基于高内涵筛选技术研究生首乌和制首乌醇提物的肝毒性机制  

High-content screen assay for studying hepatotoxicity mechanisms of ethanol extract of Radix Polygoni Multiflori and Radix Polygoni Multiflori Praeparata

作　　者：李丹丹[1,2] 汤响林[2] 龙隆[3] 许龙龙[4] 谭洪玲[2] 梁乾德[2] 肖成荣[2] 王宇光[2] 马增春[2] 王莉莉[3] 高月[1,2] 

机构地区：[1]广西医科大学药理学系,广西南宁530000 [2]军事医学科学院放射与辐射医学研究所药理学研究室,北京100850 [3]军事医学科学院毒物药物研究所药物化学研究室,北京100850 [4]安徽医科大学药理学系,安徽合肥230032

出　　处：《中国药理学与毒理学杂志》2017年第6期626-635,共10页Chinese Journal of Pharmacology and Toxicology

基　　金：北京市自然科学基金(7164291);国家科技重大专项(2014ZX09304307-001-003);国家科技重大专项(2015ZX-09501004-003-003);中医药行业科研专项(201507004)~~

摘　　要：目的应用高内涵筛选技术研究生首乌醇提物(RPM)和制首乌醇提物(RPMP)的肝毒性及其可能机制。方法 RPM(终浓度10,25,50,100,200和300 mg·L^(-1))和RPMP(终浓度10,50,100,300,600和1200 mg·L^(-1))作用于Hep G2细胞3~24 h。采用Cell Titer-GloTM荧光细胞活性检测试剂盒检测Hep G2细胞存活率;应用高内涵筛选技术进行Hep G2细胞计数,并检测线粒体内活性氧(ROS)、线粒体膜电位(MMP)、细胞内谷胱甘肽(GSH)、超氧化物歧化酶2(SOD2)、转录激活因子4(ATF4)水平及细胞凋亡和细胞周期阻滞;Western蛋白质印迹法验证Hep G2细胞SOD2和ATF4蛋白表达水平。结果与细胞对照组相比,RPM 300 mg·L^(-1)作用24 h使Hep G2细胞存活率下降约48%(P<0.01),而相同浓度RPMP对细胞存活率无显著影响;RPM和RPMP均能降低MMP(P<0.05),并升高GSH,ROS,SOD2和ATF4水平(P<0.05)。与细胞对照组相比,RPM 200 mg·L^(-1)作用3 h SOD2水平显著升高(P<0.05),6 h ATF4水平显著升高(P<0.05);RPMP 300 mg·L^(-1)作用6 h ATF4水平显著升高(P<0.05),24 h SOD2水平显著升高(P<0.05)。结论 RPM和RPMP均具有一定的细胞毒性,RPM的细胞毒性强于RPMP,两者的肝毒性可能主要与氧化应激和内质网应激导致的细胞凋亡有关。OBJECTIVE To investigate the hepatotoxicity mechanisms of ethanol extract of Radix Polygoni Multiflori（RPM） and Radix Polygoni Multiflori Praeparata（RPMP） by high-content screen assay.METHODS Hep G2 cells were treated with RPM（10, 25, 50, 100, 200 and 300 mg·L-1） and RPMP（10,50, 100, 300, 600 and 1200 mg·L-1） for 3-24 h, respectively. The cell viability was detected by a Cell TiterGloTMluminescent cell viability assay kit. Cell count, reactive oxygen species（ROS）, mitochondrial membrane potential（MMP）, glutathione（GSH）, superoxide dismutase 2（SOD2）, activating transcription factor 4（ATF4）, apoptosis, and cell cycles were investigated by high-content screen assay. Besides,SOD2 and ATF4 levels were confirmed by Western blotting. RESULTS RPM 300 mg·L-1showed nearly48 % reduction in cell viability compared with cell control（P〈0.01）, while RPMP had no significant effect at the same concentration. Both RPM and RPMP decreased the level of MMP（P〈0.05） but incresed levels of GSH, ROS, SOD2 and ATF4 significantly（P〈0.05）. Besides, RPM 200 mg·L-1significantly increased the expression of SOD2（P〈0.05） at 3 h by high-content screen assay, and the enhanced expression of ATF4 was shown at 6 h（P〈0.05）. RPMP 300 mg·L-1markedly increased the expression of ATF4 at 6 h（P〈0.05）, while the expression of SOD2 significantly increased at 24 h（P〈0.05）. CONCLUSION Both RPM and RPMP have some cytotoxicity, and the cytotoxicity of RPM is stronger than that of RPMP. The hepatotoxicity mechanisms of RPM and RPMP may be related to cell apoptosis caused by long-term oxidative stress and endoplasmic reticulum stress.

关 键 词：何首乌 肝毒性 细胞 Hep G2 高内涵筛选技术 氧化应激 内质网应激 

分 类 号：R285.1[医药卫生—中药学]