百草枯诱导AML12细胞凋亡过程中线粒体活性氧的作用

doi:10.3969/j.issn.1004-616x.2016.01.001

摘要/Abstract

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、300 μmol/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,25~300 μmol/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);300 μmol/L的百草枯作用24 h可明显诱导AML12细胞发生凋亡(P<0.05);25~300 μmol/L 百草枯处理24 h后细胞内活性氧依次增加(P<0.05),25~100 μmol/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300 μmol/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,300 μmol/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。

关键词: 百草枯, 活性氧, 线粒体活性氧, 线粒体膜电位, 凋亡

Abstract: 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 viability (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.

Key words: paraquat, reactive oxygen species, mitochondrial reactive oxygen species, mitochondrial membrane potential, apoptosis

中图分类号:

R994.6

孔德钦, 刘江正, 于卫华, 张涛, 龙子, 王欣, 张晓迪, 柏桦, 海春旭. 百草枯诱导AML12细胞凋亡过程中线粒体活性氧的作用[J]. 癌变·畸变·突变, 2016, 28(1): 1-7.

KONG Deqin, LIU Jiangzheng, YU Weihua, ZHANG Tao, LONG Zi, WANG Xin, ZHANG Xiaodi, BAI Hua, HAI Chunxu. Involvement of mitochondrial reactive oxygen species on paraquat-induced apoptosis in AML12 cells[J]. Carcinogenesis, Teratogenesis & Mutagenesis, 2016, 28(1): 1-7.

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