慢性铁过载通过铁代谢紊乱引发氧化应激与内分泌失调诱导小鼠卵巢储备功能减退

doi:10.12122/j.issn.1673-4254.2026.06.11

摘要/Abstract

摘要：

目的探讨慢性铁过载导致小鼠卵巢储备功能减退的机制。方法将40只动情周期正常的C57BL/6J雌性小鼠随机分为空白组（腹腔注射等体积生理盐水，OF组）、低铁剂组（0.1 g/kg，LF组）、中铁剂组（0.5 g/kg，MF组）、高铁剂组（1.0 g/kg，HF组），10只/组，除空白组外，其余组腹腔注射右旋糖酐铁，1次/周，持续8周，制备慢性铁过载小鼠模型。每周称量小鼠体质量、监测小鼠进食量；阴道脱落细胞涂片观察小鼠动情周期变化；称量小鼠双侧卵巢及子宫湿重；苏木精-伊红染色观察小鼠卵巢组织病理学改变；普鲁士蓝染色评估卵巢组织中铁离子的表达水平；ELISA测定血清性激素［卵泡刺激素、黄体生成素、雌二醇、抗缪勒氏管激素（AMH）］；生化试剂盒检测卵巢组织中丙二醛和超氧化物歧化酶（SOD）水平；冰冻切片小鼠卵巢组织活性氧测试；透射电镜观察小鼠卵巢组织线粒体超微结构；Rt-qPCR检测卵巢组织中铁重链蛋白1（Fth1）、铁轻链蛋白（Ftl）、转铁蛋白（Tf）、转铁蛋白受体1（Tfr1）、过氧化氢酶（Cat）、谷胱甘肽过氧化物酶1（Gpx1）基因的表达；Western blotting检测卵巢组织Ft、Ftl、Fth1、Tf、Tfr1、谷胱甘肽过氧化物酶4（Gpx4）蛋白的表达。结果与空白组相比，其余3组小鼠动情周期不同程度的紊乱，MF、HF组闭锁卵泡数量增多，卵巢指数下降、子宫指数上升（P<0.05），血清AMH、雌二醇水平下降（P<0.05），卵泡刺激素、黄体生成素水平上升（P<0.05），卵巢间质铁沉积信号显著增多，SOD活性降低（P<0.001），丙二醛水平升高（P<0.01），活性氧荧光强度上升（P<0.05），氧化应激水平显著升高，卵巢组织受损线粒体数量增多，线粒体嵴减少甚至消失，卵巢组织Tf、Tfr1、Gpx4、Gpx1、Cat表达水平均下降（P<0.05），Ft与Ftl、Fth1表达升高（P<0.05）。结论 0.5 g/kg和1.0 g/kg右旋糖酐铁成功诱导慢性浓度依赖性铁过载模型小鼠，慢性铁过载通过代偿性上调卵巢组织内Ft、Ftl和Fth1的表达，同时下调Tf和Tfr1、Gpx4的表达，导致小鼠体内铁代谢失常，铁稳态紊乱，出现氧化应激损伤和内分泌功能紊乱，进而诱发卵巢储备功能减退。

关键词: 慢性铁过载, 卵巢储备功能减退, 铁代谢, 铁紊乱, 氧化应激

Abstract:

Objective To investigate the mechanism by which chronic iron overload induces diminished ovarian reserve (DOR) in mice. Methods Forty female C57BL/6J mice with normal estrous cycles were randomly divided into 4 groups (n=10) for intraperitoneal injections of normal saline or iron dextran at 0.1, 0.5 or 1.0 g/kg once a week for 8 consecutive weeks. The changes in body weight and food intake of the mice were monitored weekly. Vaginal smears were used to assess estrous cycle changes of the mice, and wet weights of bilateral ovaries and uterus were recorded. Ovarian histopathology was evaluated by HE staining, iron deposition was detected by Prussian blue staining; serum levels of sex hormones (FSH, LH, E₂, and AMH) and the levels of MDA and SOD in the ovarian tissue were determined, and reactive oxygen species (ROS) production was assessed on frozen sections of the ovarian tissue. Mitochondrial ultrastructural changes in the ovaries of the mice were observed with transmission electron microscopy, and ovarian expression levels of Fth1, Ftl, Cat, Gpx1, Tf and Tfr1 mRNAs and Tf, Tfr1, Ft, Fth1, Ftl and Gpx4 protein were detected. Results The mice receiving injections of 0.5 and 1.0 g/kg iron dextran exhibited disrupted estrous cycles, increased atretic follicles, decreased ovarian index, and increased uterine index with reduced AMH and E₂ levels and increased FSH and LH levels. The injections caused significant iron accumulation, oxidative stress, and obvious mitochondrial damage in the ovarian tissues, resulting also in downregulation of Tf, Tfr1, Gpx4, Gpx1 and Cat and upregulation of Ft, Ftl and Fth1 expressions. Conclusion Iron dextran at 0.5 and 1.0 g/kg can induce chronic, dose-dependent iron overload in mice, which causes systemic iron metabolism disorders and disrupted iron homeostasis to trigger oxidative stress damage and endocrine dysfunction and ultimately induce DOR.

