USP18抑制GPX4泛素化降解调控蛋鸭卵巢颗粒细胞铁死亡的分子机制

doi:10.3864/j.issn.0578-1752.2026.05.016

摘要/Abstract

摘要：

【背景】 家禽卵泡发育具有独特的等级卵泡发育特性，受自分泌、旁分泌、生长因子和多种功能基因的共同调控。颗粒细胞是卵泡内数量最多的功能细胞，颗粒细胞的存活直接决定了卵泡的生长发育和成熟。铁死亡是一种新发现细胞程序性死亡方式，与卵泡发育密切相关。泛素特异性肽酶 18（ubiquitin specific peptidase 18，USP18）在铁死亡过程中发挥重要的调控作用，但其在蛋鸭卵巢颗粒细胞中的功能尚不清楚。【目的】 通过探究USP18在蛋鸭卵泡颗粒细胞铁死亡过程中的分子调控机制，为家禽产蛋性状的遗传改良提供理论基础和分子靶点。【方法】 选取300日龄高、低产蛋鸭各4只，采集等级前卵泡组织，实时荧光定量PCR和Western blotting检测高、低产蛋鸭等级前卵泡组织中USP18表达水平；分离等级前颗粒细胞，设计USP18干扰片段并转染卵巢颗粒细胞，采用CCK-8试剂盒、Calcein-AM/PI细胞双染法检测USP18对颗粒细胞活力的影响，活性氧检测试剂盒和酶联免疫吸附法分析USP18对颗粒细胞内氧化应激水平的影响；利用线粒体膜电位试剂盒、脂质过氧化荧光探针、铁离子FerroOrange探针、普鲁士蓝试剂盒检测USP18对颗粒细胞铁死亡的影响；通过免疫荧光、免疫共沉淀和Western blotting分析USP18与谷胱甘肽过氧化物酶4（glutathione peroxidase 4，GPX4）蛋白间的互作关系及GPX4蛋白表达水平。【结果】 高产蛋鸭等级前卵泡组织中USP18表达量显著高于低产蛋鸭（P<0.01）；敲降USP18显著降低颗粒细胞活力（P<0.01），抑制颗粒细胞存活（P<0.05），促进ROS积累（P<0.01），提高丙二醛（malondialdehyde，MDA）含量（P<0.01），抑制超氧化物歧化酶（superoxide dismutase，SOD）和过氧化氢酶（catalase，CAT）的活性（P<0.01）；敲降USP18显著提高颗粒细胞内脂质过氧化水平（P<0.05），降低线粒体膜电位（P<0.05），增强线粒体膜通透性（P<0.01），提高细胞内铁离子水平和铁沉积（P<0.01），促进铁死亡抑制因子A长链酰基辅酶A合成酶4（acyl-coA synthetase long chain family member 4，ACSL4）和GPX4表达并抑制铁死亡驱动因子转铁蛋白受体1（transferrin receptor protein 1，TFR1）表达（P<0.01）；免疫共沉淀和免疫荧光证实USP18可与GPX4互作；Western blotting和免疫共沉淀结果显示敲降USP18提高 GPX4泛素化水平并促进GPX4蛋白降解。【结论】 USP18在高产蛋鸭等级前卵泡组织中高表达；敲降USP18显著提高了蛋鸭卵巢颗粒细胞内氧化应激水平并诱发铁死亡；USP18可与GPX4蛋白发生互作，敲降USP18提高 GPX4泛素化水平并促进GPX4蛋白降解。综上所述，USP18通过抑制GPX4泛素化降解调控蛋鸭卵巢颗粒细胞铁死亡。

关键词: 蛋鸭, 颗粒细胞, USP18, GPX4, 铁死亡

Abstract:

