentration of 5 μmol/L was added to the model group for 24 hours.The cytotoxicity of Cer was determined by MTT assay to evaluate the cell viability.The contents of Amylase,Interleukin-1β(IL-1β)and Tumor necrosis factor-a(TNF-α)in the supernatant of the Model group and the Cer group were detected according to the ELISA kit,and the mRNA levels of IL-1β and TNF-α in the cells were measured to investigate the pro-inflammatory activity of Cer in vitro.Results:1.Metabolic characteristics of patients with hyperlipidemic AP based on untargeted metabolomics were studied:A total of 48 subjects were enrolled,including 24 HLAP and 24 healthy controls.A total of 524 lipid molecules were identified by the lipidomics platform.The OPLS-DA method was used to construct the model according to the groups,and significant differences were compared between the two groups.A total of 393 differential metabolites were found,including triacylglycerols(TG),phosphatidylcholine(PC),free fatty acid acidsx(FFA)and so on.The enriched metabolic pathways mainly included sphingolipid metabolism,fatty acid metabolism,and glycerophospholipid metabolism.Among them,the Cer molecules were the most significant pathway.2.The effect of Cer on STC-induced inflammation of pancreatic acinar cells.(1)According to the literature,we selected a final concentration of 0.5 mmol/L STC to treat murine pancreatic acinar cells AR42J.The results showed that amylase in the supernatant of the model group was significantly higher than that of the control group.To further detect the extent of inflammatory injury in pancreatic acinar cells,we detected the expression levels of inflammatory cytokine and by ELISA and Real-time PCR,respectively.The results showed that the levels of TNF-α and IL-1β in the supernatant of the model group were significantly increased.In addition,the mRNA levels of TNF-α and IL-1β were also significantly increased.These results indicated that the cell model of AP was constructed for subsequent experiments.(2)Compared with the Control group,the proportion of apoptotic cells in the Model group was increased,mainly consisted of late-phase apoptotic cells.The results of MTT assay showed that the cell had the strongest toxic effect at the concentration of 10 μmol/L.A final concentration of 5 μmol/L Cer(d 18:1/18:0)was selected as the optimal non-toxic concentration.The results showed that STC-induced AR42J cells were significantly damaged and the viability of STC-induced AR42J cells was significantly reduced by Cer(d18:1/18:0)in Cer group.(3)ELISA and Real-time PCR were used to detect the expression levels of inflammatory cytokine between the Model group and the Cer group,respectively.The levels of TNF-α and IL-1β in the supernatant of AR42J cells were significantly increased in the Cer group.Besides,the mRNA expression levels of TNF-α and IL-β in AR42J were significantly up-regulated in the Cer group.Conclusions:1.There are significant differences in lipid metabolism between HLAP patients and healthy controls.The metabolism disorders,mainly abnormalities in fatty acid metabolism,sphingolipid metabolism,and glycerophospholipid metabolism.The lipid molecule with the most significant difference was ceramide.2.Ceramide aggravated STC-induced inflammation in pancreatic acinar cells.