[关键词]
[摘要]
目的 利用正常人源肝细胞(HepaRG)和高内涵技术检测肝毒性标志物,并结合微核试验和单细胞凝胶电泳试验建立体外细胞毒性和遗传毒性的快速筛选平台。方法 选取适当的荧光探针Hoechst33342、DCFH-DA、Fluo4-AM、MitoTracker® Red CMX Ros联合高内涵技术研究不同大黄蒽醌类单体(AQs)对HepaRG细胞活性氧簇(ROS)、胞内Ca2+含量及线粒体膜完整性等肝毒性标志物的影响,并开展高内涵法胞质分裂阻断法微核试验和高通量彗星电泳试验,综合评价AQs致肝细胞毒性及染色体、DNA损伤情况。结果 与对照组比较,HepaRG细胞经25.0 μg/mL大黄素、12.5和25.0 μg/mL芦荟大黄素、50和25.0 μg/mL大黄酚处理24 h后,胞内ROS含量显著增多;12.5和25.0 μg/mL芦荟大黄素和50.0 μg/mL大黄酸可引起胞内Ca2+含量显著增多;大黄素25.0 μg/mL、芦荟大黄素25.0 μg/mL、大黄酚50.0和25.0 μg/mL、大黄酸50.0和25.0 μg/mL组导致线粒体明显损伤(P<0.05、0.01)。与对照组比较,25.0 μg/mL大黄素诱导微核率、尾DNA含量和彗星尾距(OTM)数值均显著升高(P<0.05、0.01);50.0 μg/mL大黄酚给药72 h后微核率显著升高(P<0.01)。结论 AQs的研究结果与现有文献报道基本相符。本研究成功建立肝细胞毒性和遗传毒性的联合快速筛选模型,有助于药物研发早期的毒性筛选。
[Key word]
[Abstract]
Objective To detect the hepatotoxicity biomarkers using normal human hepatocyte (HepaRG) and high-content screening, and to combine the micronucleus test and single cell gel electrophoresis to estalish a rapid screening platform for in vitro cytotoxitity and genotoxicity. Methods The effects of rhubarb anthraquinones (AQs) on the reactive oxygen species (ROS), intracellular Ca2+ concentration and mitochondrial membrane potential (MMP) in HepaRG cells were studied using appropriate fluorescent probes Hoechst33342、DCFH-DA、Fluo4-AM、Mito Tracker Red CMX Ros and high-content screening methods, and the potential genotoxiciy triggered by AQs were analyzed using the high-content based cytokinesis block micronucleus test and high throughput comet assay. Results The intracellular ROS level of HepaRG cells was significantly elevated by a 24 h treatment with Emodin (25.0 μg/mL), aloe-emodin (25.0 μg/mL) or chrysophanol (50.0 μg/mL), which are dose-concentration dependent (P < 0.05 and 0.01); the intracellular Ca2+ increased and mitochondrial damage were observed with the treatment of aloe-emodin (25.0 μg/mL) and rhein (50.0 μg/mL, P < 0.05 and 0.01). Comparing to control group, Emodin (25.0 μg/mL) induced an increased micronucleus rate (1.59% ±0.68%,P < 0.01) and significantly higher percentage tail DNA and Olive tail moment (respectively 10.155% ±2.17% and 0.510 ±0.06, P < 0.05 and 0.01) after 24 h; while the chrysophanol increased the micronucleus rate to 1.29% ±0.54% (P < 0.01) after 72 h. Conclusion The results on the cytotoxicities and genotoxicities of AQs are consistent with the literatures. In this study, a rapid screening model for both hepatotoxicity and genotoxicity was successfully established, which will help with the early screening during the drug development stage.
[中图分类号]
[基金项目]
国家"重大新药创制"科技重大专项(2015ZX09501004-002);中国食品药品检定研究院中青年发展研究基金(2014C1)