1-硝基芘通过线粒体损伤致卵巢颗粒细胞功能障碍

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

doi:10.16016/j.2097-0927.202210115

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1-硝基芘通过线粒体损伤致卵巢颗粒细胞功能障碍

基础医学 | 更新时间：2026-01-09

- 1-硝基芘通过线粒体损伤致卵巢颗粒细胞功能障碍
- 1-nitropyrene induces ovarian granulosa cell dysfunction through mitochondrial damage
- 陆军军医大学学报 2023年45卷第9期页码：947-956
- 作者机构：
  
  1.400038 重庆, 陆军军医大学(第三军医大学)军事预防医学系: 毒理学研究所
  2.400038 重庆, 陆军军医大学(第三军医大学)军事预防医学系: 环境卫生学教研室
- 作者简介：
  
  [曹佳，E-mail: caojia1962@126.comCAO Jia, E-mail: caojia1962@126.com
- 基金信息：
  
  国家重点研发计划(2017YFC1002002);国家自然科学基金面上项目(82073590)
- DOI：10.16016/j.2097-0927.202210115
  中图分类号： R322.65;R363.21;R994.6
- 收稿：2022-10-14，
  
  修回：2023-02-27，
  
  纸质出版：2023-05-15
- 稿件说明：
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崔浩楠, 何世军, 王立宏, 等. 1-硝基芘通过线粒体损伤致卵巢颗粒细胞功能障碍[J]. 陆军军医大学学报, 2023,45(9):947-956.

Haonan CUI, Shijun HE, Lihong WANG, et al. 1-nitropyrene induces ovarian granulosa cell dysfunction through mitochondrial damage[J]. Journal of Army Medical University, 2023, 45(9): 947-956.
崔浩楠, 何世军, 王立宏, 等. 1-硝基芘通过线粒体损伤致卵巢颗粒细胞功能障碍[J]. 陆军军医大学学报, 2023,45(9):947-956. DOI： 10.16016/j.2097-0927.202210115.

Haonan CUI, Shijun HE, Lihong WANG, et al. 1-nitropyrene induces ovarian granulosa cell dysfunction through mitochondrial damage[J]. Journal of Army Medical University, 2023, 45(9): 947-956. DOI： 10.16016/j.2097-0927.202210115.

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、10 μmol/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(

0.05)。2.5、5、10 μmol/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、10 μmol/L的1-NP处理后G

/M期细胞比例显著升高(

0.05)。1-NP (10 μmol/L)处理3 h即可诱导γ-H2AX表达增加(

0.01);不同浓度1-NP处理后

DNA损伤反应通路蛋白p-ATM、p53和p21

cip1

表达明显升高(

0.05)

/M期阻滞相关蛋白CDK-1和CyclinB1表达明显降低(

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(

0.05)

性激素合成相关蛋白CYP19、CYP11A1的表达也受到抑制(

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10 μmol/L 1-NP单独染毒组相比

IDE与1-NP联合处理组G

/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。

Abstract

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 10 μmol/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

1 μmol/L) and 1-NP (10 μmol/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 (0.625~10 μmol/L) treatment significantly inhibited cell proliferation (

0.05).The expression of PCNA

a marker of cell proliferation

was reduced after treatment with 1-NP (2.5

5 or 10 μmol/L)

and the percentage of cells at G

/M phase was elevated at 5 and 10 μmol/L of 1-NP (

0.05).Western blotting showed that the expression of γ-H2AX began to rise after 1-NP treatment (10 μmol/L

) for 3 h (

0.01)

and the levels of DNA damage pathway proteins

p-ATM

p53 and p21

cip1

were also obviously increased (

0.05)

while those of G

/M phase block-related proteins

CDK-1 and CyclinB1

were notably decreased at different doses of 1-NP (

0.05).In addition

the contents of estradiol and pregnenolone in the supernatant of KGN cells were remarkably diminished after 1-NP treatment (

0.05)

and the expression levels of sex hormone synthesis-related proteins CYP19 and CYP11A1 were inhibited as well (

0.05).The detection of mitochondrial and oxidative stress-related indicators indicated that the levels of ROS and MitoSOX in KGN cells were greatly up-regulated 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 G

/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 10 μmol/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.

关键词

Keywords

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