Vero E6 and IPEC-J2 cells were maintained in our laboratory and cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, Grand Island, NY, USA) under a humidified atmosphere of 5% CO₂ at 37°C.

GAPDH Polyclonal antibody (10494-1-AP), p107 (13354-1-AP), and E2F4 (10923-1-AP) were obtained from Proteintech Group (Wuhan, China). Monoclonal antibodies (MAbs) targeting the PEDV N protein (JN1401) were purchased from JNT (Beijing, China). HRP-labeled Goat Anti-Rabbit IgG (A0208) and HRP-labeled Goat Anti-Mouse IgG (A0216) were purchased from Beyotime (Shanghai, China). Antibodies against p53 (2524) and p21 (2947) were purchased from Cell Signaling Technology (Danvers, MA, USA). Antibodies targeting p130 (ab76234), cyclin A (ab32386), p130 (phospho-S672) (ab76255), and p130 (phospho-S952) (ab68136) were obtained from Abcam (Cambridge, UK). Saxifrage hay was obtained from Beijing Tongrentang (Beijing, China).

The PEDV strain LW/L was previously isolated and is maintained in our laboratory. Virus was propagated and titrated in Vero E6 cells cultured in DMEM supplemented with 10 μg/mL trypsin (Gibco, Grand Island, NY, USA) under standard incubation conditions. Briefly, 96-well plates were seeded with a monolayer of Vero E6 cells before being infected with serially diluted PEDV (10^–1 to 10^–10). The Reed-Muench method was used to calculate the viral titer, expressed as the 50% tissue culture infective dose (TCID₅₀), the virus titer of the strain used in this study was calculated to be 10^-6.11TCID₅₀/0.1 mL. For infection assays. Vero E6 cells were infected with the PEDV strain at a multiplicity of infection (MOI) of 0.1.

Dried S. stolonifera was cut into pieces and soaked in deionized water until fully saturated. The mixture was then brought to a boil, followed by a reduction to a gentle simmer to facilitate the decoction process. After the initial boiling, the liquid was filtered through gauze to separate the plant material. The remaining plant residue was subjected to a second decoction under the same conditions, and the two resulting filtrates were combined. Finally, the combined filtrate was concentrated using a vacuum rotary evaporator to obtain the final extract (Wu et al., 2015).

100 μL of cell suspension was homogeneously inoculated into 96-well plates, mixed and placed in a 5% CO₂ incubator at 37°C until monolayer; cell control wells and cell-free blank wells were also set up. The prepared S. stolonifera was diluted with DMEM medium and filtered using a 0.22 μm filter. The medium was discarded, and different concentrations of S. stolonifera were added to the 96-well plate at 100 μL/well, with three replicates for each concentration. After 12 h of drug incubation, the culture solution was discarded, D-Hanks was rinsed twice, and finally 100 μL/well of DMEM culture solution (containing 10% CCK-8 solution) was added, and the incubator was incubated in the light protected for 2 h. The absorbance was measured by an enzyme marker at 450 nm. Half cell inhibition concentration (50% inhibition concentration, IC₅₀) was calculated using GraphPad Prism 9.0 software. Cell viability (%) = (OD administration - OD blank)/(OD control - OD blank) × 100%.

We employed five distinct experimental strategies to ascertain the phase of viral inhibition by S. stolonifera during PEDV infection. In the all-treatment protocol, cells were exposed to S. stolonifera for a duration of 14 h. In the pre-treatment protocol, cells were exposed to S. stolonifera for a duration of 1 h prior to the introduction of PEDV. Subsequently, the cells were incubated for an additional 12 h. The co-treatment approach involved the simultaneous inoculation of cells with PEDV, accompanied by S. stolonifera treatment for 13 h. The post-treatment methodology commenced with the inoculation of cells with PEDV for 1 h. Post-incubation, the inoculum was aspirated, and the cells were treated with S. stolonifera for a subsequent 12 h period. The direct-treatment approach involved the simultaneous inoculation of cells with PEDV, accompanied by S. stolonifera treatment for 1 h.

