喹乙醇诱导HepG2细胞线粒体的氧化损伤

Oxidative Damage of Mitochondria by Olaquindox in HepG2 Cells

背景与目的:探讨喹乙醇(olaquindox)对人肝癌细胞(HepG2)线粒体的氧化损伤作用。材料与方法:用0、200、400和800μg/ml喹乙醇处理HepG2细胞24h后,采用噻唑蓝(MTT)法确定喹乙醇对HepG2细胞的IC50;分子探针2',7'-二氯荧光黄双乙酸盐(2',7'-dichlorodihydrofluorescein diacetate,DCFDA)和双氢溴乙啶(dihydroethidium,DHE)检测细胞内活性氧(reactive oxygen species,ROS)含量;罗丹明123检测线粒体膜电位(△Ψm)变化;钙离子荧光探针Fluo-3AM检测胞浆游离钙离子浓度并用定量PCR方法检测线粒体DNA(mtDNA)和核DNA(nDNA)的损伤情况。结果:随着喹乙醇浓度的增加,HepG2细胞的存活率逐渐降低(P均<0.01),呈时间-剂量-反应关系;不同浓度的喹乙醇作用细胞24h后,随着喹乙醇浓度的增加细胞内ROS和胞浆游离钙离子浓度显著增加(P<0.05或P<0.01),△Ψm明显降低且呈剂量-反应关系;喹乙醇能引起HepG2细胞mtDNA和nDNA损伤(P均<0.01),且mtDNA损伤程度显著高于nDNA,并呈剂量-反应关系。结论:喹乙醇在体外实验中,可诱导HepG2细胞线粒体氧化性损伤。

BACKGROUND AND AIM：To investigate oxidative damage of mitochondria induced by olaquindox in human hepatoma G2（HepG2） cells.MATERIALS AND METHODS：HepG2 cells were treated with 0,200,400 and 800 μg/ml olaquindox for 24 h.The value of IC50 of olaquindox in HepG2 cells was determined by MTT assay.The levels of reactive oxygen species（ROS） were detected by 2,7-dichlorodihydrofluorescein diacetate（DCFDA） and Dihydroethidium（DHE）.The level of mitochondrial membrane potential（△Ψm） and calcium concentration were measured by Rhodamine123（Rh-123） and Fluo-3AM, respectively. Finally, the damage ratio of mtDNA and nDNA was assessed by quantitative PCR. RESULTS： MTT assay revealed that the HepG2 cells viabilities were significantly inhibited by olaquindox in dose- and time- dependent manners. Olaquindox induced increased levels of ROS and calcium in HepG2 cells, and reduced the level of △Ψm Quantitative PCR assay showed that olaquindox led to a dose-dependent decrease in the amplification of both mtDNA and nDNA. These data also suggested that the olaquindox-induced damage to mtDNA was more extensive than its damage to nDNA. CONCLUSION： Olaquindox could cause oxidative damage of mitochondria in HepG2 cells.