脂质过氧化和铁死亡参与调节衰老卵巢颗粒细胞的实验研究

doi:10.12280/gjszjk.20260180

摘要/Abstract

摘要：

目的： 通过体内和体外实验比较正常和衰老卵巢颗粒细胞的脂质过氧化、铁累积水平和铁死亡敏感性。方法： 通过体内实验检测3月龄（生殖活跃期）和12月龄（生殖衰老期）时雌性C57BL/6小鼠卵巢组织中的线粒体和铁累积情况。通过体外实验检测人卵巢颗粒细胞系SVOG用毒胡萝卜素（thapsigargin，TG）诱导衰老前后细胞衰老相关指标以及脂质过氧化、铁死亡相关的指标。衰老相关蛋白即P16、P21及TP53；衰老相关分泌表型（senescence-associated secretory phenotype，SASP）中的促炎性细胞因子，包括白细胞介素-1A（interleukin-1A，IL-1A）、IL-1B、IL-6及C-X-C趋化因子配体8（C-X-C chemokine ligand 8，CXCL8）。铁死亡及脂质过氧化指标包括：铁死亡相关蛋白[谷胱甘肽过氧化物酶4（glutathione peroxidase 4，GPX4）、转铁蛋白受体（transferrin receptor，TFRC）、核受体共激活因子4（nuclear receptor coactivator 4，NCOA4）]的表达水平，以及活性氧（reactive oxygen species，ROS）、丙二醛（malondialdehyde，MDA）和亚铁离子（Fe²⁺）水平。结果： 体内实验证实12月龄小鼠卵巢中铁累积增加（P<0.01）。体外实验证实TG成功诱导SVOG细胞衰老并降低了细胞活力（P<0.05）。衰老SVOG细胞中铁死亡相关蛋白GPX4表达下降（P<0.000 1），TFRC和NCOA4表达升高（均P<0.01），ROS、MDA、Fe²⁺水平均升高（均P<0.01）。结论： 衰老卵巢颗粒细胞存在明显的铁累积和脂质过氧化增高，铁死亡敏感性增强，提示铁死亡参与调控卵巢颗粒细胞衰老过程。

关键词: 卵巢, 细胞衰老, 铁死亡, 脂质过氧化作用, 颗粒细胞

Abstract:

Objective: To compare the levels of lipid peroxidation, iron accumulation and ferroptosis sensitivity between normal and senescent ovarian granulosa cells by the in vivo and in vitro experiments. Methods: The mitochondrial status and iron accumulation in the ovarian tissues of female C57BL/6 mice aged 3 months (reproductive active period) and 12 months (reproductive senescence) were detected in the in vivo experiment. In the in vitro experiment, the SVOG cell, the human ovarian granulosa cell line, was used to develope the senescence model by thapsigargin (TG). The cellular senescence-related markers, lipid peroxidation and ferroptosis-related markers were detected. Senescence-associated proteins such as P16, P21 and TP53 were included. Pro-inflammatory cytokines of the senescence-associated secretory phenotype (SASP), including interleukin-1A (IL-1A), IL-1B, IL-6 and C-X-C chemokine ligand 8 (CXCL8) were analyzed. The ferroptosis and lipid peroxidation indicators were also tested, including the expression levels of ferroptosis-related proteins [glutathione peroxidase 4 (GPX4), transferrin receptor (TFRC), and nuclear receptor coactivator 4 (NCOA4)], and the levels of reactive oxygen species (ROS), malondialdehyde (MDA), and ferrous ion (Fe²⁺). Results: The in vivo experiment confirmed a significant increase in the iron accumulation in senescence ovaries (P<0.01). The in vitro experiment confirmed that TG successfully induced the senescence model of SVOG cells and reduced cell viability (P<0.05). In the senescent cells, the expression of the ferroptosis-related protein GPX4 was decreased (P<0.000 1), while the expressions of TFRC and NCOA4 were increased (P<0.01). Furthermore, the levels of ROS, MDA and Fe²⁺, were all increased (P<0.01). Conclusions: Senescent ovarian granulosa cells display the iron accumulation and the elevated lipid peroxidation, and the increased ferroptosis sensitivity, indicating that ferroptosis participates in the regulation of ovarian granulosa cell aging.

Key words: Ovary, Cellular senescence, Ferroptosis, Lipid peroxidation, Granulosa cells

董思睿, 孟艳. 脂质过氧化和铁死亡参与调节衰老卵巢颗粒细胞的实验研究[J]. 国际生殖健康/计划生育杂志, 2026, 45(3): 177-183.

DONG Si-rui, MENG Yan. Experimental Study on the Involvement of Lipid Peroxidation and Ferroptosis in the Regulation of Senescent Ovarian Granulosa Cells[J]. Journal of International Reproductive Health/Family Planning, 2026, 45(3): 177-183.