Key words: chronic iron overload, diminished ovarian reserve, iron metabolism, iron dyshomeostasis, oxidative stress

于静, 储继军, 梁程程. 慢性铁过载通过铁代谢紊乱引发氧化应激与内分泌失调诱导小鼠卵巢储备功能减退[J]. 南方医科大学学报, 2026, 46(6): 1313-1322.

Jing YU, Jijun CHU, Chengcheng LIANG. Chronic iron overload induces diminished ovarian reserve in mice via iron metabolism dysregulation-mediated oxidative stress and endocrine dysfunction[J]. Journal of Southern Medical University, 2026, 46(6): 1313-1322.

图/表 10

表1 RT-qPCR引物序列

Tab.1 Primer sequences for RT-qPCR

Primer name	Sequence (5' to 3')	Length (bp)
Tf	F:GAGACGGAGACCTTGTGATTC R:AACTGCTGAATTACTGGCTGTCC	24
Tf	F:GAGACGGAGACCTTGTGATTC R:AACTGCTGAATTACTGGCTGTCC	23
Tfr1	F:GGCTCTGGCTCTCACACTCTC R:AACTGCTGAATTACTGGCTGTCC	24
Tfr1	F:GGCTCTGGCTCTCACACTCTC R:AACTGCTGAATTACTGGCTGTCC	23
Fth1	F:CAACTTACGCAAGATGGGTG R:CTTAGCTCTCATCACCGTGT	20
Fth1	F:CAACTTACGCAAGATGGGTG R:CTTAGCTCTCATCACCGTGT	20
Ftl	F:CGTCAGAATTATTCCACCGAGG	22
Ftl	R:GCCACGTCATCCCGATCAAA	20
Gpx1	F:TCAGTTCGGACACCAGAATG R:CACTTCGCACTTCTCAAAC	20
Gpx1	F:TCAGTTCGGACACCAGAATG R:CACTTCGCACTTCTCAAAC	19
Cat	F:AAGTCACCACTCCAGC R:GGTCAGGACATCAGGTCT	16
Cat	F:AAGTCACCACTCCAGC R:GGTCAGGACATCAGGTCT	18
β-actin	F:AGTGTGACGTTGACATCC	20
β-actin	R:TGCTAGGAGCCAGAGCAGTA	20

图1 小鼠阴道脱落细胞涂片

Fig.1 Smear of exfoliated cells from the vagina of mice (HE staining, original magnification: ×100). A: Proestrus. B: Estrus. C: Metestrus. D: Diestrus.

图2 慢性铁过载对小鼠子宫、卵巢的影响

Fig.2 Effect of chronic iron overload on the uterus and ovaries of the mice. OF: Control group with intraperitoneal injection of normal saline; LF: Low-dose iron group (0.1 g/kg); MF: Medium-dose iron group (0.5 g/kg); HF: High-dose iron group (1.0 g/kg). *P<0.05, **P<0.01 vs OF group (n=10).

图3 小鼠卵巢组织及卵巢周围脂肪组织HE染色

Fig.3 HE staining of mouse ovarian tissue and periovarian adipose tissue. A: Ovarian tissue (×50, ×200). B: Periovarian adipose tissue (×200).

图4 小鼠卵巢组织及卵巢周围脂肪组织普鲁士蓝染色

Fig.4 Prussian blue staining of mouse ovarian tissue and periovarian adipose tissue. A: Ovarian tissue (×50, ×200). B: Periovarian adipose tissue (×200).

图5 慢性铁过载对小鼠血清性激素水平的影响

Fig.5 Effect of chronic iron overload on serum sex hormone levels in the mice. A: Serum AMH levels. B: Serum E2 levels. C: Serum FSH levels. D: Serum LH levels. *P<0.05, **P<0.01, ***P<0.001 vs OF group (n=5).

图6 慢性铁过载对小鼠卵巢组织氧化应激水平的影响

Fig.6 Effect of chronic iron overload on oxidative stress levels in ovarian tissue of the mice. A: ROS fluorescence images of mouse ovarian tissue. B: Quantitative analysis of ROS fluorescence intensity. C: MDA content in mouse ovarian tissue. D: SOD activity. *P<0.05, **P<0.01, ***P<0.001 vs OF group (n=3).

图7 慢性铁过载对小鼠卵巢线粒体形态的影响

Fig.7 Effects of chronic iron overload on mitochondrial morphology in the ovaries of the mice (Transmission electron microscopy).

