基于蛋白质组学探讨绿原酸抗玉米赤霉烯酮致卵泡颗粒细胞死亡的作用机制

doi:10.11843/j.issn.0366-6964.2025.12.048

摘要/Abstract

摘要：

玉米赤霉烯酮(zearalenone，ZEA)是一种常见的霉菌毒素，对人和动物有严重的生殖毒性。本团队前期研究发现绿原酸(chlorogenic acid)可缓解ZEA诱导的卵巢颗粒细胞毒性，然而其作用机制并不清楚。本试验旨在通过蛋白质组学技术解析绿原酸拮抗ZEA诱导小鼠卵泡颗粒细胞死亡的分子机制。将原代分离培养的小鼠卵巢颗粒细胞分为两组：ZEA组和绿原酸干预组，处理24 h后，采用定量蛋白质组学技术，结合生物信息学分析和RT-qPCR验证，解析绿原酸的调控网络。结果表明，绿原酸干预组与ZEA处理组相比，共鉴定出32个差异表达蛋白(differentially expressed proteins，DEPs)，其中15个显著上调，17个显著下调。通过GO功能注释和KEGG通路富集分析，发现DEPs主要参与有丝分裂调控、细胞骨架重构等生物学过程，并显著富集于凋亡、黏着斑、Toll样受体信号通路和NF-κB信号通路等关键调控途径。进一步通过RT-qPCR验证关键调控因子RIPK1的表达变化，发现ZEA处理显著抑制RIPK1表达(P < 0.001)，而绿原酸干预可部分恢复其表达水平(P < 0.01)。绿原酸通过调控细胞周期进程和凋亡、维持细胞结构稳态等多靶点多通路协同作用，从而拮抗ZEA诱导的生殖细胞毒性。此外，RIPK1作为RIPK1作为细胞生存和死亡的关键调控开关可能发挥关键作用。本研究从蛋白层面揭示绿原酸拮抗ZEA生殖毒性的作用机制，为开发基于绿原酸的ZEA解毒剂或功能性饲料添加剂提供了新的分子靶点和理论依据。

关键词: 玉米赤霉烯酮, 绿原酸, 卵泡颗粒细胞, 蛋白质组学

Abstract:

Zearalenone (ZEA) is a common mycotoxin that can cause severe reproductive toxicity. In our previous study, we found that chlorogenic acid could alleviate ZEA-induced ovarian granulosa cell toxicity, however, the mechanism of action is not clear. The aim of this study was to investigate the molecular mechanism by which chlorogenic acid antagonizes ZEA-induced follicular granulosa cell death in mice using proteomics techniques. Primary isolated and cultured mouse ovarian granulosa cells were divided into two groups: the ZEA group and the chlorogenic acid intervention group. After 24 h of treatment, the regulatory network of chlorogenic acid was systematically analyzed using quantitative proteomics technology combined with bioinformatics analysis and RT-qPCR validation. The results showed that a total of 32 differentially expressed proteins (DEPs) were identified in the chlorogenic acid-treated group compared with the ZEA-treated group, of which 15 were significantly up-regulated and 17 were significantly down-regulated. Through GO functional annotation and KEGG pathway enrichment analysis, the DEPs were mainly involved in biological processes such as mitotic regulation and cytoskeletal remodeling, and were significantly enriched in key regulatory pathways such as apoptosis, focal adhesion, Toll-like receptor signaling pathway and NF-κB signaling pathway. The changes in the expression of the key regulator RIPK1 were further verified by RT-qPCR and it was found that ZEA treatment significantly inhibited the expression of RIPK1, whereas chlorogenic acid intervention partially restored its expression level. Chlorogenic acid acts synergistically through multi-targets and multi-pathways such as regulation of cell cycle progression and apoptosis and maintenance of cellular structural homeostasis to antagonize ZEA-induced reproductive toxicity. In addition, RIPK1 may play a critical role as a key regulatory switch for cell survival and death. The present study reveals the mechanism of action of chlorogenic acid in antagonizing reproductive toxicity of ZEA at the protein level, providing new molecular targets and theoretical basis for the development of chlorogenic acid-based ZEA detoxifying agents or functional feed additives.

Key words: zearalenone, chlorogenic acid, ovarian granulosa cells, proteomics

中图分类号:

扆妍妍, 孙盼盼, 孙娜, 李宏全. 基于蛋白质组学探讨绿原酸抗玉米赤霉烯酮致卵泡颗粒细胞死亡的作用机制[J]. 畜牧兽医学报, 2025, 56(12): 6477-6486.

YI Yanyan, SUN Panpan, SUN Na, LI Hongquan. Proteomic Profiling Unveils the Protective Mechanism of Chlorogenic Acid against Zearalenone-induced Ovarian Granulosa Cell Death[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(12): 6477-6486.

