纳米氧化锌诱发人心肌细胞AC16氧化应激损伤及转录组分析改变  

Oxidative stress injury and transcriptome analysis of human myocardial AC16 cells induced by zinc oxide nanoparticles

作　　者：鲁方梅 李宇杰 郭家彬 徐德祥[1] 刘超 Lu Fangmei;Li Yujie;Guo Jiabin;Xu Dexiang;Liu Chao(Dept of Health Toxicology,School of Public Health,Anhui Medical University,Hefei 230032;Chinese People's Liberation Army Center for Disease Control and Prevention,Beijing 100071)

机构地区：[1]安徽医科大学公共卫生学院卫生毒理学系,合肥230032 [2]中国人民解放军疾病预防控制中心,北京100071

出　　处：《安徽医科大学学报》2023年第3期450-456,共7页Acta Universitatis Medicinalis Anhui

基　　金：国家自然科学基金(编号:82003880)。

摘　　要：目的探讨纳米氧化锌(ZnO NPs)对人心肌细胞AC16的氧化应激损伤,并从转录组层面分析ZnO NPs作用机制。方法利用动态光散射法(DLS)对ZnO NPs进行表征检测。将AC16细胞暴露于不同剂量、不同时间的ZnO NPs后,使用CCK-8法测定细胞存活率。将AC16细胞分为对照组、ZnO NPs(50、100、200μmol/L)暴露组,处理6 h后检测细胞线粒体膜电位(MMP)及活性氧自由基(ROS)。将AC16细胞分为对照组、50μmol/L ZnO NPs组、200μmol/L ZnO NPs组,暴露6 h后使用TRIzol提取细胞总RNA,进行转录组分析,并对差异表达基因进行基因本体(GO)、京都基因和基因组百科全书(KEGG)富集分析。结果DLS法结果显示,流体动力学直径为(192.2±1.63)nm,Zeta电位为(-23.26±1.05)m V。CCK-8结果显示,随着ZnO NPs暴露的剂量与时间的增加,AC16细胞存活率下降。荧光定量法观察显示,随着ZnO NPs暴露剂量的增加,MMP在100μmol/L ZnO NPs时下降(P<0.05),ROS在50μmol/L ZnO NPs时升高(P<0.05)。转录组分析结果显示,50μmol/L ZnO NPs组与对照组相比共富集到1071个基因,其中上调基因561个,下调基因510个;200μmol/L ZnO NPs组与对照组相比共富集到7164个基因,其中上调基因4098个,下调基因3066个。GO与KEGG分析结果显示,差异基因主要富集于活性氧、抗氧化活性、线粒体细胞色素C的释放、凋亡等信号通路。结论ZnO NPs可导致AC16细胞存活率下降,诱发细胞线粒体损伤和氧化应激,其中ROS介导的氧化应激与线粒体功能改变是ZnO NPs致AC16细胞毒性的重要毒作用机制。Objective To investigate the oxidative stress injury of nano zinc oxide nanoparticles(ZnO NPs)on human myocardial cells(AC16),and to analyze the mechanism of ZnO NPs from the transcriptome level.Methods Dynamic light scattering(DLS)was used to characterize and detect ZnO NPs.After AC16 cells were exposed to ZnO NPs at different doses and at different times,the cell survival rate was determined by CCK-8 method.AC16 cells were divided into control group,ZnO NPs(50,100,200μmol/L),after 6 h treatment,the mitochondrial membrane potential(MMP)and reactive oxygen species(ROS)were measured.AC16 cells were divided into control group,50μmol/L ZnO NPs group and 200μmol/L ZnO NPs group.After 6 h exposure,total RNA was extracted by TRIzol for transcriptome analysis,and the differentially expressed genes were enriched by gene body(GO),Kyoto Encyclopedia of Genes and Genomes(KEGG).Results The results of DLS showed that the hydrodynamic diameter was(192.2±1.63)nm and the Zeta potential was(-23.26±1.05)mV.CCK-8 results showed that the survival rate of AC16 cells decreased with the increase of dose and time of exposure to ZnO NPs.Fluorescence quantification showed that with the increase of ZnO NPs exposure dose,MMP significantly decreased at 100μmol/L ZnO NPs(P<0.05),and ROS significantly increased at 50μmol/L ZnO NPs(P<0.05).Using the multifunctional microplate reader,it was observed that MMP and ROS were statistically significant at 100 and 50μmol/L ZnO NPs,respectively,showing a decrease in MMP and an increase in ROS.Transcriptome analysis showed that 1071 genes were enriched in the 50μmol/L ZnO NPs group compared with the control group,including 561 up-regulated genes and 510 down-regulated genes.Compared with the control group,7164 genes were enriched in 200μmol/L ZnO NPs group,including 4098 up-regulated genes and 3066 down-regulated genes.GO and KEGG analysis showed that the differential genes were mainly concentrated in ROS,antioxidant activity,mitochondrial cytochrome C release,apoptosis and other signaling path

关 键 词：纳米氧化锌 心肌毒性 转录组学 氧化应激 线粒体损伤 

分 类 号：R114[医药卫生—卫生毒理学]