For virus absorption assessment, cells were seeded in 6-well plates and after pre-cooling at 4°C for 30 min in a refrigerator, the virus maintenance solution was used to infiltrate three times, and a mixture of PEDV LW/L and 200 μg/mL S. stolonifera was added and adsorbed for 2 h at 4°C. To evaluate virus internalization, after pre-cooling at 4°C refrigerator for 30 min, the virus maintenance solution was used to infiltrate 3 times, PEDV LW/L was added and incubated at 4°C for 2 h. D-Hank’s was used to infiltrate 3 times, and 200 μg/mL of S. stolonifera was added and incubated for 3 h at 37°C. For viral replication assessment, virus maintenance solution was first used to moisten and wash 3 times, inoculated with PEDV LW/L for 1 h. D-Hank’s was used to infiltrate 3 times, and the virus maintenance solution was incubated for 3 h. The virus was discarded, and then added to 200 μg/mL S. stolonifera for 12 h of incubation. Viral release was evaluated by using virus maintenance solution to infiltrate 3 times, inoculated with PEDV LW/L for 1 h, washed 3 times with D-Hank’s, incubated with virus maintenance solution for 5 h and discarded, and then added with 200 μg/mL S. stolonifera and incubated up to 8 h. Negative and positive control groups were set up at the same time, and cells were collected at the end of the treatment, and the relative expression of PEDV N mRNA was detected using fluorescence quantitative PCR method.

The proteins were extracted with RIPA lysate at a ratio of 1:100 (V/V), separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and transferred onto a PVDF membrane. After the transfer, the membrane was slightly cooled to room temperature, and the PVDF membrane was first washed with ultrapure water for a suitable period of time and then discarded, and then selected to add 5% BSA blocking solution and incubated at room temperature for 1 h, or 1× rapid blocking solution and incubated at room temperature for 20 min, respectively. the primary antibody was incubated overnight at 4°C. The primary antibody was incubated with TBST solution and then incubated with TBST solution. The primary antibody was incubated overnight at 4°C and was then washed three times with TBST solutio. Finally, the corresponding secondary antibody was added (1:1000) and incubated on a shaker at room temperature for 1 h. The membranes were then washed using TBST solution, and protein visualization was achieved by using the enhanced chemiluminescence (ELC) reagents. Target protein expression levels were analysed using ImageJ software (National Institutes of Health, Bethesda, MD, USA).

The relevant components of S. stolonifera were searched through the Herbal Etymology Review (HERB) platform(http://herb.ac.cn/), and eligible compounds were selected through the SWISSADME platform(http://www.swissadme.ch/) on the basis of the Linpinski 5 principle; The target proteins were searched in TCMSP (https://lilab-ecust.cn/pharmmapper/index.html) and PharmMapper (https://lilab-ecust.cn/pharmmapper/index.html) databases according to their composition, and the database information was summarized and duplicates were deleted. Filtering target information in the UniProt protein database (https://www.uniprot.org/). The GeneCards database(https://www.genecards.org/) was used to obtain the disease-related targets. After identifying the overlapping genes, protein-protein interactions were studied using the STRING protein interaction database(https://cn.string-db.org/) to exclude proteins that do not connect to other proteins, and the strength of the protein interactions was used as a basis for identifying core protein targets. GO and KEGG enrichment analyses of potential targets of S. stolonifera were performed and visualized using the Microbiotics online website(https://www.bioinformatics.com.cn/).

The whole cell lysates were added lysis/rinsing buffer (containing 1% protease inhibitor mixture) and mix thoroughly, and were then centrifuged to collect the supernatant (4°C, 12,000 × g, 10 min) for the preparation of Input protein samples, in which the antibody was added and incubated overnight at 4°C to allow the formation of antigen-antibody complexes. Protein A/G magnetic bead suspensions were prepared in clean 1.5 mL centrifuge tubes by adding lysis/rinsing buffer and gently blowing to resuspend the beads. The tubes were then placed on a magnetic rack to ensure complete adsorption of the beads to the tube side, after which the supernatant was discarded. The antigen-antibody complexes were added to the pre-treated magnetic beads and incubate at 4°C for 4~6 h. After incubation, the magnetic beads were fully adsorbed and separated, and the supernatant was discarded. The remaining sample constituted the antigen-antibody-magnetic bead complex. The beads were resuspended in lysis/rinsing buffer, placed on the magnetic rack to adsorb to the side wall of the tube, and the supernatant was discarded. This wash step was repeated twice. To elute the proteins, an appropriate volume of PBS and 5× SDS-PAGE sample buffer was added to the washed complex, mixed well, and heated in a metal bath for 10 minutes. After cooling to room temperature, the tube was placed on a magnetic rack to adsorb the magnetic beads, and the supernatant was collected for Western blot analysis.

