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

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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"

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