DON和PRRSV共暴露通过PI3K/AKT/mTOR信号通路影响PAM-KNU细胞自噬

doi:10.3864/j.issn.0578-1752.2025.13.015

摘要/Abstract

摘要：

【目的】环境因素脱氧雪腐镰刀菌烯醇（DON）和传染性病原体猪繁殖与呼吸综合征病毒（PRRSV）作为对我国养猪业造成严重危害的两种因素，两者在临床中常同时发生，但鲜有两者共暴露的研究，其具体机制也尚不清楚。通过探究DON和PRRSV双重暴露下对宿主细胞猪肺泡巨噬细胞（PAM-KNU）的影响，为养猪业的环境要求提供理论参考。【方法】将扩增收集后的PRRSV进行梯度稀释，通过显微镜观察PRRSV感染后的Marc-145细胞的病变现象（CPE），计算出病毒的滴度。用MOI=1的PRRSV感染PAM-KNU细胞，通过蛋白免疫印迹分别在0、12、24、36、48、60和72 h检测细胞中PRRSV-N的表达量及上清液病毒滴度，筛选出合适的时间。在选定时间下，通过CCK-8法分别检测0、8、16、32、64、128、256、512、1 024、2 048 nmol·L^-1浓度的DON对PAM-KNU细胞活力的影响，以此选出合适的4个DON浓度进行后续试验。将4个不同浓度DON与PRRSV共暴露于PAM-KNU细胞中，通过蛋白免疫印迹检测PRRSV-N蛋白表达量，选定最终DON浓度。在此基础上建立DON和PRRSV共暴露细胞模型，具体分组为对照组（C组）、DON组（D组）、PRRSV组（V组）和DON+PRRSV组（VD组）。通过电镜观察PAM-KNU细胞内自噬情况，Western Blot和间接免疫荧光对LC3-Ⅱ、Beclin 1、p62、ULK 1蛋白进行自噬水平检测，应用Astral DIA技术进行蛋白质组测序，对差异蛋白进行KEGG通路富集分析，并对富集到的PI3K/AKT/mTOR通路进行蛋白表达验证。【结果】通过观察CPE现象，计算出PRRSV的滴度为10^7.3 TCID₅₀/mL。PRRSV在PAM-KNU细胞的生长动力曲线显示，细胞中PRRSV-N蛋白的表达量在24 h时达到指数增长期。CCK-8结果显示，DON浓度为8、16、32、64 nmol·L^-1时，对PAM-KNU细胞活力无显著影响，当DON浓度大于等于128 nmol·L^-1时，对细胞活力有显著影响，且细胞活力逐渐下降。将其中的4个DON浓度（32、128、256、512 nmol·L^-1）与PRRSV共暴露于PAM-KNU细胞中，Western Blot结果显示DON浓度为128 nmol·L^-1时，PRRSV-N蛋白表达量开始显著下降。在此基础上建立PAM-KNU细胞模型。透射电镜结果显示，与D组和V组相比，VD组自噬小体减少，同时LC3-Ⅱ、Beclin 1、p62、ULK 1蛋白表达量显著降低，表明自噬水平被抑制。通过蛋白质组学测序筛选差异蛋白，并进行KEGG分析，富集到多条通路，本实验对PI3K/AKT/mTOR通路进行验证，Western Blot结果显示，p-PI3K/PI3K、p-AKT/AKT、p-mTOR/mTOR表达量上升，与间接免疫荧光结果一致。【结论】 DON与PRRSV共暴露通过上调PI3K/AKT/mTOR通路显著抑制PAM-KNU细胞的自噬水平。这种抑制可能导致宿主细胞免疫反应失调，从而影响宿主细胞对病原体的反应能力。

关键词: 脱氧雪腐镰刀菌烯醇, 猪繁殖与呼吸综合征病毒, 自噬, PI3K/AKT/mTOR信号通路

Abstract:

