非细胞毒性浓度AFB1暴露下H1N1型猪流感病毒感染3D4/21细胞的蛋白质组分析

doi:10.11843/j.issn.0366-6964.2025.04.041

摘要/Abstract

摘要：

世界范围内普遍存在黄曲霉毒素B₁(aflatoxin B₁，AFB₁)污染，严重危害畜禽健康。目前，全世界超过34个国家和地区对畜禽饲料中AFB₁的含量制定了限量标准。然而，即使低于限量标准的AFB₁暴露仍具有促进某些病原微生物感染的风险。因此，本研究以3D4/21细胞为模型，首先筛选对细胞无毒性的AFB₁浓度，然后使用蛋白转印和TCID₅₀法分别检测猪流感病毒(swine influenza virus，SIV)的NP蛋白相对表达量和滴度，确定非毒性浓度AFB₁暴露对SIV复制的影响。接下来，应用蛋白质组学平台下的TMT技术检测SIV感染组和SIV + AFB₁共同处理组细胞之间的差异表达蛋白和富集通路，探索AFB₁促进SIV复制的可能机制。结果显示，0.01 μg·mL^-1 AFB₁对3D4/21细胞无细胞毒性，且能够显著促进SIV复制；TMT检测显示，与SIV感染组相比SIV + AFB₁共同处理组细胞中242个蛋白表达上调，327个蛋白表达下调；进一步通路富集分析筛选出影响AFB₁促进SIV复制的差异通路137条，前5名分别为蛋白酶体通路、坏死性凋亡通路、多物种细胞凋亡通路、军团杆菌病通路和药物代谢-其他酶通路。其中细胞凋亡相关通路可能是调控AFB₁促进SIV复制的关键性通路，随后的DAPI染色试验也证实了这一点。本研究将为早期预防和控制因AFB₁污染而加剧的SIV感染奠定基础，并为探讨其他感染性疾病增加的原因提供参考。

关键词: 黄曲霉毒素B₁, H1N1, 猪流感病毒, 差异表达蛋白, 细胞凋亡

Abstract:

Aflatoxin B₁ (AFB₁) contamination is wide spread worldwide and seriously endangers the health of livestock and poultry. At present, more than 34 countries and regions in the world have established limit standards for the content of AFB₁ in livestock and poultry feed. However, even exposure to AFB₁ below the limit standard may still have the risk to promote infections of certain pathogens. Therefore, in this study, 3D4/21 cells were used as a model. Firstly, the non-toxic concentrations of AFB₁ were screened, and then the relative NP protein expression and titers of swine influenza virus (SIV) were detected by Western blots and TCID₅₀, respectively, thereby determining the effect of non-toxic concentrations of AFB₁ exposures on SIV replication. Next, TMT technology under the proteomics platform was used to detect the differentially expressed proteins and enrichment pathways between SIV infection and SIV + AFB₁ groups, in order to explore the possible mechanism of AFB₁ promoting SIV replication. The results showed that 0.01 μg·mL^-1 AFB₁ had no cytotoxicities on 3D4/21 cells and significantly promoted the replication of SIV in 3D4/21 cells; TMT results showed that 242 proteins were differentially up-regulated while 327 proteins were differentially down-regulated in the SIV + AFB₁ group compared to SIV infection group. Further pathway enrichment analysis screened 137 pathways that affect the difference of AFB₁-induced SIV replication. The top five enrichment pathways were proteasome pathway, necroptosis pathway, multi-species apoptosis pathway, legionella disease pathway, and drug metabolism-other enzyme pathway, respectively. Among them, the apoptosis-related pathway may be the key pathway regulating AFB₁ promoting SIV replication, and the subsequent DAPI staining test confirmed that. The research results will lay a foundation for early prevention and control of SIV infection aggravated by AFB₁ pollution, and provide a reference for exploring the causes of the increase of other infectious diseases.

