百草枯诱导AML12细胞凋亡过程中线粒体活性氧的作用  

Involvement of mitochondrial reactive oxygen species on paraquat-induced apoptosis in AML12 cells

作　　者：孔德钦[1] 刘江正[1] 于卫华[1] 张涛[1] 龙子 王欣[1] 张晓迪[1] 柏桦[1] 海春旭[1] 

机构地区：[1]第四军医大学军事预防医学系毒理学教研室,陕西西安710032

出　　处：《癌变．畸变．突变》2016年第1期1-7,共7页Carcinogenesis,Teratogenesis & Mutagenesis

摘　　要：目的：探讨百草枯诱导小鼠肝细胞系AML12细胞凋亡过程中线粒体活性氧的作用。方法：AML12细胞分别给予0、25、50、100、200、300μmol/L的百草枯处理。采用四甲基噻唑蓝（MTT）法检测细胞活力;AnnexinV/PI双染法结合流式细胞术检测细胞凋亡率;DCFH-DA荧光探针经流式细胞仪测定细胞内活性氧水平;MitoSOX荧光探针检测线粒体活性氧变化;激光共聚焦测定JC-1探针荧光强度以检测线粒体膜电位变化。结果：与对照组比较,25~300μmol/L的百草枯处理后可以诱导AML12细胞活力下降,且具有剂量效应关系（r=-0.94,P〈0.01）;300μmol/L的百草枯作用24 h可明显诱导AML12细胞发生凋亡（P〈0.05）;25~300μmol/L百草枯处理24 h后细胞内活性氧依次增加（P〈0.05）,25~100μmol/L百草枯作用24 h时线粒体活性氧增加且呈剂量效应关系（r=0.98,P〈0.05）,而200和300μmol/L的百草枯作用24 h时线粒体活性氧水平降低（P〈0.05）。细胞活力下降与细胞内活性氧升高呈负相关（r=-0.90,P〈0.05）;晚期凋亡细胞的比例与细胞内活性氧呈正相关（r=0.96,P〈0.01）;JC-1检测线粒体膜电位结果显示,与对照组相比,300μmol/L的百草枯处理24 h时线粒体膜电位显著降低（P〈0.05）。结论：百草枯诱导AML12细胞凋亡过程主要由细胞整体水平的活性氧介导,提示线粒体活性氧并未直接参与百草枯诱导的肝损伤过程。OBJECTIVE：To investigate the role of mitochondrial reactive oxygen species（mtROS） on the apoptosis of a mouse liver cell line-AML12 cells after their exposure to paraquat.METHODS：AML12 cells were treated with paraquat at concentrations of 0,25,50,100,200 and 300 μmol/L for 24 h,and cell viability was measured by MTT assay.Apoptosis was assessed by flow cytometry using Annexin V-FITC apoptosis detection kit.DCFH-DA fluorescent probe and MitoSOX were used to determine the level of ROS in whole cell and in mitochondria by flow cytometry,respectively.Mitochondrial membrane potential was evaluated by confocal microscope using JC-1 probe.RESULTS：Paraquat from 25 to 300 μmol/L induced dose-dependent decrease of AML12 cells viability.Apoptosis was significantly induced by paraquat at 300 μmol/L for 24 h.Treatment with 25-100 μmol/L paraquat raised the level of mitochondrial reactive oxygen species.However,200 and 300 μmol/L paraquat decreased the level of mtROS.The increase of ROS level was closely related to the decrease of AML12 cells viabihty（r=-0.90,P〈0.05）,and the ROS level was positively correlated with the proportion of the late apoptotic cells（r=0.96,P〈0.01）.Additionally,300 μmol/L paraquat markedly reduced mitochondrial membrane potential.CONCLUSION：Paraquat could induce apoptosis of AML12 cells via the induction of whole cell ROS which suggest that mtROS may not be directly involved in paraquat-induced liver injury.

关 键 词：百草枯 活性氧 线粒体活性氧 线粒体膜电位 凋亡 

分 类 号：R730.231[医药卫生—肿瘤]