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

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.