图/表 8

表 1

图 1

表 2

表 3

差异蛋白"

序号 Number	蛋白 Protein	描述 Description	基因 Gene	CA vs. ZEA FC	CA vs. ZEA P-value	CA vs.ZEA up. down
1	Q60855	Receptor-interacting serine/threonine-protein kinase 1	Ripk1	6.838 597 732	0.000 908	up
2	P20918	Plasminogen	Plg	1.936 831 791	0.000 809	up
3	P52825	Carnitine O-palmitoyltransferase 2, mitochondrial	Cpt2	1.875 604 744	0.028 423	up
4	Q9EST3	Eukaryotic translation initiation factor 4E transporter	Eif4enif1	1.741 499 162	0.008 496	up
5	A0A0A0MQ89	Actin-related protein 5	Actr5	1.660 375 215	0.022 784	up
6	Q8C8K1	Ephrin type-B receptor 4	Ephb4	1.656 338 654	0.018 795	up
7	Q80TA1	Ethanolaminephosphotransferase 1	Selenoi	1.643 163 895	0.042 674	up
8	Q9CWX4	Mitochondrial RNA pseudouridine synthase Rpusd4	Rpusd4	1.601 582 318	0.024 808	up
9	Q9D3E6	Cohesin subunit SA-1	Stag1	1.601 233 098	0.010 973	up
10	Q3TST1	Gremlin	Grem2	1.590 010 117	0.001 580	up
11	Q3T9M2	Uncharacterized protein	2410002F23Rik	1.581 384 943	0.012 780	up
12	Q8BH57	WD repeat-containing protein 48	Wdr48	1.554 648 198	0.004 134	up
13	Q8CI75	DIS3-like exonuclease 2	Dis3l2	1.532 886 977	0.002 319	up
14	D3YYC3	Histone-lysine N-methyltransferase	Setdb1	1.522 323 567	0.011 408	up
15	Q9JI13	Something about silencing protein 10	Utp3	1.515 893 567	0.015 876	up
16	Q80VP8	Transmembrane protein 106C	Tmem106c	0.666 359 885	0.038 453	down
17	Q9R061	Cytosolic Fe-S cluster assembly factor NUBP2	Nubp2	0.665 644 345	0.010 295	down
18	Q6NSP9	High mobility group protein HMGI-C	Hmga2	0.664 658 291	0.008 745	down
19	A0A1L1SQ51	Talin-2	Tln2	0.663 968 51	0.028 922	down
20	Q6DID3	Protein SCAF8	Scaf8	0.662 149 128	0.024 783	down
21	B2RSR2	SEC22 vesicle trafficking protein homologe A (S. cerevisiae)	Sec22a	0.650 305 429	0.001 730	down
22	A0A0A0MQN9	Tetratricopeptide repeat protein 28	Ttc28	0.628 430 984	0.002 058	down
23	Q3T9S3	SET translocation	Set	0.623 105 049	0.004 339	down
24	Q7TPH6	E3 ubiquitin-protein ligase MYCBP2	Mycbp2	0.614 511 875	0.003 391	down
25	Q3THK3	General transcription factor IIF subunit 1	Gtf2f1	0.583 172 060	0.000 440	down
26	Q6NXZ5	ADP-ribosylation factor-like 5B	Arl5b	0.555 110 976	0.005 276	down
27	Q3TD85	Uncharacterized protein		0.552 628 993	0.000 079	down
28	P02798	Metallothionein-2	Mt2	0.465 704 189	0.045 177	down
29	B1B0E8	U2 small nuclear ribonucleoprotein auxiliary factor 35 ku subunit-related protein 2	Zrsr2	0.433 118 824	0.005 291	down
30	P63260	Actin, cytoplasmic 2	Actg1	0.348 302 060	0.000 224	down
31	E9QNG6	Sorting nexin-13	Snx13	0.296 579 413	0.008 409	down
32	E9QMV2	Costars family protein ABRACL	Abracl	0.280 216 784	0.002 122	down

表 3

图 2

图 3

图 4

图 5

参考文献 30

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基因 Gene	引物序列(5′→3′) Primers sequence
RIPK1	F: TAAAGACCCAGATAGATGTCC R: CCTTAGTCAGTTTGCTCCA
β-actin	F: CTGTCCCTGTATGCCTCTG R: ATGTCACGCACGATTTCC

比较对 Compared samples	总蛋白数量 Number of total quantity	总差异蛋白数量 Number of total significantly	上调蛋白数量 Number of significantly up	下调蛋白数量 Number of significantly down
CA vs. ZEA	4 998	32	15	17

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