【Background】 Follicle development is characterized by a unique hierarchical progression in poultry, regulated by autocrine, paracrine, growth factors, and multiple functional genes. Granulosa cells are the most abundant functional somatic cells within the follicle, and their survival directly determines follicular growth, development, and maturation. Ferroptosis, a newly identified type of regulated cell death, has been closely linked to follicular development. USP18 plays an important regulatory role in ferroptosis, however, its function in duck granulosa cells remains unclear. 【Objective】 The objective of this study was to investigate the molecular regulatory mechanisms of USP18 in the process of ferroptosis in duck granulosa cells of laying ducks, so as to provide a theoretical basis and molecular targets for the genetic improvement of egg production traits in poultry. 【Method】 Four high-yield and four low-yield laying ducks aged 300-day-old were selected. Prehierarchical follicular tissues were collected from each group. The expression level of USP18 was assessed using quantitative real-time PCR (qRT-PCR) and Western blotting in follicles of high-yield and low-yield laying ducks. Granulosa cells isolated from prehierarchical follicles were transfected with the interference fragment of USP18. The effects of USP18 on cell viability were evaluated using the CCK-8 assay and Calcein-AM/PI double staining. The effects of USP18 on intracellular oxidative status were evaluated using the reactive oxygen species (ROS) detection kit and enzyme-linked immunosorbent assay (ELISA). The impact of USP18 on ferroptosis was examined using the mitochondrial membrane potential assay kit, the lipid peroxidation fluorescent probe, FerroOrange iron probe, and Prussian blue staining. Furthermore, the interaction between USP18 and GPX4 protein, as well as GPX4 protein expression, were analyzed by immunofluorescence, co-immunoprecipitation, and Western blotting.【Result】 The expression level of USP18 in prehierarchical follicles of high-yield ducks was significantly higher than that in low-yield ducks (P<0.01); USP18 knockdown significantly reduced granulosa cell viability (P<0.01), inhibited cell survival (P<0.05), promoted ROS accumulation (P<0.01), increased malondialdehyde (MDA) content (P<0.01), and suppressed the activities of superoxide dismutase (SOD) and catalase (CAT) (P<0.01); USP18 knockdown notably elevated lipid peroxidation levels (P<0.05), decreased mitochondrial membrane potential (P<0.05), enhanced mitochondrial membrane permeability (P<0.01), increased intracellular iron levels and iron deposition (P<0.01), increased the expression of ferroptosis suppressor markers ACSL4 and GPX4, and suppressed the expression of TFR1 (P<0.01); the interaction relationship between USP18 and GPX4 was confirmed by co-immunoprecipitation and immunofluorescence assays. Western blotting and co-immunoprecipitation analyses further demonstrated that USP18 knockdown promoted ubiquitination and degradation of GPX4 protein.【Conclusion】 USP18 was highly expressed in the prehierarchical follicles of high-yield laying ducks. USP18 knockdown significantly increased oxidative stress levels and induced ferroptosis in duck granulosa cells. Moreover, USP18 was found to interact with GPX4 protein, and USP18 knockdown promoted ubiquitination and degradation of GPX4 protein. In summary, USP18 modulated ferroptosis in duck granulosa cells by suppressing the ubiquitination and degradation of GPX4.

Key words: duck, granulosa cells, USP18, GPX4, ferroptosis

陈亚茹, 王磊, 付明, 黄涛, 张昊, 梁振华, 皮劲松, 吴艳. USP18抑制GPX4泛素化降解调控蛋鸭卵巢颗粒细胞铁死亡的分子机制[J]. 中国农业科学, 2026, 59(5): 1128-1140.

CHEN YaRu, WANG Lei, FU Ming, HUANG Tao, ZHANG Hao, LIANG ZhenHua, PI JinSong, WU Yan. Molecular Mechanism of USP18 Facilitating Ferroptosis by Suppressing GPX4 Ubiquitination and Degradation in Duck Granulosa Cells[J]. Scientia Agricultura Sinica, 2026, 59(5): 1128-1140.

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试验样本 Test sample	300 d产蛋数 Egg production in 300 days (No.)	体重 Bodyweight (kg)
高产蛋鸭1 High-yield laying duck 1	130	1.65
高产蛋鸭2 High-yield laying duck 2	145	1.79
高产蛋鸭3 High-yield laying duck 3	138	1.68
高产蛋鸭4 High-yield laying duck 4	140	1.58
低产蛋鸭1 Low-yield laying duck 1	84	1.75
低产蛋鸭2 Low-yield laying duck 2	86	1.64
低产蛋鸭3 Low-yield laying duck 3	80	1.60
低产蛋鸭4 Low-yield laying duck 4	75	1.67

基因Gene	引物序列Primer sequence	产物长度Product length (bp)
β-actin	F: CCGCTCTATGAAGGCTACGC R: CTCTCGGCTGTGGTGGTGAA	105
FLAG-USP18	F: CCTGAGGCAAATCTGTCAGTC R: CGAACACCTGAATCAAGGAGTTAATGTGCGCCGTGCCGGCCC	1281
HA-GPX4	F: TAGCCCGGGCGGATCCAAGCTTATGAGCTTTAG R: GACTCGAGAGATCTGTCGACGATATCGAATTCGTGCTACCTCTAG	594
ACSL4	F: CATCCCTGGAGCAGATACTCT R: TCACTTAGGATTTCCCTGGTCC	98
GPX4	F: CCAGCCCCTACACCTACCTC R: GCTGGGCTTGTCAGTTTCTC	106
TFR1	F: AACTCAGCAAAGTCTGGCGT R: GACCCCCAATACACCGCATA	87
USP18	F: CCTGAGGCAAATCTGTCAGTC R: CGAACACCTGAATCAAGGAGTTA	124