图8 小鼠卵巢组织Fth1、Ftl、Cat、Gpx1、Tf和Tfr1 mRNA表达

Fig.8 Relative mRNA expression of Fth1 (A), Ftl (B), Cat (C), Gpx1 (D), Tf (E)and Tfr1 (F)in mouse ovarian tissues. *P<0.05, **P<0.01, ***P<0.001 vs OF group (n=3).

图9 小鼠卵巢组织Tf、Tfr1、Ft、Fth1、Ftl和Gpx4蛋白表达

Fig.9 Protein expression of Tf, Tfr1, Ft, Fth1, Ftl and Gpx4 in mouse ovarian tissue. *P<0.05, **P<0.01, ***P<0.001 vs OF group (n=3).

参考文献 32

[1]	Steiner AZ, Pritchard D, Stanczyk FZ, et al. Association between biomarkers of ovarian reserve and infertility among older women of reproductive age[J]. Jama, 2017, 318(14): 1367. doi：10.1001/jama.2017.14588
[2]	卵巢储备功能减退临床诊治专家共识专家组, 中华预防医学会生育力保护分会生殖内分泌生育保护学组. 卵巢储备功能减退临床诊治专家共识[J]. 生殖医学杂志, 2022, 31(04): 425-34.
[3]	李文燕, 丁澍, 熊湘蕾, 等. 卵巢储备功能减退的代谢异常及干预策略[J]. 中国生育健康杂志，2025, 36(03): 297-302.
[4]	Li SS, Zhou YE, Huang QX, et al. Iron overload in endometriosis peritoneal fluid induces early embryo ferroptosis mediated by HMOX1[J]. Cell Death Discov, 2021, 7: 355. doi：10.1038/s41420-021-00751-2
[5]	Ni ZX, Li YS, Song D, et al. Iron-overloaded follicular fluid increases the risk of endometriosis-related infertility by triggering granulosa cell ferroptosis and oocyte dysmaturity[J]. Cell Death Dis, 2022, 13(7): 579. doi：10.1038/s41419-022-05037-8
[6]	Voros C, Chatzinikolaou F, Papadimas G, et al. Ferroptosis in the ovarian follicular microenvironment: a redox-dependent cell death pathway with emerging roles in PCOS, oocyte quality, and IVF outcomes[J]. Int J Mol Sci, 2025, 26(21): 10381. doi：10.3390/ijms262110381
[7]	Huang Y, Cheng Y, Zhang M, et al. Oxidative stress and inflammatory markers in ovarian follicular fluid of women with diminished ovarian reserve during in vitro fertilization[J]. J Ovarian Res, 2023, 16(1): 206. doi：10.1186/s13048-023-01293-0
[8]	梁程程, 章晓乐, 齐聪, 等. 卵巢储备功能下降患者卵泡液中8-OHdG和4-HNE的水平及其与体外受精相关指标的关系[J]. 中国生育健康杂志, 2022, 33(6): 525-31. doi：10.3969/j.issn.1671-878X.2022.06.005
[9]	Liang CC, Zhang XL, Qi C, et al. UHPLC-MS-MS analysis of oxylipins metabolomics components of follicular fluid in infertile individuals with diminished ovarian reserve[J]. Reprod Biol Endocrinol, 2021, 19(1): 143. doi：10.1186/S12958-021-00825-X
[10]	Liao CY, Cao Q, Zhang Y, et al. A dietary antioxidant nanodrug with sustained antioxidation for premature ovarian failure by rescuing hormone metabolism of granulosa cells via WNT7A/CYP19 pathway[J]. Nano Res, 2025, 18(8): 94907638. doi：10.26599/NR.2025.94907638
[11]	Shang YJ, Li YJ, Han D, et al. LRRC4 deficiency drives premature ovarian insufficiency by disrupting metabolic homeostasis in granulosa cells[J]. Adv Sci, 2025, 12(23): 2417717. doi：10.1002/advs.202417717
[12]	程萌, 孔令伶俐, 许良智, 等. 卵巢储备功能减退临床诊治专家共识解读[J]. 实用妇产科杂志, 2022, 38(10): 743-5.
[13]	Loy SL, Cheung YB, Fortier MV, et al. Age-related nomograms for antral follicle count and anti-Mullerian hormone for subfertile Chinese women in Singapore[J]. PLoS One, 2017, 12(12): e0189830. doi：10.1371/journal.pone.0189830
[14]	江楠, 赵晓丽, 李凯茜, 等. 始基卵泡过度激活与卵巢储备功能减退的相关性及其机制[J]. 国际生殖健康/计划生育杂志, 2025, 44(2): 132-6. doi：10.12280/gjszjk20040540
[15]	Zhang L, Dong Z, Jiang F, et al. Ferrostatin-1 ameliorates Cis-dichlorodiammineplatinum(II)-induced ovarian toxicity by inhibiting ferroptosis[J]. Mol Med, 2024, 30(1): 150. doi：10.1186/s10020-024-00923-7
