去甲斑蝥素作用早期对人肝癌细胞活性氧及NF-E2相关因子2/抗氧化反应元件信号通路的激活  

Early events of norcantharidin reactive oxygen species and NF-E2-related factor 2/antioxidant response element pathway of HepG2 cells

作　　者：陈静[1] 崔宝弟 孙震晓[1] 

机构地区：[1]北京中医药大学中药学院,北京100102

出　　处：《中国药理学与毒理学杂志》2017年第1期94-100,共7页Chinese Journal of Pharmacology and Toxicology

基　　金：国家自然科学基金项目(81473418);国家林业局野生动植物保护项目(2012-2016)~~

摘　　要：目的探究去甲斑螯素(NCTD)诱导人肝癌Hep G2细胞凋亡及G2/M期阻滞的早期事件,分析NCTD作用早期Hep G2细胞内活性氧自由基(ROS)的变化规律及NCTD对NF-E2相关因子/抗氧化反应元件(Nrf2/ARE)信号通路的影响。方法 NCTD30,60和120μmol·L^(-1)分别作用于体外培养的人肝癌Hep G2细胞3,6,12,24,48和72 h,MTT法检测NCTD对细胞存活的影响;流式细胞术检测NCTD60μmol·L^(-1)作用细胞12,24和48 h对细胞周期和细胞凋亡的影响;DCFH-DA探针结合流式细胞术检测NCTD 30,60和120μmol·L^(-1)作用于3,6和12 h对Hep G2细胞内ROS的影响;荧光素酶法测定NCTD同时转染ARE和荧光素酶报告基因的Hep G2C8细胞的荧光强度;实时荧光定量PCR检测对血红素氧合酶-1(HO-1)和醌氧化还原酶-1(NQO1)m RNA表达的影响。结果 NCTD 30,60和120μmol·L^(-1)作用3和6 h对Hep G2细胞存活无明显影响,而作用24,48和72 h对Hep G2细胞有明显生长抑制作用(P<0.01);NCTD 60μmol·L^(-1)作用12 h后可诱导Hep G2细胞发生凋亡及G2/M期阻滞,12,24和48 h凋亡细胞比例分别由12 h细胞对照组(4.00±1.98)%增加到(12.10±1.70)%,24 h对照组(4.05±0.21)%增加到(31.80±6.50)%,48 h对照组(3.90±0.85)%增加到(33.30±1.41)%;12,24和48 h G2/M期细胞比例分别由12 h对照组的(16.51±1.58)%增加到(40.89±0.18)%,24 h对照组的(16.99±1.32)%增加到(55.29±3.99)%,48 h对照组的(14.45±0.59)%增加到(50.66±5.88)%,相应各时相NCTD处理组G1期细胞比例明显下降(P<0.01);NCTD 30,60和120μmol·L^(-1)作用Hep G2细胞3,6和12 h,ROS无明显变化,作用Hep G2C8细胞6和12 h可明显激活Nrf2/ARE信号通路,下游基因HO-1和NQO1 m RNA表达显著上调(P<0.05)。结论NCTD作用Hep G2细胞早期可明显激活Nrf2/ARE信号通路;ROS激活可能不是NCTD诱导人肝癌Hep G2细胞凋亡及G2/M期阻滞的主要原因。OBJECTIVE To investigate the early events of norcantharidin （NCTD） induced cell apoptosis and cell cycle arrest, the variation of reactive oxygen species （ROS） and the NF-E2-relate-dactor 2/antioxidant response element（Nrf2/ARE） pathway in human HepG2 cells. METHODS The cytotoxicity was measured by MTT assay. Apoptosis and cell cycle was analyzed by flow cytometry. The intra toxicity ROS production was evaluated by flow cytometry analysis with DCFH-DA probe and the effect of NCTD on Nrf2/ARE pathway was detected by luciferase assay in HepG2C8 cells under the same condition. The mRNA expression of heme oxygenase-1（HO-1） and NAD（P）H： quinone oxidoreductase 1（NQO1） antioxidase gene in Nrf2/ARE pathway downstream was evaluated by quantitative real-time PCR. RESULTS No significant cytotoxicity was detected after HepG2 cells were treated with NCTD 30, 60 and 120 μmol· L-1 for 3 and 6 h, but cellular viability was inhibited significantly by NCTD 30, 60 and 120 μmol· L-1 for 24, 48 and 72 h（P〈0.01）. Cell apoptosis and G2/M phase arrest occurred after HepG2 cells were treated with NCTD 60 μmol· L-1 for 12, 24 and 48 h. The percentage of apoptosis increased from （4.00±1.98）% to （12.10±1.70）% for 12 h, from （4.05±0.21）% to （31.8±6.50）% for 24 h, and from （3.90±0.85）% to （33.30±1.41）% for 48 h, respectively. The percentage of G2/M phase increased from （16.51±1.58）% to （40.89±0.18）% for 12 h, from （16.99±1.32）% to （55.29±3.99）% for 24 h, and from （14.45 ± 0.59）% to （50.66 ± 5.88）% for 48 h, respectively. Compared with cell control group, the percentage of G1 phase had a significant decrease in the group with NCTD treated at different time points（P〈0.01）. No significant change in ROS in HepG2 cells was detected after the treatment with NCTD 30, 60 and 120 μmol· L-1 for 3, 6 and 12 h. Nrf2/ARE pathway in HepG2C8 cells was activated by NCTD 30, 60 and 120 μmol·L-1 for 6 and 12 h. mRNA expression of HO-1 and NQO1 had a sign

关 键 词：去甲斑蝥素 细胞凋亡 细胞周期 Nrf2/ARE信号通路 活性氧自由基 血红素加氧酶-1 NAD(P)H:醌氧化还原酶-1 

分 类 号：R285[医药卫生—中药学]