关键词: 绿原酸; 游离脂肪酸(FFA); 犊牛原代肝细胞; 胆固醇; 胆汁酸

Abstract: 【Objective】 The objective of this study was to investigate the effects of chlorogenic acid (CGA) on cholesterol and bile acid metabolism in calf hepatocytes treated with high free fatty acid (FFA).【Method】 The primary calve hepatocytes were separated by two-step collagenase perfusion method,and the cells were identified by immunofluorescence and divided into 4 groups.Cells in control group were cultured with RPMI-1640 medium containing 2% BSA.The cells in FFA group were cultured after adding 1.2 mmol/L FFA in RPMI-1640 medium containing 2% BSA,and CGA group were cultured after adding 20 μg/mL CGA in RPMI-1640 medium containing 2% BSA.Cells in CGA＋FFA group were cultured after adding 1.2 mmol/L FFA and 20 μg/mL CGA in RPMI-1640 medium containing 2% BSA.Cells were collected after 12 h,and the contents of triglyceride (TAG) and total cholesterol (TC) were detected by the kit.Real-time quantitative PCR and Western blotting were used to detect the mRNA and proteins expression levels of cholesterol synthesis-related factors,including sterol regulatory element binding transcription factor 2 (SREBF2) and 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR),and cholesterol effection-related factors,including acetyl-CoA acetyltransferase 2(ACAT2),ATP-binding box subfamily A member 1(ABCA1),ATP-binding box subfamily G member 5(ABCG5),and bile acid metabolism related factors,including cholesterol 7α-hydroxylase (CYP7A1),cholesterol 12α-hydroxylase (CYP8B1),cholesterol of 27 α-hydroxylase (CYP27A1),farnesol X receptor (FXR) and fibroblast growth factor receptor 4(FGFR4).【Result】 After different treatments of calf hepatocytes,compared with control group,the TC content of calf hepatocytes in FFA group was extremely significantly reduced (P＜0.01),and the TAG content was extremely significantly increased (P＜0.01),and the mRNA expression levels of HMGCR,ABCA1,ABCG5,ABCG8,APOA1,ACAT1,NPC1L1,FXR and FGFR4 genes and the protein expression levels of SREBF2,ACAT2,ABCA1 and ABCG5 were extremely significant or significantly reduced (P＜0.01 or P＜0.05),the mRNA expression of CYP8B1 gene and the protein expression of CYP7A1 and CYP8B1 were extremely significant or significantly increased (P＜0.01 or P＜0.05).The TC content of calf hepatocytes in CGA group was extremely significantly increased (P＜0.01),the protein expression levels of SREBF2,ABCA1,CYP27A1,FXR and FGFR4 were extremely significantly increased (P＜0.01),the protein expression of CYP7A1 was extremely significantly reduced (P＜0.01).Compared with FFA group,the TC content of calf hepatocytes in CGA＋FFA group was significantly increased (P＜0.05),and the TAG content was significantly reduced (P＜0.05),the mRNA expression levels of HMGCR,ACAT2,ABCA1,ABCG5 and APOA1 genes and the protein expression levels of SREBF2,ACAT2,ABCA1,ABCG5,FXR and FGFR4 were extremely significant or significantly increased (P＜0.01 or P＜0.05),the protein expression levels of CYP7A1 and CYP8B1 were extremely significantly reduced (P＜0.01).【Conclusion】 CGA could participate in the regulation of intracellular cholesterol homeostasis in calf hepatocytes while activating FXR and FGFR4,which in turn alleviates high FFA-treated bile acid accumulation.

Key words: chlorogenic acid; free fatty acid (FFA); primary calve hepatocytes; cholesterol; bile acids