[16]	Li FF, Zhu FY, Wang SY, et al. Icariin alleviates cisplatin-induced premature ovarian failure by inhibiting ferroptosis through activation of the Nrf2/ARE pathway[J]. Sci Rep, 2024, 14: 17318. doi：10.1038/s41598-024-67557-x
[17]	林华丹, 王旭, 陈春宏, 等. 三种卵巢储备功能减退小鼠模型的效果比较[J]. 中国比较医学杂志, 2025, 35(5): 51-9.
[18]	丁婷, 王世宣. 卵巢衰老的评估和预警[J]. 实用妇产科杂志, 2022, 38(2): 86-9.
[19]	Wu YQ, Yang R, Lan J, et al. Iron overload modulates follicular microenvironment via ROS/HIF-1α/FSHR signaling[J]. Free Radic Biol Med, 2023, 196: 37-52. doi：10.1016/j.freeradbiomed.2022.12.105
[20]	胡恒, 储继军, 李哲, 等. 基于TLR4/MyD88/NF-κB信号通路探讨益经汤改善卵巢储备功能减退大鼠卵巢炎症反应的作用机制[J]. 中国实验方剂学杂志, 2025, 31(11): 20-30.
[21]	Wei MK, Han CY, Zhou XJ, et al. Filamentous morphology engineering of bacteria by iron metabolism modulation through MagR expression[J]. Synth Syst Biotechnol, 2024, 9(3): 522-30. doi：10.1016/j.synbio.2024.04.009
[22]	Qin X, Liang DK, Hu M, et al. Chronic overload of concentration-dependent iron exerts different effects on ovarian function in C57BL/6J mice[J]. Biol Reprod, 2021, 104(6): 1347-59. doi：10.1093/biolre/ioab040
[23]	Zhang SH, Liu Q, Chang MY, et al. Chemotherapy impairs ovarian function through excessive ROS-induced ferroptosis[J]. Cell Death Dis, 2023, 14(5): 340. doi：10.1038/s41419-023-05859-0
[24]	Gulec S, Anderson GJ, Collins JF. Mechanistic and regulatory aspects of intestinal iron absorption[J]. Am J Physiol Gastrointest Liver Physiol, 2014, 307(4): G397-409. doi：10.1152/ajpgi.00348.2013
[25]	Wang M, Zhang BQ, Ma S, et al. Broadening horizons: the role of ferroptosis in polycystic ovary syndrome[J]. Front Endocrinol, 2024, 15: 1390013. doi：10.3389/fendo.2024.1390013
[26]	Zhao JR, Dong LP, Gong X, et al. Hepcidin/HIF-1α is involved in the endogenous recovery and ferroptosis after cerebral ischemia in mice[J]. Biochim Biophys Acta BBA Mol Basis Dis, 2025, 1871(8): 168022. doi：10.1016/j.bbadis.2025.168022
[27]	Yang CJ, Wu AM, Tan LQ, et al. Epigallocatechin-3-gallate alleviates liver oxidative damage caused by iron overload in mice through inhibiting ferroptosis[J]. Nutrients, 2023, 15(8): 1993. doi：10.3390/nu15081993
[28]	Wang ZX, Ma J, Li XY, et al. Quercetin induces p53-independent cancer cell death through lysosome activation by the transcription factor EB and Reactive Oxygen Species-dependent ferroptosis[J]. British J Pharmacology, 2021, 178(5): 1133-48. doi：10.1111/bph.15350
[29]	Asperti M, Bellini S, Grillo E, et al. H-ferritin suppression and pronounced mitochondrial respiration make Hepatocellular Carcinoma cells sensitive to RSL3-induced ferroptosis[J]. Free Radic Biol Med, 2021, 169: 294-303. doi：10.1016/j.freeradbiomed.2021.04.024
[30]	Wang FX, Liu YF, Ni FD, et al. BNC1 deficiency-triggered ferroptosis through the NF2-YAP pathway induces primary ovarian insufficiency[J]. Nat Commun, 2022, 13: 5871. doi：10.1038/s41467-022-33323-8
[31]	梁程程, 于静, 胡恒, 等. 益经汤含药血清对人卵巢颗粒细胞铁过载诱导氧化应激的影响[J].中国中医药信息杂志, 2025, 32(9): 105-12.
[32]	Chen Y, Zhang JQ, Tian Y, et al. Iron accumulation in ovarian microenvironment damages the local redox balance and oocyte quality in aging mice[J]. Redox Biol, 2024, 73: 103195. doi：10.1016/j.redox.2024.103195