At the end of the cell treatment, the culture medium was discarded, and the total RNA was extracted according to the Accurate Biology Steady Pure Rapid RNA Extraction Kit, and the concentration was measured using the Nano Drop 2000. The extracted total RNA was reverse transcribed into cDNA using Accurate Biology Reverse Transcription Premix Kit. cDNA was obtained at the end of the reaction and can be stored at -20°C. The real-time fluorescence quantitative PCR was performed using a premixed qPCR kit from Accurate Biology. All primers for the test were commissioned to be synthesised by Sangon Biotech, as shown in Table 1 .

The 3D structure of the PEDV N protein was predicted using I-TASSER (https://zhanggroup.org/I-TASSER/), The corresponding small-molecule ligand was retrieved from the PubChem database (https://pubchem.ncbi.nlm.nih.gov/). The protein structure was preprocessed by adding hydrogen atoms and modifying amino acid residues using AutoDock Vina 1.2.0., The docking search space was defined using the Grid module, with the center coordinates set to (X=120, Y=134, Z=109) and the grid box size set to 165×135×108. A semi-flexible docking approach was employed, in which the ligand was flexible and the receptor was kept rigid. The docking conformation with the lowest binding energy was visualized and analyzed by PyMOL 2.3.0 and LigPlot 2.2.8 software. Sequence alignment analysis by using WeMol (https://wemol.wecomput.com/ui/#/frontend/home/navigator-board) (Katoh et al., 2005).

All experiments were repeated at least three times and the calculated results were presented as means ± standard error of mean (SEM). Data were analysed and processed using GraphPad Prism 9.0 and statistical analyses were performed using one-way ANOVA or student’s t-test. p<0.05 indicates a significant difference, p<0.01 indicates a highly significant difference and p>0.05 indicates no statistical significance.

To evaluate the effects of S. stolonifera on PEDV infection, Vero E6 and IPEC-J2 cell lines were employed as in vitro models. IPEC-J2 cells, derived from the piglet small intestine, were chosen for their relevance to the primary infection sites, where the virus peaks, causing severe intestinal cell necrosis and detachment. Cytotoxicity assays showed a dose-dependent relationship between S. stolonifera concentration and cytotoxic effects on Vero E6 and IPEC-J2 cells after 12 h of treatment. Concentrations of S. stolonifera above 400 μg/mL significantly reduced cell viability, while treatments within the range of 0–400 μg/mL were well-tolerated. Both cell types maintained their morphological integrity and high viability, with Vero E6 cell viability consistently exceeding 80% ( Figure 1A ). The IC₅₀ of S. stolonifera in Vero E6 cells was determined to be 1091 μg/mL ( Figure 1B ), highlighting the lack of significant cytotoxicity at lower concentrations. Similarly, IPEC-J2 cells showed no significant morphological changes at S. stolonifera concentrations below 400 μg/mL after 12 h of treatment, with cell viability consistently exceeding 80% ( Figure 1C ). The IC₅₀ of S. stolonifera in IPEC-J2 cells was calculated as 1015 μg/mL ( Figure 1D ).

Several studies have demonstrated that various drug administration modes influence the antiviral efficacy of treatments (Chen et al., 2020; Liu et al., 2021). To determine the most effective method of S. stolonifera treatment, various administration modes were evaluated based on their antiviral efficacy ( Figure 1E ). The All-treatment, Co-treatment, and Post-treatment administration modes significantly reduced PEDV N mRNA expression levels (p<0.01), while Pre-treatment and Direct-treatment showed no significant inhibitory effects on PEDV (p>0.05) ( Figures 1F–J ). The results suggest that the Post-treatment mode has the strongest inhibitory effect on viral replication, demonstrating its efficacy in the shortest intervention period. Consequently, this administration mode was identified as optimal for managing PEDV infection in subsequent experiments.

