摘要
目的探索三氯异氰尿酸(TCCA)对小鼠睾丸精原细胞GC-1氧化应激和DNA甲基化的影响。方法以TCCA 0(细胞对照),97,194和387μmol·L^(-1)处理GC-1细胞24 h;CCK-8法检测细胞存活率;Hoechst 33342染色检测各组细胞形态和凋亡率;流式法检测细胞周期;实时荧光定量PCR(RT-qPCR)检测细胞凋亡相关基因Bax和Fas、氧化应激相关基因线粒体超氧化物歧化酶(SOD2)、铁死亡相关基因谷胱甘肽过氧化酶4(GPX4)、核因子红细胞2相关因子2(Nrf2)、溶质载体家族7成员11(SLC7A11)、二氢乳清酸脱氢酶(DHODH)、DNA甲基化转移酶3A(DNMT3A)和DNA甲基转移酶3L(DNMT3L)mRNA表达水平;Griess法测定GC-1细胞一氧化氮(NO)含量,比色法测定丙二醛(MDA)水平;DCFH-DA荧光探针法测定GC-1细胞活性氧(ROS)水平,DNTB比色法测定还原型谷胱甘肽(GSH)含量,WST-8法测定还原型辅酶Ⅱ(NADPH)含量。结果与细胞对照组相比,TCCA 194和387μmol·L^(-1)组细胞存活率降低(P<0.01),出现细胞核固缩及核碎裂,细胞凋亡率显著增加(P<0.01),细胞被阻滞在G2/M期(P<0.05);TCCA 387μmol·L^(-1)组细胞NO和MDA含量增加(P<0.01),ROS水平升高(P<0.01),GSH和NADPH含量降低(P<0.01),SOD2,GPX4,Nrf2,SLC7A11和DHODH mRNA表达减少(P<0.05,P<0.01);Bax,Fas,DNMT3L和DNMT3A mRNA表达增加(P<0.05,P<0.01)。结论TCCA可降低GC-1细胞存活率,抑制细胞增殖,引起细胞凋亡。其机制可能与其破坏抗氧化系统、增强氧化应激、诱导铁死亡及干扰GC-1细胞甲基化有关。
OBJECTIVE To explore the effects of trichloroisocyanuric acid(TCCA)on the prolifera⁃tion of spermatogonia by inducing oxidative stress and ferroptosis.METHODS GC-1 cells were cultured in DMEM-F12 medium,and cell proliferation was plotted according to the growth curve.GC-1 cells were treated with TCCA at concentrations of 0(cell control),97,194,and 387μmol·L^(-1) for 24 h.Cell viability was detected using the CCK-8 method,apoptosis cells were stained with Hoechst 33342,cell cycle was examined by PI staining method,RT-qPCR was performed to measure the mRNA expres⁃sion levels of apoptosis-related genes Bax,Fas,oxidative stress-related genes superoxide dismutase 2(SOD2),glutathione peroxidase 4(GPX4),nuclear factor erythroid 2-related factor 2(Nrf2),solute carrier family 7 member 11(SLC7A11),dihydroorotate dehydrogenase(DHODH),DNA methyltransferase 3A(DNMT3A),and DNA methyltransferase 3L(DNMT3L).The Griess method was used to determine the nitric oxide(NO)content,colorimetric method for the malondialdehyde(MDA)level,DCFH-DA fluores⁃cence probe method for the reactive oxygen species(ROS)level,DNTB colorimetric method for the reduced glutathione(GSH)content,and WST-8 method for the reduced coenzymeⅡ(NADPH)content.RESULTS Compared with the cell control group,the cell survival rates in the TCCA 194 and 387μmol·L^(-1) groups decreased significantly(P<0.01),accompanied by nuclear condensation and fragmentation,a significant increase in apoptosis rate(P<0.01),and cell arrest in the G2/M phase(P<0.05).Additionally,in the TCCA 387μmol·L^(-1) group,the levels of NO,MDA and ROS increased(P<0.01),while the levels of GSH and NADPH decreased(P<0.01).Moreover,the mRNA expressions of SOD2,GPX4,Nrf2,SLC7A11,and DHODH decreased(P<0.05,P<0.01),while the expressions of Bax,Fas,DNMT3L,and DNMT3A increased(P<0.05,P<0.01).CONCLUSION TCCA exposure reduces the viability of GC-1 cells,inhibits cell proliferation,induces apoptosis of GC-1 cells.The mechanism may be related to the ability of TCCA to enhance oxidate stress,induce fe
作者
蒋莉
韩雪
吴德生
黄海燕
刘建军
JIANG Li;HAN Xue;WU Desheng;HUANG Haiyan;LIU Jianjun(College of Public Health,Shanxi Medical University,Taiyuan 030000,China;Shenzhen Key Labora-tory of Modern Toxicology,Shenzhen Medical Key Discipline of Health Toxicology(2020-2024),Shenzhen Center for Disease Control and Prevention,Shenzhen 518055,China)
出处
《中国药理学与毒理学杂志》
CAS
北大核心
2024年第6期426-435,共10页
Chinese Journal of Pharmacology and Toxicology
基金
深圳市医学重点学科建设经费资助(SZXK069)。
关键词
三氯异氰尿酸
精原细胞
氧化应激
DNA甲基化
铁死亡
trichloroisocyanuric acid
spermatogonia
oxidative stress
DNA methylation
ferroptosis