【Objective】 Environmental factor deoxynivalenol (DON) and infectious agent Porcine reproductive and respiratory syndrome virus (PRRSV), as two factors that cause serious harm to the pig industry in China, often occur simultaneously in clinical practice, but few studies have been conducted on their co-exposure, and their specific mechanisms are still unclear. The aim of this study was to study the effects of DON and PRRSV on host cell porcine alveolar macrophages (PAM-KNU), and to provide theoretical reference for the environmental requirements of pig industry. 【Method】 In this experiment, the PRRSV collected after amplification was diluted by gradient, and the lesion phenomenon (CPE) of Marc-145 cells infected with PRRSV was observed by microscope, and the titer of the virus was calculated. PAM-KNU cells were infected with PRRSV with MOI=1, and the expression level of PRRSV-N and virus titer of supernatant were detected by western blot at 0, 12, 24, 36, 48, 60 and 72 h, respectively, and the appropriate time was selected. At the selected time, the effects of DON concentrations of 0, 8, 16, 32, 64, 128, 256, 512, 1 024 and 2 048 nmol·L^-1 on the cell viability of PAM-KNU were detected by CCK-8 method. Therefore, the appropriate 4 DON concentrations were selected for follow-up tests. Four different concentrations of DON and PRRSV were exposed to PAM-KNU cells, and the protein expression of PRRSV-N was detected by western blot, and the final DON concentration was selected. On this basis, DON and PRRSV co-exposed cell models were established, which were divided into control group (group C), DON group (group D), PRRSV group (group V) and DON+PRRSV group (group VD). Autophagy in PAM-KNU cells was examined using electron microscopy. The levels of autophagy-related proteins LC3-II, Beclin1, p62, and ULK 1 were quantified by Western Blot and indirect immunofluorescence. Proteomic sequencing was conducted using Astral DIA technology, followed by KEGG pathway enrichment analysis of differentially expressed proteins. The expression of proteins involved in the PI3K/AKT/mTOR pathway was validated. 【Result】 By observing the CPE phenomenon, the titer of PRRSV is calculated to be 10^7.3 TCID₅₀/mL. The growth dynamic curve of PRRSV in PAM-KNU cells showed that the expression of PRRSV-N protein reached an exponential growth period at 24 h. The results of CCK-8 showed that DON concentration at 8, 16, 32 and 64 nmol·L^-1 had no significant effect on PAM-KNU cell viability. When the DON concentration is greater than or equal to 128 nmol·L^-1, it has a significant impact on cell viability, and the cell viability gradually decreases. Four of the DON concentrations (32, 128, 256, 512 nmol·L^-1) were exposed to PAM-KNU cells together with PRRSV. Western Blot results showed that when the DON concentration was 128 nmol·L^-1, the expression of PRRSV-N protein began to decrease significantly. On this basis, PAM-KNU cell model was established. The results of transmission electron microscopy indicated a decrease in the number of autophagosomes in the VD group compared to the D and V groups. Additionally, the expression levels of LC3-Ⅱ, Beclin1, p62, and ULK 1 were significantly reduced, suggesting that the level of autophagy was inhibited. Differential proteins were identified through proteomic sequencing and subsequently enriched in multiple pathways as determined by KEGG analysis. In this study, the PI3K/AKT/mTOR pathways were specifically validated. Western blot analysis revealed increased expression levels of p-PI3K/PI3K, p-AKT/AKT, and p-mTOR/mTOR, corroborating the findings from indirect immunofluorescence. 【Conclusion】 Co-exposure to DON and PRRSV significantly inhibited autophagy in PAM-KNU cells by upregulating the PI3K/AKT/mTOR pathway. This inhibition may result in the dysregulation of the host cell immune response, thereby affecting the host cell's ability to respond to pathogens.

Key words: deoxynivalenol, porcine reproductive respiratory syndrome virus, autophagy, PI3K/AKT/mTOR signaling pathway

王琳媛, 宣晶焱, 杜禹, 陈彤, 牛瑞燕. DON和PRRSV共暴露通过PI3K/AKT/mTOR信号通路影响PAM-KNU细胞自噬[J]. 中国农业科学, 2025, 58(13): 2693-2706.

WANG LinYuan, XUAN JingYan, DU Yu, CHEN Tong, NIU RuiYan. Co-Exposure of DON and PRRSV Induces Autophagy in PAM-KNU Cells Through the PI3K/AKT/mTOR Signaling Pathway[J]. Scientia Agricultura Sinica, 2025, 58(13): 2693-2706.