To investigate the effects of varying S. stolonifera concentrations on PEDV N gene expression, in vitro assays were performed with different S. stolonifera concentrations applied to Vero E6 cells after a 12-hour exposure period. S. stolonifera significantly suppressed PEDV N gene expression at 200 and 400 μg/mL compared to the PEDV-infected control group (p<0.01) ( Figure 2A ). This suppressive effect was corroborated in IPEC-J2 cells, where S. stolonifera at 100, 200, and 400 μg/mL significantly reduced PEDV N gene expression (p<0.05) ( Figure 2B ). Collectively, these findings highlight that S. stolonifera at 200–400 μg/mL exhibits a potent in vitro inhibitory effect on PEDV, reducing PEDV N gene expression in a dose-dependent manner. To systematically evaluate the effect of S. stolonifera on PEDV N protein expression, comparative analyses were performed at varying concentrations. In Vero E6 cells, S. stolonifera at 200 and 400 μg/mL significantly reduced PEDV N protein expression compared to the PEDV-infected controls (P<0.05) ( Figures 2C, D ). This inhibitory effect was substantiated in IPEC-J2 cells ( Figures 2E, F ). In summary, S. stolonifera exerts a dose-dependent inhibitory effect on PEDV N protein expression in vitro, demonstrating significant antiviral activity.

To investigate the antiviral efficacy of S. stolonifera and its mechanism of action in the PEDV life cycle, we quantified viral N gene expression during key phases: adsorption, invasion, replication, and release ( Figure 3A ). Results revealed that 200 μg/mL S. stolonifera significantly inhibited the replication stage, reducing PEDV N gene mRNA expression in Vero E6 cells (p<0.01) ( Figures 3B–E ). However, this concentration showed no significant antiviral activity during adsorption, invasion, or release stages. Similar results were found in IPEC-J2 cells, where 200 μg/mL S. stolonifera significantly suppressed PEDV replication (p<0.05) but had negligible effects on other viral life cycle stages ( Figures 3F–I ). Next, we examined the effect of S. stolonifera on N protein expression during PEDV replication. Data showed that 200 μg/mL S. stolonifera significantly reduced PEDV N protein expression in Vero E6 cells (p<0.0001) ( Figures 3J, K ). This inhibitory effect was also observed in IPEC-J2 cells, where 200 μg/mL S. stolonifera significantly reduced PEDV N protein expression during replication (p=0.0010) ( Figures 3L, M ).

Viral replication relies on host cells as the main energy source. Infected cells, in turn, activate the host’s innate immune system through a cascade of responses. This activation alters the expression of inflammatory cytokines and chemokines (Nguyen et al., 2021). Research shows that PEDV infection induces distinct cytokine profiles in host cells. Identifying the specific cytokines and chemokines involved is crucial for understanding the host’s immune response to viral infection (Huang et al., 2024). To investigate S. stolonifera’s antiviral mechanism, we used Real-time PCR to measure the transcriptional levels of key inflammatory cytokines— (TNF-α, IL-1β, IL-6, IL-8, and IL-10)—in PEDV-infected cells after S. stolonifera treatment. Results showed significant upregulation of TNF-α, IL-1β, IL-6, and IL-8 mRNA levels in Vero E6 cells post-infection. Notably, S. stolonifera at 200 μg/mL significantly reduced this upregulation (p<0.01) ( Figures 4A–D ). Moreover, the same concentration of S. stolonifera significantly increased the expression level of IL-10 mRNA ( Figure 4E ). Validation in IPEC-J2 cells confirmed the upregulation of TNF-α, IL-1β, IL-6, and IL-8 mRNA levels following PEDV infection. S. stolonifera at 200 μg/mL significantly reduced TNF-α and IL-6 mRNA levels but had no significant effect on IL-1β and IL-8 expression ( Figures 4F–I ). Additionally, S. stolonifera significantly upregulated IL-10 expression, highlighting its immunomodulatory potential ( Figure 4J ). These results suggest that PEDV induces pro-inflammatory cytokine expression, while S. stolonifera modulates inflammation by reducing pro-inflammatory factors and increasing IL-10 levels, demonstrating its potential anti-inflammatory effects.

