细胞能量代谢分析法评价对乙酰氨基酚对人肝细胞线粒体有氧呼吸的毒性作用  

Evaluation of Toxicity of Acetaminophen on Human Hepatocyte Mitochondrial Aerobic Respiration by Cellular Energy Metabolism Analysis

作　　者：李梅 刘宁[1] 张凡[1] 孙华[1] LI Mei;LIU Ning;ZHANG Fan;SUN Hua(Institute of Materia Medica,Chinese Academy of Medical Sciences and Peking Union Medical College,Beijing 100050,China)

机构地区：[1]中国医学科学院北京协和医学院药物研究所,北京100050

出　　处：《中国药物警戒》2019年第4期193-198,207,共7页Chinese Journal of Pharmacovigilance

基　　金：国家自然科学基金面上项目(81573487):二氢神经酰胺类鞘脂及其代谢通路在慢加急性肝衰竭发生发展中的作用及防治措施研究;中国医学科学院医学与健康科技创新工程重大协同创新项目(2017-12M-1-013):中药安全风险预警及防控;国际合作项目(Grant ID4084):Applications and core technology university research(ACT-UR)proposal form

摘　　要：目的利用Seahorse XF^e24海马细胞能量代谢分析系统分析对乙酰氨基酚(APAP)对人肝细胞线粒体有氧呼吸的整体影响,为APAP在能量代谢角度的毒性防控及毒性机制研究提供方法学的参考。方法体外培养人肝细胞系(HepG2),分别以1、2、4、8、16 mM APAP处理细胞24 h,MTT法检测细胞增殖情况,倒置显微镜观察细胞形态;BCA法定蛋白含量。采用Seahorse XF^e24海马细胞能量代谢分析系统建立HepG2细胞实验方法,并检测不同浓度APAP对人肝细胞线粒体有氧呼吸的影响和特点。结果 Seahorse XF^e24海马细胞能量代谢分析系统检测HepG2细胞能量代谢的最佳条件为:24孔板细胞密度2×10~4/孔,FCCP浓度为2μM。MTT结果中对人肝细胞增殖有显著抑制作用的APAP(4、8、16 mM)能干扰人肝细胞的线粒体有氧呼吸,其中4、8 mM APAP有降低肝细胞基础呼吸和ATP生成的趋势,16 mM APAP能显著抑制肝细胞的基础呼吸、最大呼吸和ATP合成。结论高剂量APAP(16 mM)在人肝细胞活细胞体系中能够显著抑制细胞能量代谢,对活细胞线粒体有氧呼吸显示显著毒性作用。Objective This study aims to observe the effects of APAP on human hepatocyte mitochondrial aerobic respiration utilizing Seahorse XFe24 cell energy metabolism analysis system,thus to provide a methodological reference for the study of APAP toxicity prevention and mechanism in an energy metabolism way.Methods Human hepatocytes(HepG2)were treated with 1,2,4,8,and 16 mM APAP for 24 h in vitro.Cell proliferation was detected by MTT assay and cell morphology was observed under an inverted microscope;cell protein was quantified by BCA assay.Experiment method in HepG2 cells was established by Seahorse XFe24 cell energy metabolism analysis system,effects and characteristics of different concentrations of APAP on human hepatocyte mitochondrial aerobic respiration were detected.Results The best conditions of Seahorse XFe24 cell energy metabolism analysis system for HepG2 were:2×104 cells per well in 24 wells plate and the concentration of FCCP was 2μM.APAP(4,8,16 mM)which could suppress cell proliferation in MTT results could interrupt mitochondrial aerobic respiration in human hepatocytes.APAP in 4 mM and 8 mM had a tendency to reduce basal respiration and ATP production in human hepatocytes,and 16 mM APAP can significantly inhibit basal respiration,maximal respiration and ATP synthesis.Conclusion High-dose APAP(16 mM)significantly inhibited cell energy metabolism in human hepatocytes live system and showed significant toxicity on live cell mitochondrial aerobic respiration.

关 键 词：对乙酰氨基酚 SEAHORSE XFe24 线粒体有氧呼吸 肝毒性 

分 类 号：R992[医药卫生—毒理学]