体外评价马兜铃酸对HepG2细胞线粒体功能的影响  

In vitro assessment of mitochondrial dysfunction of Aristolochic acid on HepG2 cells

作　　者：富欣[1,2] 潘晓靓[1,2] 李华[1,2] 汤纳平[1,2] 王雁[1,2] 惠涛涛 马璟[1,2] 张泽安[1,2] 

机构地区：[1]上海中医药大学药物安全评价研究中心,上海201203 [2]中国医药工业研究总院国家上海新药安全评价研究中心,上海201203

出　　处：《世界临床药物》2015年第4期236-242,共7页World Clinical Drug

基　　金：国家"十二五"重大专项基金(2012ZX09302002;2012ZX09505-001-003)

摘　　要：目的评价马兜铃酸的线粒体毒性,从而探讨马兜铃酸的可能毒性机制。方法 HepG2细胞培养基中分别加入齐多夫定8 000~20 000μmol/L,或加入马兜铃酸25~500μmol/L,均培养24 h,以CCK8细胞计数法测定细胞存活率。同时比较不同浓度齐多夫定（8 000、16 000和20 000μmol/L）和马兜铃酸（25、200和500μmol/L）培养24 h的胞内ATP合成水平和胞内钙离子浓度、活性氧及线粒体膜通透性转换孔（MPTP）变化,透射电镜观察线粒体超微结构。结果齐多夫定8 000~20 000μmol/L可抑制细胞存活,IC50为12 713μmol/L;马兜铃酸25~500μmol/L可抑制细胞存活,IC50为214.6μmol/L。与溶媒对照组（DMEM）相比,齐多夫定≥8 000μmol/L细胞内活性氧水平显著升高（P〈0.01）;≥16 000μmol/L线粒体ATP显著下降（P〈0.01）、钙离子浓度明显升高（P〈0.01）,并可见线粒体结构发生病理改变;20 000μmol/L组MPTP开放水平显著升高（P〈0.01）。与溶媒对照组（DMSO）相比,马兜铃酸≥25μmol/L,ATP合成水平明显下降（P〈0.01）、MPTP开放水平显著升高（P〈0.01）;≥200μmol/L细胞内钙离子浓度明显升高（P〈0.01）;500μmol/L细胞内活性氧水平显著升高（P〈0.01）、并可见线粒体结构发生病变。结论马兜铃酸可以通过干扰破坏线粒体代谢功能和结构而诱导线粒体损伤。Objective To evaluate the potential mitochondrial toxicity of Aristolochic acid and to explore its toxic mechanism. Methods HepG2 cells were cultured in zidovudine 8 000~20 000 μmol/L or Aristolochic acid 25~500 μmol/L respectively for 24 h. The cell viability was assessed by CCK8 assay and the levels of cellular ATP were detected by luciferase assay. The levels of reactive oxygen species（ROS）, intracellular free Ca2＋ and mitochondrial permeability transition pore opening was determined by flow cytometer using different fluorescent dyes（DCFH, Fluo-3, and calcein AM/CoCl2, respectively）. Mitochondria ultra-structure was observed by transmission electron microscope. Results Zidovudine at 8 000~20 000 μmol/L markedly inhibited cell viability（IC50=12 713 μmol/L）, Aristolochic acid at 25~500 μmol/L markedly inhibited cell viability（IC50=214.6 μmol/L）. After zidovudine≥8 000 μmol/L treatment, compared with normal HepG2 cells, the level of ROS was significantly increased（P〈0.01）. While ≥16 000 μmol/L treatment, ATP levels were significantly decreased（P〈0.01）, intracellular free Ca2＋ was significantly increased（P〈0.01） and mitochondrial ultrastructural changes were observed. At 20 000 μmol/L, the MPTP opening was significantly increased（P〈0.01）. After the treatment of aristolochic acid at ≥25 μmol/L, cellular ATP levels were decreased significantly（P〈0.01） and MPTP opening was increased significantly（P〈0.01）. At the treatment with ≥200 μmol/L, the intracellular Ca2＋ level was significantly increased（P〈0.01）. And at 500 μmol/L, ROS level was significantly increased（P〈0.01） and mitochondrial ultra-structural changes were observed. Conclusion Aristolochic acid can induce mitochondrial toxicity by interfering with mitochondrial metabolism and damaging mitochondrial ultra-structure.

关 键 词：马兜铃酸 HEPG2细胞 线粒体毒性 齐多夫定 

分 类 号：R969[医药卫生—药理学]