Effects of Chlorogenic Acid on Cholesterol and Bile Acid Metabolism in Calf Hepatocytes Treated with High FFA
[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 7a-hydroxylase(CYP7A1),cholesterol 12a-hydroxylase(CYP8B1),cholesterol of 27 a-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.
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