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参考文献 32

[1]	李睿, 梁跃, 白杨, 张桂悦, 王楠楠, 乔松林, 张改平. 自噬在猪繁殖与呼吸综合征病毒感染中的作用机制研究进展. 中国农业科学, 2025, 58(4): 792-801.doi:10.3864/j.issn.0578-1752.2025.04.013.
	LI R, LIANG Y, BAI Y, ZHANG G Y, WANG N N, QIAO S L, ZHANG G P. Research progress on the roles and mechanisms of autophagy involved in porcine reproductive and respiratory syndrome virus infection. Scientia Agricultura Sinica, 2025, 58(4): 792-801. doi:10.3864/j.issn.0578-1752.2025.04.013. (in Chinese)
[2]	LIAO Y, WANG H, LIAO H Y, SUN Y J, TAN L, SONG C P, QIU X S, DING C. Classification, replication, and transcription of nidovirales. Frontiers in Microbiology, 2024, 14: 1291761.
[3]	ZHAO S S, QIAN Q S, CHEN X X, LU Q X, XING G X, QIAO S L, LI R, ZHANG G P. Porcine reproductive and respiratory syndrome virus triggers Golgi apparatus fragmentation-mediated autophagy to facilitate viral self-replication. Journal of Virology, 2024, 98(2): e0184223.
[4]	史建荣, 刘馨, 仇剑波, 祭芳, 徐剑宏, 董飞, 殷宪超, 冉军舰. 小麦中镰刀菌毒素脱氧雪腐镰刀菌烯醇污染现状与防控研究进展. 中国农业科学, 2014, 47(18): 3641-3654. doi:10.3864/j.issn.0578-1752.2014.18.012.
	SHI J R, LIU X, QIU J B, JI F, XU J H, DONG F, YIN X C, RAN J J. Deoxynivalenol contamination in wheat and its management. Scientia Agricultura Sinica, 2014, 47(18): 3641-3654. doi:10.3864/j.issn.0578-1752.2014.18.012. (in Chinese)
[5]	KOZIEŁ M J, KOWALSKA K, PIASTOWSKA-CIESIELSKA A W. Nrf2: A main responsive element in cells to mycotoxin-induced toxicity. Archives of Toxicology, 2021, 95(5): 1521-1533. doi: 10.1007/s00204-021-02995-4 pmid: 33554281
[6]	HE P Z, ZHAO Z Y, TAN Y L, E H C, ZUO M H, WANG J H, YANG J H, CUI S X, YANG X L. Photocatalytic degradation of deoxynivalenol using cerium doped titanium dioxide under ultraviolet light irradiation. Toxins, 2021, 13(7): 481.
[7]	ADUGNA C, WANG K, DU J, LI C M. Deoxynivalenol mycotoxin dietary exposure on broiler performance and small intestine health: a comprehensive meta-analysis. Poultry Science, 2024, 103(12): 104412.
[8]	ZHANG H, DENG X W, ZHOU C, WU W D, ZHANG H B. Deoxynivalenol induces inflammation in IPEC-J2 cells by activating P38 mapk and Erk1/2. Toxins, 2020, 12(3): 180.
[9]	PANISSON J C, WELLINGTON M O, BOSOMPEM M A, NAGL V, SCHWARTZ-ZIMMERMANN H E, COLUMBUS D A. Urinary and serum concentration of deoxynivalenol (DON) and DON metabolites as an indicator of DON contamination in swine diets. Toxins, 2023, 15(2): 120.
[10]	LIU Q, HE Q, ZHU W Y. Deoxynivalenol mycotoxin inhibits rabies virus replication in vitro. International Journal of Molecular Sciences, 2023, 24(9): 7793.
[11]	LIU J T, LUMSDEN J S. Impact of feed restriction, chloroquine and deoxynivalenol on viral haemorrhagic septicaemia virus IVb in fathead minnow Pimephales promelas Rafinesque. Journal of Fish Diseases, 2021, 44(2): 217-220.
[12]	LIU D D, WANG Q, HE W M, GE L, HUANG K H. Deoxynivalenol aggravates the immunosuppression in piglets and PAMs under the condition of PEDV infection through inhibiting TLR4/NLRP3 signaling pathway. Ecotoxicology and Environmental Safety, 2022, 231: 113209.
