Scientia Agricultura Sinica ›› 2025, Vol. 58 ›› Issue (13): 2693-2706.doi: 10.3864/j.issn.0578-1752.2025.13.015

• ANIMAL SCIENCE·VETERINARY SCIENCE • Previous Articles    

Co-Exposure of DON and PRRSV Induces Autophagy in PAM-KNU Cells Through the PI3K/AKT/mTOR Signaling Pathway

WANG LinYuan(), XUAN JingYan, DU Yu, CHEN Tong, NIU RuiYan()   

  1. Laboratory of Environmental Veterinary Medicine, College of Veterinary Medicine, Shanxi Agricultural University, Taigu 030801, Shanxi
  • Received:2025-02-01 Accepted:2025-05-30 Online:2025-07-01 Published:2025-07-05

RichHTML

6

PDF

13

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 107.3 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

Fig. 1

PRRSV virus identification a: Marc-145 cells (1 μm) uninoculated with PRRSV; b: Marc-145 cells (1 μm) inoculated with PRRSV; c: Marc-145 cell supernatant PRRSV-N nucleic acid gel electrophoresis assay; d: Western Blot analysis of Marc-145 cell supernatant; e: Western Blot analysis of Marc-145 cell precipitate"

Table 1

The virulence test results of PRRSV on PAM-KNU cells"

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 (%)
CPE孔数
Number of CPE holes		 无CPE孔数
None number of CPE holes
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）

Fig. 2

Growth kinetics of PRRSV in PAM-KNU cells a: Western Blot of protein bands; b: The statistics of protein bands; c: Virus titer in supernatant"

Fig. 3

Effect of DON on cell viability of PAM-KNU a: DON's half toxic concentration of PAM-KNU; b: Effects of different concentrations of DON on the viability of PAM-KNU cells"

Fig. 4

Effect of DON on PRRSV replication in PAM-KNU cells a: Effect of different concentrations of DON on the activity of PRRSV in PAM-KNU cells; b: Western Blot of protein bands; c: The statistics of protein bands; d: Virus titer in supernatant; e: Number of virus copies in the supernatant"

Fig. 5

Effects of DON and PRRSV co-exposure on autophagy of PAM-KNU under electron microscopy (The DON concentration was 128 nmol·L-1) a: Autophagy of group C (1 μm); b: Local magnification of autophagy in group C (500 nm); c: Autophagy of group D (1 μm); d: Local magnification of autophagy in group D (500 nm); e: Autophagy diagram of group V (1 μm); f: Local magnification of autophagy in group V (500 nm); g: Autophagy of VD group (1 μm); h: Partial magnification of autophagy in VD group (500 nm)"

Fig. 6

Western Blot analysis of the effects of co-exposure with DON and PRRSV on autophagy associated proteins of PAM-KNU a: The statistics of LC3-Ⅱ protein bands; b: The statistics of Beclin 1 protein bands; c: The statistics of p62 protein bands; d: The statistics of ULK 1 protein bands"

Fig. 7

Indirect immunofluorescence detection of the effects of co-exposure with DON and PRRSV on autophagy associated proteins of PAM-KNU a: Indirect immunofluorescence map of LC3 protein; b: Indirect immunofluorescence map of Beclin 1 protein"

Fig. 8

Proteome sequencing a: Total protein quantity identification map; b: PCA principal component analysis diagram; c: C.vs.D, C.vs.V, V.vs.VD differential protein volcano map (red for high expression, green for low expression, gray for no significant difference); KEGG enrichment analysis of d: C.vs.D, C.vs.V, V.vs.VD differential proteins"

Fig. 9

Western Blot analysis of the effects of co-exposure with DON and PRRSV on PI3K/AKT/mTOR pathway-related proteins a: The statistics of p-PI3K/PI3K protein bands; b: The statistics of p-AKT protein bands; c: The statistics of p-mTOR/mTOR protein bands"

Fig. 10

Indirect immunofluorescence detection of the effects of co-exposure with DON and PRRSV on PI3K/AKT/mTOR pathway-related proteins a: Indirect immunofluorescence map of p-PI3K protein; b: Indirect immunofluorescence map of p-AKT protein; c: Indirect immunofluorescence image of p-mTOR protein"