图/表 5

表1 实时荧光定量PCR引物序列

基因名称	序列
IL-1A	上游：5'-AATCATCAAGCCTAGGTCAGC-3'
	下游：5'-CTTCATCTTGGGCAGTCACA-3'
IL-1B	上游：5'-GTTGAAAGATGATAAGCCCACT-3'
	下游：5'-GTTATATCCTGGCCGCCTT-3'
IL-6	上游：5'-CACACAGACAGCCACTCACC-3'
	下游：5'-ATTTTCACCAGGCAAGTCTCC-3'
CXCL8	上游：5'-CAGTTTTGCCAAGGAGTGCTA-3'
	下游：5'-TTTTCCTTGGGGTCCAGACA-3'
GAPDH	上游：5'-GGAGCGAGATCCCTCCAAAAT-3'
	下游：5'-GGCTGTTGTCATACTTCTCATGG-3'

图1 衰老卵巢比年轻卵巢表现出更多的铁死亡注：A 代表性透射电子显微镜图像，红色箭头所示为线粒体，12月龄小鼠卵巢中的线粒体体积减小，线粒体嵴减少或消失，提示铁死亡发生，×6 000，标尺=500 nm；B 小鼠卵巢组织切片的普鲁士蓝铁染色代表性图像，以识别富含铁的细胞，×40，标尺=500 μm；C 普鲁士蓝铁染色的阳性面积定量分析，与年轻小鼠（3月龄，n=3）相比，衰老小鼠（12月龄，n=3）卵巢铁沉积显著增多，** P<0.01。

图2 利用TG构建SVOG细胞衰老模型注：A 不同浓度（0、125、250 nmol/L）TG处理SVOG细胞24 h后的IL-1A、IL-1B、IL-6、CXCL8的mRNA表达水平的定量分析（n=3）；B 不同浓度（0、125、250 nmol/L）TG处理SVOG细胞24 h后的P16、P21、TP53的Western blotting代表性图像；C、D、E 分别为不同浓度（0、125、250 nmol/L）TG处理SVOG细胞24 h后P16、P21、TP53蛋白表达的定量分析（n=3）；F 不同浓度（0、125、250 nmol/L）TG处理SVOG细胞不同时间（0、12、24、48、72 h）的细胞增殖曲线（n=4）；G 不同浓度（0、125、250 nmol/L）TG处理SVOG细胞24 h后的细胞活力定量分析（n=4）；* P<0.05，** P<0.01，*** P<0.001，**** P<0.000 1，ns 差异无统计学意义。

图3 衰老SVOG细胞脂质过氧化水平升高注：A 正常与衰老SVOG细胞中MDA的定量分析（n=3）；B 正常与衰老SVOG细胞中ROS水平的代表性图像，绿色为ROS荧光，×100，标尺=50 μm；C 正常与衰老SVOG细胞中ROS水平的定量分析（n=3）；*** P<0.001。

图4 衰老SVOG细胞铁死亡敏感性增加注：A 正常与衰老SVOG细胞中Fe2+含量的定量分析（n=3）；B 正常与衰老SVOG细胞GPX4、NCOA4、TFRC的Western blotting代表性图像；C 正常与衰老SVOG细胞中GPX4蛋白水平定量分析（n=3）；D 正常与衰老SVOG细胞中NCOA4蛋白水平定量分析（n=3）；E 正常与衰老SVOG细胞中TFRC蛋白水平定量分析（n=3）；** P<0.01，**** P<0.000 1。