[13]	RÜCKNER A, PLAGGE L, HEENEMANN K, HARZER M, THAA B, WINKLER J, DÄNICKE S, KAUFFOLD J, VAHLENKAMP T W. The mycotoxin deoxynivalenol (DON) can deteriorate vaccination efficacy against porcine reproductive and respiratory syndrome virus (PRRSV) at subtoxic levels. Porcine Health Management, 2022, 8(1): 13. doi: 10.1186/s40813-022-00254-1 pmid: 35307023
[14]	XIAO H P, QIN Z H, XU B C, LONG M, WU Q H, GUO X Y, ZHANG H Y, LI Z L, WU W D. Bacillus amyloliquefaciens B10 alleviates the immunosuppressive effects of deoxynivalenol and porcine circovirus type 2 infection. Toxins, 2024, 16(1): 14.
[15]	LIU D D, GE L, WANG Q, SU J R, CHEN X X, WANG C F, HUANG K H. Low-level contamination of deoxynivalenol: A threat from environmental toxins to porcine epidemic diarrhea virus infection. Environment International, 2020, 143: 105949.
[16]	HU L L, LIU Y X, YU X T, SUN S C, YANG F L. Deoxynivalenol exposure disturbs the cytoplasmic maturation in porcine oocytes. Ecotoxicology and Environmental Safety, 2024, 285: 117137.
[17]	GU X L, GUO W Y, ZHAO Y J, LIU G, WU J E, CHANG C. Deoxynivalenol-induced cytotoxicity and apoptosis in IPEC-J2 cells through the activation of autophagy by inhibiting PI3K-AKT-mTOR signaling pathway. ACS Omega, 2019, 4(19): 18478-18486. doi: 10.1021/acsomega.9b03208 pmid: 31720552
[18]	LI C, LIU J X, ZHANG X, WEI S N, HUANG X H, HUANG Y H, WEI J G, QIN Q W. Fish autophagy protein 5 exerts negative regulation on antiviral immune response against iridovirus and nodavirus. Frontiers in Immunology, 2019, 10: 517. doi: 10.3389/fimmu.2019.00517 pmid: 30941145
[19]	KHALID T, HASAN A, FATIMA J E, AHMAD FARIDI S, KHAN A F, MIR S S. Therapeutic role of mTOR inhibitors in control of SARS-CoV-2 viral replication. Molecular Biology Reports, 2023, 50(3): 2701-2711.
[20]	DIAO F F, JIANG C L, SUN Y Y, GAO Y N, BAI J, NAUWYNCK H, WANG X W, YANG Y Q, JIANG P, LIU X. Porcine reproductive and respiratory syndrome virus infection triggers autophagy via ER stress-induced calcium signaling to facilitate virus replication. PLoS Pathogens, 2023, 19(3): e1011295.
[21]	GU H, QIU H, YANG H T, DENG Z F, ZHANG S K, DU L Y, HE F. PRRSV utilizes MALT1-regulated autophagy flux to switch virus spread and reserve. Autophagy, 2024, 20(12): 2697-2718.
[22]	LI J, ZHOU Y R, ZHAO W K, LIU J, ULLAH R, FANG P X, FANG L R, XIAO S B. Porcine reproductive and respiratory syndrome virus degrades DDX10 via SQSTM1/p62-dependent selective autophagy to antagonize its antiviral activity. Autophagy, 2023, 19(8): 2257-2274.
[23]	KOLETSI P, SCHRAMA J W, GRAAT E A M, WIEGERTJES G F, LYONS P, PIETSCH C. The occurrence of mycotoxins in raw materials and fish feeds in Europe and the potential effects of deoxynivalenol (DON) on the health and growth of farmed fish species-a review. Toxins, 2021, 13(6): 403.