The shared targets between the drug components and disease were depicted using Venn diagrams, identifying 55 common targets, which were subsequently imported into the String database ( Figure 5A ). Cytoscape software was utilized to visualize the protein-protein interaction (PPI) network of S. stolonifera and identify the direct and indirect regulation of the targets ( Figure 5B ). Using the Cytoscape plugin CentiScaPe 2.2, core targets including IL10, CCL2, IL6, IFNG, IL1B, CRP, CASP3, HMOX1, and PPARG were identified based on Betweenness unDir, Closeness unDir, and Degree unDir metrics ( Figure 5C ). The corresponding components of these targets were deduced as focal gallic acid, quercetin, coumarin, caffeic acid, arbutin, succinic acid, 6,7-dihydroxycoumarin, gallic acid, and hydroquinone. GO and KEGG pathway enrichment analyses revealed that S. stolonifera combats PEDV infection via the MAPK, TNF, PI3K-Akt, JAK-STAT, and p53 signaling pathways ( Figures 5D, E ).

Previous studies have shown that the binding of the PEDV N protein to p53 sustains elevated nuclear p53 levels, mediating S-phase arrest through activation of the p53-DREAM pathway (Su et al., 2021). This interaction highlights a potential antiviral strategy targeting the host-virus interface. Using a Co-Immunoprecipitation (Co-IP) assay, we explored the effect of S. stolonifera on the molecular interaction between the PEDV N protein and the tumor suppressor p53 in Vero E6 cells. We found that 200 μg/mL S. stolonifera significantly reduced the formation of the complex between the PEDV N protein and p53 (p<0.0003) ( Figures 6A, B ). Moreover, this treatment effectively inhibited PEDV replication, as shown by comparison with the PEDV-infected control group, highlighting the antiviral potential of S. stolonifera. The inhibitory effect of S. stolonifera on the interaction between the PEDV N protein and p53 was further confirmed in IPEC-J2 cells ( Figures 6C, D ). These results demonstrate the broad applicability of the antiviral mechanism of S. stolonifera, emphasizing its potential as a therapeutic option for various cell types affected by PEDV infection.

We conducted a thorough evaluation of S. stolonifera effects on the expression dynamics of proteins involved in the p53-DREAM signaling cascade using Western blot analysis ( Figure 7A ). Our analysis revealed a marked upregulation of p53 and its downstream effector p21 upon PEDV infection ( Figures 7B, C ). We also observed a significant downregulation in the levels of phosphorylated p130 at Ser672, E2F4, and Cyclin A following infection ( Figures 7F, H, I ). In contrast, the expression of p107, total p130, and p130 phosphorylated at Ser952 remained unchanged after PEDV infection (p>0.05) ( Figures 7D, E, G ). Interestingly, treatment with 200 μg/mL S. stolonifera significantly reduced the expression levels of p53 and p21 while robustly upregulating phosphorylated p130 (Ser672), E2F4, and Cyclin A, suggesting a potent regulatory effect on the p53-DREAM signaling pathway. These effects of S. stolonifera were also characterized by downregulation of p53 and p21 in IPEC-J2 cells ( Figures 8A–C ), alongside upregulation of phosphorylated p130 (Ser672), E2F4, and Cyclin A ( Figures 8F, H, I ). These coordinated actions suggest a complex mechanism by which S. stolonifera may suppress viral replication via the p53-DREAM signaling pathway.

The PEDV N protein is abundantly expressed during the early stages of viral infection, and pigs produce high levels of anti-N protein antibodies, which are highly conserved across different PEDV strains (Ma et al., 2019). Consequently, the N protein serves as a valuable tool for exploring the mechanisms by which host antiviral genes inhibit PEDV replication, offering new insights for PEDV prevention and control. Molecular docking was employed to investigate the binding sites of core S. stolonifera components with the N protein. The results were visualized using Pymol, with smaller free binding energy indicating a more stable bound conformation and a higher likelihood of interaction. Binding activity was considered favorable when the energy was ≤-5.0 kcal-mol^-1. The analysis revealed that arbutin, quercetin, caffeic acid, coumarin, gallic acid, pyrogallol, and esculetin exhibited strong binding activity with the N protein. Additionally, the amino acid residues at the docking sites were visualized using Ligplot software ( Figures 9A–G ). Five N gene sequences were selected for comparison (Wang et al., 2021; Guo et al., 2024). The CV777 and SC1402 strains belong to genotype GI, while the other three strains are classified as epidemic or virulent strains of genotype GII. We found that the amino acids involved in hydrogen bonding at the docking site between S. stolonifera and the N protein are conserved across these sequences ( Figure 9H ). These findings suggest that S. stolonifera may bind to conserved amino acids via hydrogen bonding with the N protein, thereby inhibiting infection.