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

作　　者：田艳 刘佳晨 王爽[1] 范文文 常雅奇 杨天娇 张泽鑫 张冰冰[2] 徐闯 杨威[1] TIAN Yan;LIU Jiachen;WANG Shuang;FAN Wenwen;CHANG Yaqi;YANG Tianjiao;ZHANG Zexin;ZHANG Bingbing;XU Chuang;YANG Wei(College of Animal Science and Veterinary Medicine,Heilongjiang Bayi Agricultural University,Daqing 163319,China;College of Life Science and Technology,Heilongjiang Bayi Agricultural University,Daqing 163319,China;College of Veterinary Medicine,China Agricultural University,Beijing 100193,China)

机构地区：[1]黑龙江八一农垦大学动物科技学院,大庆163319 [2]黑龙江八一农垦大学生命科学技术学院,大庆163319 [3]中国农业大学动物医学院,北京100193

出　　处：《中国畜牧兽医》2024年第7期2766-2775,共10页China Animal Husbandry & Veterinary Medicine

基　　金：黑龙江省自然基金优秀青年项目(YQ2022C029)。

摘　　要：【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【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

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 

分 类 号：S823[农业科学—畜牧学]