[24]	GONG X Y, LIANG Y, WANG J J, PANG Y P, WANG F, CHEN X H, ZHANG Q Y, SONG C C, WANG Y H, ZHANG C L, FANG X T, CHEN X. Highly pathogenic PRRSV upregulates IL-13 production through nonstructural protein 9-mediated inhibition of N6-methyladenosine demethylase FTO. Journal of Biological Chemistry, 2024, 300(4): 107199.
[25]	DUAN H, CHEN X, ZHANG Z W, ZHANG Z J, LI Z H, WANG X J, ZHAO J K, NAN Y C, LIU B Y, ZHANG A K, SUN Y N, ZHAO Q. A nanobody inhibiting porcine reproductive and respiratory syndrome virus replication via blocking self-interaction of viral nucleocapsid protein. Journal of Virology, 2024, 98(1): e0131923.
[26]	PIERRON A, VATZIA E, STADLER M, MAIR K H, SCHMIDT S, STAS M R, DÜRLINGER S, KREUTZMANN H, KNECHT C, BALKA G, LAGLER J, ZARUBA M, RÜMENAPF T, SAALMÜLLER A, MAYER E, LADINIG A, GERNER W. Influence of deoxynivalenol-contaminated feed on the immune response of pigs after PRRSV vaccination and infection. Archives of Toxicology, 2023, 97(4): 1079-1089. doi: 10.1007/s00204-023-03449-9 pmid: 36781434
[27]	LI S F, ZHOU A, WANG J X, ZHANG S J. Interplay of autophagy and apoptosis during PRRSV infection of Marc145 cell. Infection, Genetics and Evolution, 2016, 39: 51-54. doi: S1567-1348(16)30010-7 pmid: 26774368
[28]	ZHAO X F, AN X D, YANG C Q, SUN W J, JI H Y, LIAN F M. The crucial role and mechanism of insulin resistance in metabolic disease. Frontiers in Endocrinology, 2023, 14: 1149239.
[29]	KOWALSKA K, KOZIEŁ M J, HABROWSKA-GÓRCZYŃSKA D E, URBANEK K A, DOMIŃSKA K, PIASTOWSKA-CIESIELSKA A W. Deoxynivalenol induces apoptosis and autophagy in human prostate epithelial cells via PI3K/Akt signaling pathway. Archives of Toxicology, 2022, 96(1): 231-241.
[30]	ZHUO J J, WANG C, KAI Y L, XU Y Y, CHENG K J. The role of autophagy regulated by the PI3K/AKT/mTOR pathway and innate lymphoid cells in eosinophilic chronic sinusitis with nasal polyps. Immunity, Inflammation and Disease, 2024, 12(6): e1310.
[31]	ZHAO Y J, GUO W Y, GU X L, CHANG C, WU J E. Repression of deoxynivalenol-triggered cytotoxicity and apoptosis by mannan/β- glucans from yeast cell wall: Involvement of autophagy and PI3K- AKT-mTOR signaling pathway. International Journal of Biological Macromolecules, 2020, 164: 1413-1421.
[32]	LIU B J, LUO L Z, SHI Z Q, JU H B, YU L X, LI G X, CUI J. Research progress of porcine reproductive and respiratory syndrome virus NSP2 protein. Viruses, 2023, 15(12): 2310.

稀释梯度 Dilution number	出现CPE孔数 Number of CPE holes	未出现CPE孔数 The number of CPE holes did not appear	累计Total		出现CPE孔占比 The percentage of CPE holes appeared (%)
稀释梯度 Dilution number	出现CPE孔数 Number of CPE holes	未出现CPE孔数 The number of CPE holes did not appear	CPE孔数 Number of CPE holes	无CPE孔数 None number of CPE holes	出现CPE孔占比 The percentage of CPE holes appeared (%)
10^-1	8	0	46	0	100（46/46）
10^-2	8	0	38	0	100（38/38）
10^-3	8	0	30	0	100（30/30）
10^-4	8	0	22	0	100（22/22）
10^-5	8	0	14	0	100（14/14）
10^-6	5	3	6	3	67（6/9）
10^-7	1	7	1	10	9（1/11）
10^-8	0	8	0	18	0（0/18）