Key words: aflatoxin B₁, H1N1, swine influenza virus, differentially expressed proteins, apoptosis

中图分类号:

庞思瑶, 张金龙, 孙雨航. 非细胞毒性浓度AFB₁暴露下H1N1型猪流感病毒感染3D4/21细胞的蛋白质组分析[J]. 畜牧兽医学报, 2025, 56(4): 1947-1957.

PANG Siyao, ZHANG Jinlong, SUN Yuhang. Proteomic Analysis of 3D4/21 Cells Infected with H1N1 Swine Influenza Virus under Non-cytotoxic Concentration of AFB₁ Exposure[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(4): 1947-1957.

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蛋白序号 Protein number	蛋白名称 Protein name	P值 P value	上/下调 Up-regulation/down-regulation
P62895	细胞色素c Cytochrome c	2.327×10^-5	上
A0A480M1Q1	E3泛素蛋白连接酶HERC6亚型1 Putative E3 ubiquitin-protein ligase HERC6 isoform 1	2.731×10^-5	上
A0A481BZ00	羧酸酯水解酶 Carboxylic ester hydrolase	7.838×10^-5	上
Q079S8	NADH-泛醌氧化还原酶链 NADH-ubiquinone oxidoreductase chain 1	13.402×10^-5	上
A0A480X9B8	F-box蛋白21 F-box only protein 21	36.782×10^-5	上
A0A8D1E4F1	S100蛋白 Protein S100	37.440×10^-5	上
A0A8D1V8L7	磷酸肌肽磷脂酶C Phosphoinositide phospholipase C	52.343×10^-5	上
A0A4X1W7V3	t复合体蛋白1亚基 T-complex protein 1 subunit eta	53.854×10^-5	上
Q2VTP6	肽基脯氨酰异构酶 Peptidylprolyl isomerase	80.059×10^-5	上
A0A8D1MRD3	无特征的蛋白质 Uncharacterized protein	115.198×10^-5	上
A0A4X1U844	丝氨酸蛋白酶抑制剂E家族成员2 Serpin family E member 2	5.344×10^-5	下
A0A8D0QDG2	含有RRM结构域的蛋白质 RRM domain-containing protein	5.949×10^-5	下
A0A8D1KDY7	无特征的蛋白质 Uncharacterized protein	6.075×10^-5	下
F1RW43	蛋白脂蛋白2 Proteolipid protein 2	9.699×10^-5	下
A0A8D0YI95	无特征的蛋白质 Uncharacterized protein	10.818×10^-5	下
A0A8D0XN07	蛋白SPT2同源物 Protein SPT2 homolog	14.590×10^-5	下
A0A8D1TV96	前mRNA 3′端加工因子FIP1 Pre-mRNA 3′-end-processing factor FIP1	15.115×10^-5	下
A0A8D1GKW2	集成复杂亚单位12 Integrator complex subunit 12	24.120×10^-5	下
Q9TR35	120 ku POG-2抗原(片段) 120 kuPOG-2 antigen (Fragment)	25.941×10^-5	下
A0A4X1WBN6	含中频棒结构域蛋白 IF rod domain-containing protein	30.558×10^-5	下

通路ID号 Pathway ID number	通路名称 Pathway name	P值 P value
map03050	蛋白酶体 Proteasome	656.951×10^-6
map04217	坏死性凋亡 Necroptosis	3 748.277×10^-6
map04215	多物种细胞凋亡 Apoptosis-multiple species	442.416×10^-5
map05134	军团杆菌病 Legionellosis	7346.851×10^-6
map00983	药物代谢-其他酶 Drug metabolism-other enzymes	1 0765.069×10^-6

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非细胞毒性浓度AFB₁暴露下H1N1型猪流感病毒感染3D4/21细胞的蛋白质组分析

Proteomic Analysis of 3D4/21 Cells Infected with H1N1 Swine Influenza Virus under Non-cytotoxic Concentration of AFB₁ Exposure

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