参考文献 22

[1]	Johnson JA, Tough S. No-271-Delayed Child-Bearing[J]. J Obstet Gynaecol Can, 2017, 39(11):e500-e515. doi: 10.1016/j.jogc.2017.09.007. pmid: 29080737
[2]	Broekmans FJ, Knauff EA, te Velde ER, et al. Female reproductive ageing: current knowledge and future trends[J]. Trends Endocrinol Metab, 2007, 18(2):58-65. doi: 10.1016/j.tem.2007.01.004.
[3]	Levine ME, Lu AT, Chen BH, et al. Menopause accelerates biological aging[J]. Proc Natl Acad Sci U S A, 2016, 113(33):9327-9332. doi: 10.1073/pnas.1604558113.
[4]	Muka T, Oliver-Williams C, Kunutsor S, et al. Association of Age at Onset of Menopause and Time Since Onset of Menopause With Cardiovascular Outcomes, Intermediate Vascular Traits, and All-Cause Mortality: A Systematic Review and Meta-analysis[J]. JAMA Cardiol, 2016, 1(7):767-776. doi: 10.1001/jamacardio.2016.2415. pmid: 27627190
[5]	Kordus RJ, LaVoie HA. Granulosa cell biomarkers to predict pregnancy in ART: pieces to solve the puzzle[J]. Reproduction, 2017, 153(2):R69-R83. doi: 10.1530/REP-16-0500.
[6]	Liu C, Zuo W, Yan G, et al. Granulosa cell mevalonate pathway abnormalities contribute to oocyte meiotic defects and aneuploidy[J]. Nat Aging, 2023, 3(6):670-687. doi: 10.1038/s43587-023-00419-9.
[7]	Tatone C, Amicarelli F. The aging ovary--the poor granulosa cells[J]. Fertil Steril, 2013, 99(1):12-17. doi: 10.1016/j.fertnstert.2012.11.029. pmid: 23273984
[8]	Chen X, Kang R, Kroemer G, et al. Ferroptosis in infection, inflammation, and immunity[J]. J Exp Med, 2021, 218(6):e20210518. doi: 10.1084/jem.20210518.
[9]	Meng Y, Sun HY, He Y, et al. BET inhibitors potentiate melanoma ferroptosis and immunotherapy through AKR1C2 inhibition[J]. Mil Med Res, 2023, 10(1):61. doi: 10.1186/s40779-023-00497-1. pmid: 38049916
[10]	Zhao WP, Wang HW, Liu J, et al. Mitochondrial respiratory chain complex abnormal expressions and fusion disorder are involved in fluoride-induced mitochondrial dysfunction in ovarian granulosa cells[J]. Chemosphere, 2019, 215:619-625. doi: 10.1016/j.chemosphere.2018.10.043.
[11]	Wang F, Liu Y, Ni F, et al. BNC1 deficiency-triggered ferroptosis through the NF2-YAP pathway induces primary ovarian insufficiency[J]. Nat Commun, 2022, 13(1):5871. doi: 10.1038/s41467-022-33323-8. pmid: 36198708
[12]	Ma J, Chen S, Liu J, et al. Cryptochrome 1 regulates ovarian granulosa cell senescence through NCOA4-mediated ferritinophagy[J]. Free Radic Biol Med, 2024, 217:1-14. doi: 10.1016/j.freeradbiomed.2024.03.015.
[13]	Babayev E, Duncan FE. Age-associated changes in cumulus cells and follicular fluid: the local oocyte microenvironment as a determinant of gamete quality[J]. Biol Reprod, 2022, 106(2):351-365. doi: 10.1093/biolre/ioab241. pmid: 34982142
[14]	Hennet ML, Combelles CM. The antral follicle: a microenvironment for oocyte differentiation[J]. Int J Dev Biol, 2012, 56(10/11/12):819-831. doi: 10.1387/ijdb.120133cc.
[15]	Wang S, Zheng Y, Li J, et al. Single-Cell Transcriptomic Atlas of Primate Ovarian Aging[J]. Cell, 2020, 180(3):585-600.e19. doi: 10.1016/j.cell.2020.01.009. pmid: 32004457
[16]	Dixon SJ, Lemberg KM, Lamprecht MR, et al. Ferroptosis: an iron-dependent form of nonapoptotic cell death[J]. Cell, 2012, 149(5):1060-1072. doi: 10.1016/j.cell.2012.03.042. pmid: 22632970
[17]	Hu J, Wang H, Fang J, et al. Ovarian aging-associated downregulation of GPX4 expression regulates ovarian follicular development by affecting granulosa cell functions and oocyte quality[J]. FASEB J, 2025, 39(6):e70469. doi: 10.1096/fj.202401580RR.
[18]	Chen H, Wang S, Ding Z, et al. Rbbp7-mediated deacetylation of Acsl4 promotes ovarian aging by enhancing ferroptosis[J]. Int J Biol Macromol, 2026, 339(Pt 2):149976. doi: 10.1016/j.ijbiomac.2025.149976.
[19]	Chen D, Wang Y, Wang X, et al. Corrigendum to "Nuclear receptor coactivator 4 linked to follicular dysplasia in polycystic ovary syndrome: A key regulator that aggravates ovarian granulosa cells ferritinophagy and ferroptosis" [Biochim Biophys Acta Mol Basis Dis. 1871 (7) (Oct 2025) 167955 (BBADIS 167955)][J]. Biochim Biophys Acta Mol Basis Dis, 2026, 1872(5):168183. doi: 10.1016/j.bbadis.2026.168183.
[20]	Hu L, Hong T, He Y, et al. Chromosome Segregation-1-like Gene Participates in Ferroptosis in Human Ovarian Granulosa Cells via Nucleocytoplasmic Transport[J]. Antioxidants(Basel), 2024, 13(8):911. doi: 10.3390/antiox13080911.
[21]	Qin X, Zhao Y, Zhang T, et al. TrkB agonist antibody ameliorates fertility deficits in aged and cyclophosphamide-induced premature ovarian failure model mice[J]. Nat Commun, 2022, 13(1):914. doi: 10.1038/s41467-022-28611-2. pmid: 35177657
[22]	Zhou C, Guo Q, Lin J, et al. Single-Cell Atlas of Human Ovaries Reveals The Role Of The Pyroptotic Macrophage in Ovarian Aging[J]. Adv Sci(Weinh), 2024, 11(4):e2305175. doi: 10.1002/advs.202305175.