非细胞毒性浓度AFB_(1)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 AFB_(1) Exposure

作　　者：庞思瑶 张金龙 孙雨航 PANG Siyao;ZHANG Jinlong;SUN Yuhang(College of Animal Science and Medicine,Shenyang Agricultural University,Shenyang 110866,China)

机构地区：[1]沈阳农业大学动物科学与医学学院,沈阳110866

出　　处：《畜牧兽医学报》2025年第4期1947-1957,共11页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：辽宁省科技厅博士科研启动项目(2024BS094);辽宁省教育厅基本科研项目青年项目(Z20240295);国家自然科学基金青年项目(32002347);中国博士后基金面上项目(2023M741095,2021M692231)。

摘　　要：世界范围内普遍存在黄曲霉毒素B_(1)(aflatoxin B_(1),AFB_(1))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AFB_(1)的含量制定了限量标准。然而,即使低于限量标准的AFB_(1)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AFB_(1)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AFB_(1)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+AFB_(1)共同处理组细胞之间的差异表达蛋白和富集通路,探索AFB_(1)促进SIV复制的可能机制。结果显示,0.01μg·mL^(-1)AFB_(1)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+AFB_(1)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AFB_(1)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AFB_(1)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AFB_(1)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。Aflatoxin B_(1)(AFB_(1))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 AFB_(1) in livestock and poultry feed.However,even exposure to AFB_(1) 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 nontoxic concentrations of AFB_(1) 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 AFB_(1) exposures on SIV replication.Next,TMT technology under the proteomics platform was used to detect the differentially ex-pressed proteins and enrichment pathways between SIV infection and SIV+AFB_(1) groups,in or-der to explore the possible mechanism of AFB_(1) promoting SIV replication.The results showed that 0.01μg·mL^(-1) AFB_(1) 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+AFB_(1) group com-pared to SIV infection group.Further pathway enrichment analysis screened 137 pathways that affect the difference of AFB_(1)-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 apopto-sis-related pathway may be the key pathway regulating AFB_(1) 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 AFB_(1) pollution,and provide a reference for exploring the causes of the increase of other infectious diseases.

关 键 词：黄曲霉毒素B_(1) H1N1 猪流感病毒 差异表达蛋白 细胞凋亡 

分 类 号：S852.65[农业科学—基础兽医学] S856.9[农业科学—兽医学]