[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.
[1] CHEN LongYun, HU JunQiang, HE Can, SHI JianRong, XU JianHong, WANG Gang. Purification of Deoxynivalenol-3-Glucoside by Using Macroporous Adsorption Resin Combined with High-Speed Counter-Current Chromatography [J]. Scientia Agricultura Sinica, 2025, 58(8): 1627-1637.
[2] LI Rui, LIANG Yue, BAI Yang, ZHANG GuiYue, WANG NanNan, QIAO SongLin, ZHANG GaiPing. Research Progress on the Roles and Mechanisms of Autophagy Involved in Porcine Reproductive and Respiratory Syndrome Virus Infection [J]. Scientia Agricultura Sinica, 2025, 58(4): 792-801.
[3] XU YuanYuan, HUANG LiQing, LU XingRong, FENG Chao, SHANG JiangHua. Effects of β-mercaptoethanol on Rapamycin Induced Autophagy, Apoptosis and Steroid Hormone Synthesis in Buffalo Granulosa Cells [J]. Scientia Agricultura Sinica, 2025, 58(11): 2265-2274.
[4] GE Yi, ZHENG QiuLing, CHEN MengXia, XIA JiaXin, FANG Xiang, TANG MeiLing, FANG JingGui, SHANGGUAN LingFei. Cloning and Functional Analysis of the Autophagy Gene ATG8f in the Grapevine [J]. Scientia Agricultura Sinica, 2025, 58(1): 156-169.
[5] LI WenHui,HE YiJing,JIANG Yao,ZHAO HongYu,PENG Lei,LI Jia,RUI Rong,JU ShiQiang. Effects of FB1 on Apoptosis and Autophagy of Porcine Oocytes in vitro Maturation [J]. Scientia Agricultura Sinica, 2022, 55(6): 1241-1252.
[6] Xin ZHANG,KongLin HUO,XingXing SONG,DuoNi ZHANG,Wen HU,ChuanHuo HU,Xun LI. Effects of GnIH on Autophagy and Apoptosis of Porcine Ovarian Granulosa Cells via p38MAPK Signaling Pathway [J]. Scientia Agricultura Sinica, 2020, 53(9): 1904-1912.
[7] ZHANG Yong, HAO Yuan-feng, ZHANG Yan, HE Xin-yao, XIA Xian-chun, HE Zhong-hu. Progress in Research on Genetic Improvement of Nutrition and Health Qualities in Wheat [J]. Scientia Agricultura Sinica, 2016, 49(22): 4284-4298.
[8] SUN Hong, ZHANG Wei, WEI Xiao-Jing, WANG Hua-Zhong. Identification of Wheat Key Autophagy-Related Factor ATG18 and Profiling of Their Expression Under Biotic and Abiotic Stresses [J]. Scientia Agricultura Sinica, 2014, 47(9): 1657-1669.
[9] SHI Jian-rong, LIU Xin, QIU Jian-bo, JI Fang, XU Jian-hong, DONG Fei, YIN Xian-chao, RAN Jun-jian. Deoxynivalenol Contamination in Wheat and Its Management [J]. Scientia Agricultura Sinica, 2014, 47(18): 3641-3654.
[10] CHENG Xing-An, QIN Xiang-Jing, JIANG Xu-Hong, Sammy ZHENG, LIU Zhan-Mei, LIU Xiang-Dong. Cytological Mechanism of Autophagosome Biogenesis During Cell Autophagic Apoptosis in Rice and Insect Cell [J]. Scientia Agricultura Sinica, 2013, 46(5): 871-880.
[11] XU Jian-Hong, PAN Yan-Mei, HU Xiao-Dan, JI Fang, YIN Xian-Chao, SHI Jian-Rong. Enzymatic Characteristics of 3-Acetyl Deoxynivalenol Oxidase by Devosia sp. DDS-1 [J]. Scientia Agricultura Sinica, 2013, 46(11): 2240-2248.
[12] XU Jian-hong,JI Fang,WANG Hong-jie,WANG Jian-wei,LIN Fan-yun,SHI Jian-rong
. Isolation and Identification of Deoxynivalenol Degradation Strains
 [J]. Scientia Agricultura Sinica, 2010, 43(22): 4635-4641 .
Viewed
Full text


Abstract

Cited
  Shared   
  Discussed   
No Suggested Reading articles found!
京ICP备09089781号-30
Copyright 2018 © Editorial office of Scientia Agricultura Sinica
Tel: 86-010-82106279    E-mail: zgnykx@mail.caas.net.cn
Supported by Beijing Magtech Co.Ltd