猪流行性腹泻病毒诱导细胞铁死亡的分析

doi:10.11843/j.issn.0366-6964.2025.11.041

摘要/Abstract

摘要：

本文旨在探究猪流行性腹泻病毒(porcine epidemic diarrhea virus，PEDV)感染后是否引起铁死亡及对其复制水平的影响。首先通过PEDV感染IPEC-J2(porcine intestinal epithelial cells, IPEC-J2)细胞后的蛋白组学差异蛋白、GO和KEGG富集分析推测IPEC-J2细胞在感染PEDV后会发生铁死亡，通过测定细胞中MDA、GSH含量，铁死亡关键蛋白TFRC、xCT、GPX4、ACSL4、LPCAT3和Ferritin蛋白水平及倒置荧光显微镜观察Fe²⁺、脂质ROS和线粒体膜电位荧光强度变化确定了会发生铁死亡。进一步通过添加铁死亡抑制剂Fer-1(ferrostatin-1)探讨了PEDV感染IPEC-J2细胞后引起的铁死亡在其感染过程中N蛋白的表达变化和病毒滴度的变化。蛋白组学的筛选结果：PEDV感染组Ferritin下调最显著；GO和KEGG富集分析结果均显示，铁代谢紊乱和脂代谢失调途径最显著，推测PEDV感染IPEC-J2后可能引起了铁死亡；感染细胞中MDA含量极显著升高，GSH含量极显著降低(P < 0.01)；Ferritin、GPX4、ACSL4和LPCAT3蛋白水平均极显著下调(P < 0.01)，并且TFRC蛋白水平极显著上调(P < 0.01)；荧光结果显示，Fe²⁺相对荧光强度极显著增强、氧化型脂质ROS荧光强度极显著增强及线粒体膜电位极显著降低(P < 0.01)。在添加Fer-1后PEDV的N蛋白水平和基因水平均极显著下调(P < 0.01)，并且Fer-1处理后病毒滴度也极显著下降(P < 0.01)。PEDV感染IPEC-J2细胞可以引起细胞发生铁死亡，这可能是促进其复制的机制之一。

关键词: 猪流行性腹泻病毒, 猪小肠上皮细胞, 铁死亡

Abstract:

This study aimed to investigate whether porcine epidemic diarrhea virus (PEDV) infection induces ferroptosis and its impact on viral replication. Proteomic analysis of differentially expressed proteins in IPEC-J2 cells (porcine intestinal epithelial cells) post-PEDV infection, combined with Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses, initially suggested ferroptosis occurrence. Subsequent validation included measuring malondialdehyde (MDA) and glutathione (GSH) levels, assessing protein expression of ferroptosis-related markers (TFRC, xCT, GPX4, ACSL4, LPCAT3, and Ferritin), and observing Fe²⁺ accumulation, lipid reactive oxygen species (ROS), and mitochondrial membrane potential via inverted fluorescence microscopy. Further, by adding the ferroptosis inhibitor Fer-1, the expression changes of N protein and the changes of viral titer during the infection process of PEC-J2 cells caused by PEDV infection were investigated. Proteomic analysis revealed significant downregulation of Ferritin in PEDV-infected cells. GO and KEGG analyses highlighted prominent enrichment in iron metabolism dysregulation and lipid metabolism disruption, indicating ferroptosis induction. PEDV infection markedly elevated MDA levels (P < 0.01) and reduced GSH content (P < 0.01). Protein expression of Ferritin, GPX4, ACSL4, and LPCAT3 was significantly downregulated (P < 0.01), while TFRC was upregulated (P < 0.01). Fluorescence assays demonstrated intensified Fe²⁺ and lipid ROS signals (P < 0.01) and diminished mitochondrial membrane potential (P < 0.01). After the addition of Fer-1, both the N protein level and the gene level of PEDV were extremely significantly downregulated (P < 0.01), and the viral titer also decreased extremely significantly after Fer-1 treatment (P < 0.01). PEDV infection induces ferroptosis in IPEC-J2 cells, a process that may mechanistically contribute to enhanced viral replication by disrupting cellular iron homeostasis and lipid peroxidation signaling pathways.

Key words: porcine epidemic diarrhea virus, porcine small intestinal epithelial cells, ferroptosis

中图分类号:

S852.6

王公民, 伍钢, 陈雪清, 陈希文, 徐佳靖, 张源淑. 猪流行性腹泻病毒诱导细胞铁死亡的分析[J]. 畜牧兽医学报, 2025, 56(11): 5852-5863.

WANG Gongmin, WU Gang, CHEN Xueqing, CHEN Xiwen, XU Jiajing, ZHANG Yuanshu. Analysis of Ferroptosis Induced by Porcine Epidemic Diarrhea Virus[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(11): 5852-5863.

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组别Group	处理Treatment
Con	病毒维持液作用48 h
PEDV	接种PEDV，吸附2 h后换病毒维持液作用48 h
PEDV+Fer-1	接种PEDV前6 h加入Fer-1处理6 h，接种PEDV，吸附2 h后换病毒维持液并在加入10 μmol·L^-1的Fer-1，作用48 h

引物名称Primer name	引物序列(5′→3′) Primer sequence	产物长度/bp Length
PEDV N-F	GGAGTCGTGGTAATGGCAAC
PEDV N-R	CTCTGTTCTGGGAAGCTCCA	172

NCBI accession	蛋白质Protein	变化Change	差异倍数Fold change
F1SCY2	干扰素诱导的含四肽重复序列的蛋白3	上调	24.92
I3LS55	表皮生长因子受体途径底物8样蛋1	上调	22.58
A0A287AXN9	角蛋白17	上调	14.33
F1RGY8	内质网氧化还原酶1β	上调	13.63
A0A5K1UFA2	干扰素诱导的含四肽重复序列的蛋白3	上调	10.58
A0A5G2REI9	F-盒蛋白28	下调	-6.80
I3LII4	丝氨酸/苏氨酸蛋白激酶	下调	-7.21
F1RT08	腺苷甲硫氨酸脱羧酶	下调	-14.28
A0A8W4FQ04	铁蛋白	下调	-37.26
P19133	铁蛋白轻链	下调	-38.84

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