摘要
目的探讨1-硝基芘(1-nitropyrene,1-NP)通过诱导线粒体损伤对人卵巢颗粒细胞KGN的增殖、周期进展及性激素合成的影响。方法采用不同浓度(0.625、1.25、2.5、5、10μmol/L)1-NP染毒KGN细胞48 h,EdU掺入法检测细胞增殖,流式细胞仪检测细胞周期,ELISA检测细胞培养上清中雌二醇及孕烯醇酮浓度,DCFH-DA探针检测细胞活性氧(reactive oxygen species,ROS)水平,MitoSOX探针检测线粒体超氧化物(mitochondrial superoxides,MitoSOX)水平,JC-1探针检测线粒体膜电位,ATP试剂盒检测细胞内ATP含量,Western blot检测细胞增殖、周期进展、DNA损伤反应以及性激素合成相关蛋白的表达。采用线粒体抗氧化剂IDE、1-NP单独和联合处理细胞,再次检测上述指标。结果不同浓度1-NP染毒后KGN细胞增殖显著降低(P<0.05)。2.5、5、10μmol/L的1-NP处理细胞后,细胞增殖标志物PCNA蛋白表达显著降低,5、10μmol/L的1-NP处理后G2/M期细胞比例显著升高(P<0.05)。1-NP(10μmol/L)处理3 h即可诱导γ-H2AX表达增加(P<0.01);不同浓度1-NP处理后,DNA损伤反应通路蛋白p-ATM、p53和p21cip1表达明显升高(P<0.05),G2/M期阻滞相关蛋白CDK-1和CyclinB1表达明显降低(P<0.05)。此外,1-NP处理后KGN细胞的雌二醇、孕烯醇酮浓度显著下降(P<0.05),性激素合成相关蛋白CYP19、CYP11A1的表达也受到抑制(P<0.05)。线粒体及氧化应激相关指标检测结果显示,1-NP导致KGN细胞中ROS、MitoSOX水平显著升高,线粒体膜电位和ATP水平呈剂量依赖性下降。与10μmol/L 1-NP单独染毒组相比,IDE与1-NP联合处理组G2/M期细胞比例显著下降,雌二醇、孕烯醇酮水平出现一定程度升高,DNA损伤反应、细胞周期进展以及性激素合成相关蛋白的表达也出现明显恢复。同时,IDE与1-NP联合处理显著改善1-NP诱导的线粒体损伤,包括降低细胞ROS和MitoSOX水平,提高线粒体膜电位和ATP水平。结论1-NP可诱导线粒体损伤,造成卵巢颗粒细胞KGN的增殖抑制、周期阻滞、性激素合成障碍,而提高线粒体功能则能部分缓解1-NP诱导的KGN细胞功能障碍。
Objective To investigate the effect of 1-nitropyrene(1-NP)on the proliferation,cycle progression and sex hormone synthesis of human ovarian granulosa cell line KGN via inducing mitochondrial damage.Methods After KGN cells were treated with 1-NP at different concentrations(0.625,1.25,2.5,5 and 10μmol/L)for 48 h,cell proliferation,cell cycle,and contents of estradiol and pregnenolone in the supernatant were detected by EdU assay,flow cytometry and ELISA,respectively.And the levels of reactive oxygen species(ROS),mitochondrial superoxide(MitoSOX)and mitochondrial membrane potential were examined with specific fluorescent probes including DCFH-DA probe,MitoSOX probe and JC-1 probe,respectively.In addition,ATP kit was adopted to measure the cellular ATP level,and Western blotting was also conducted to determine the expression of proteins related with the cell proliferation,cycle progression,DNA damage response and synthesis of sex hormones.Finally,the mitochondrial antioxidant idebenone(IDE,1μmol/L)and 1-NP(10μmol/L)were combined to treat the cells for 48 h,and then the above indicators were determined correspondingly again.Result As compared with the control cells,1-NP(0.625~10μmol/L)treatment significantly inhibited cell proliferation(P<0.05).The expression of PCNA,a marker of cell proliferation,was reduced after treatment with 1-NP(2.5,5 or 10μmol/L),and the percentage of cells at G2/M phase was elevated at 5 and 10μmol/L of 1-NP(P<0.05).Western blotting showed that the expression ofγ-H2AX began to rise after 1-NP treatment(10μmol/L)for 3 h(P<0.01),and the levels of DNA damage pathway proteins,p-ATM,p53 and p21cip1 were also obviously increased(P<0.05),while those of G2/M phase block-related proteins,CDK-1 and CyclinB1,were notably decreased at different doses of 1-NP(P<0.05).In addition,the contents of estradiol and pregnenolone in the supernatant of KGN cells were remarkably diminished after 1-NP treatment(P<0.05),and the expression levels of sex hormone synthesisrelated proteins CYP19 and CYP11A1 were inhibited as well(P<0.05).The detection of mitochondrial and oxidative stress-related indicators indicated that the levels of ROS and MitoSOX in KGN cells were greatly upregulated due to 1-NP treatment,while the mitochondrial membrane potential and ATP level were decreased in a dose-dependent manner.However,the combination of 1-NP and IDE significantly declined the percentage of G2/M-phase cells,elevated the contents of estradiol and pregnenolone,and restored the expression levels of proteins related to DNA damage,cell cycle as well as sex hormone synthesis when compared with the cells treated with 10μmol/L 1-NP.Moreover,the combination treatment also ameliorated the mitochondrial damage induced by 1-NP,including lowering ROS and MitoSOX levels and improving the mitochondrial membrane potential and ATP level.Conclusion 1-NP can cause mitochondrial damage,which leads to proliferation inhibition,cycle arrest and disorder of sex hormone synthesis,improve mitochondrial function,and thus partially alleviate 1-NP-induced dysfunction in KGN cells.
作者
崔浩楠
何世军
王立宏
杨望
杨彬伟
凌曦
邹鹏
张国伟
敖琳
曹佳
CUI Haonan;HE Shijun;WANG Lihong;YANG Wang;YANG Binwei;LING Xi;ZOU Peng;ZHANG Guowei;AO Lin;CAO Jia(Institute of Toxicology,Faculty of Military Preventive Medicine,Army Medical University(Third Military Medical University),Chongqing,400038,China;Department of Environmental Hygiene,Faculty of Military Preventive Medicine,Army Medical University(Third Military Medical University),Chongqing,400038,China)
出处
《陆军军医大学学报》
CAS
CSCD
北大核心
2023年第9期947-956,共10页
Journal of Army Medical University
基金
国家重点研发计划(2017YFC1002002)
国家自然科学基金面上项目(82073590)。
关键词
1-硝基芘
人卵巢颗粒细胞系
周期阻滞
性激素
线粒体
1-nitropyrene
human ovarian granulosa cell line
cycle arrest
sex hormone
mitochondria
作者简介
通信作者:曹佳,E-mail:caojia1962@126.com。
