绿原酸对高游离脂肪酸处理犊牛肝细胞胆固醇和胆汁酸代谢的影响

Effects of Chlorogenic Acid on Cholesterol and Bile Acid Metabolism in Calf Hepatocytes Treated with High FFA

【目的】探究绿原酸(CGA)对高游离脂肪酸(FFA)处理的犊牛肝细胞胆固醇和胆汁酸代谢的影响。【方法】采用两步胶原酶灌注法分离犊牛原代肝细胞,利用免疫荧光鉴定细胞后将其分为4组,对照组细胞用含2%BSA的RPMI-1640培养基培养;FFA组细胞在含2%BSA的RPMI-1640培养基中添加1.2 mmol/L FFA后培养;CGA组细胞在含2%BSA的RPMI-1640培养基中添加20μg/mL CGA后培养;CGA+FFA组细胞在含2%BSA的RPMI-1640培养基中添加1.2 mmol/L FFA和20μg/mL CGA后培养。培养12 h后收集细胞,通过试剂盒检测细胞中甘油三酯(TAG)和总胆固醇(TC)含量;利用实时荧光定量PCR、Western blotting分别检测肝细胞中胆固醇合成相关因子甾醇调节元件结合转录因子2(SREBF2)、3-羟基-3-甲基戊二酰辅酶A还原酶(HMGCR),胆固醇外排相关因子乙酰辅酶A乙酰转移酶2(ACAT2)、ATP结合盒亚家族A成员1(ABCA1)、ATP结合盒亚家族G成员5(ABCG5)以及胆汁酸代谢相关因子胆固醇7α-羟化酶(CYP7A1)、胆固醇12α-羟化酶(CYP8B1)、胆固醇27α-羟化酶(CYP27A1)、法尼醇X受体(FXR)、成纤维细胞生长因子受体4(FGFR4)等因子的mRNA和蛋白表达水平。【结果】犊牛肝细胞经过不同处理后,与对照组相比,FFA组犊牛肝细胞中TC含量极显著降低(P<0.01),TAG含量极显著升高(P<0.01),HMGCR、ABCA1、ABCG5、ABCG8、APOA1、ACAT1、NPC1L1、FXR、FGFR4基因mRNA表达量和SREBF2、ACAT2、ABCA1、ABCG5蛋白表达量均极显著或显著降低(P<0.01;P<0.05),CYP8B1基因mRNA表达量和CYP7A1、CYP8B1蛋白表达量均极显著或显著升高(P<0.01;P<0.05);CGA组犊牛肝细胞中TC含量极显著升高(P<0.01),SREBF2、ABCA1、CYP27A1、FXR、FGFR4蛋白表达量均极显著升高(P<0.01),CYP7A1蛋白表达量极显著降低(P<0.01)。与FFA组相比,CGA+FFA组犊牛肝细胞中TC含量显著升高(P<0.05),TAG含量显著降低(P<0.05),HMGCR、ACAT2、ABCA1、ABCG5、APOA1基因mRNA表达量和SREBF2、ACAT2、ABCA1、ABCG5、FXR、FGFR4蛋白表达量均极显著或显著升高(P<0.01;P<0.05),CYP7A1、CYP8B1蛋白表达量均极显著降低(P<0.01)。【结论】CGA能参与调节犊牛肝细胞内胆固醇稳态,同时激活FXR和FGFR4,进而缓解高FFA处理的犊牛肝细胞胆汁酸蓄积。

【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 20μg/mL CGA in RPMI-1640 medium containing 2%BSA.Cells in CGA+FFA group were cultured after adding 1.2 mmol/L FFA and 20μg/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 27α-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,ABCA 1,ABCG 5,ABCG 8,APOA 1,ACAT 1,NPC 1 L 1,FXR and FGFR 4 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 CYP 8 B 1 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,ACAT 2,ABCA 1,ABCG 5 and APOA 1 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.