Mechanism research of Punicalagin in treating representative strains of enterovirus A and B types based on systems pharmacology and experimental validation

Abstract

Background

Enteroviruses (EVs), particularly types A (e.g., EV-A71) and B (e.g., CVB3), cause severe complications in vulnerable populations. Limited vaccines and no antivirals underscore the need for broad-spectrum therapies. Punicalagin, a natural anti-inflammatory compound, was investigated for its pan-enteroviral therapeutic potential.

Objective

To evaluate punicalagin’s efficacy and mechanisms against multiple EV serotypes via integrated systems pharmacology and experimental validation.

Methods

Network pharmacology identified punicalagin’s targets and pathways. In vitro antiviral activity was assessed in Vero/A549 cells infected with EV-A71/CVB3. Neonatal mice were intraperitoneally inoculated with these viruses to test in vivo efficacy. Molecular docking, apoptosis assays, and inflammatory factor analyses elucidated mechanisms.

Results

Punicalagin inhibited EV-A71 and CVB3 replication in vitro and improved survival in infected mice. Systems pharmacology linked its effects to anti-apoptotic and anti-inflammatory pathways. Molecular docking confirmed interactions with apoptosis/inflammation regulators (e.g., CASP3, TNF-α). Experimental validation demonstrated reduced viral-induced apoptosis and suppressed IL-6/TNF-α levels.

Conclusion

Punicalagin exhibits broad-spectrum anti-enteroviral activity through dual inhibition of apoptosis and inflammation, validated across in vitro, in vivo, and computational models. This study provides a systems-level framework for repurposing natural compounds against phylogenetically diverse EVs, addressing critical therapeutic gaps for high-risk populations.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12985-025-02845-0.

Highlights

In this study, we employed network pharmacology analysis to demonstrate that the targets of Punicalagin were highly correlated with severe complications caused by enterovirus A and B. The predicted target results were further analyzed using GO and KEGG pathways, revealing its potential influence on the inflammatory response and apoptosis process in host cells following viral infection. Through experimental validation, Punicalagin was found to significantly inhibit the cytopathic effects induced by representative strains EV-A71 and CVB3 of enterovirus A and B types in vitro, as well as mitigate clinical symptoms in neonatal mice models in vivo.

Through analyzing the correlation between Punicalagin targets and the top ten predicted pathways, this study identified key action molecules including CASP3, BAD, MAPK14, PRKCA, AKT1, IL1B, TNF, and IL6. Molecular docking analysis was conducted to align these molecules with the ligand-binding pockets of the native proteins, thereby validating that Punicalagin demonstrated varying degrees of affinity for these molecular targets. These findings suggested that its anti-apoptotic and anti-inflammatory activities may underlie its efficacy against major enteroviruses viruses. Experimental validation confirmed that Punicalagin effectively inhibits apoptosis in host cells infected by EV-A71 and CVB3, and reduces the expression of various pro-inflammatory factors.

Through molecular docking analysis of the viral VP1 protein, this study demonstrated that Punicalagin exhibits significant binding affinity with the VP1 protein of EV-A71 and CVB3, indicating its potential to inhibit viral replication. Experimental validation confirmed that Punicalagin effectively suppresses the expression of the viral capsid protein VP1 in host cells infected with both representative strains of Enterovirus A (EV-A71) and Enterovirus B (CVB3).

Supplementary Information

The online version contains supplementary material available at 10.1186/s12985-025-02845-0.

Introduction

The Enterovirus genus, a type of RNA virus capable of encoding RNA-dependent RNA polymerase, which encompasses multiple species, including Enterovirus species A through L and Rhinovirus species A through C [1, 2]. Over one hundred serotypes of human enteroviruses have been identified, some of which are associated with severe diseases such as poliovirus and Coxsackievirus infections. Enteroviruses are non-enveloped viral particles approximately 30 nanometers in diameter, exhibiting stability in acidic environments [3, 4]. Transmission occurs primarily via fecal-oral or respiratory routes. While most adult infections are asymptomatic, enteroviruses can cause severe illnesses such as hand-foot-and-mouth disease and central nervous system disorders in infants, young children, and immunocompromised individuals. In light of the recurrent outbreaks of enteroviruses, the development of vaccines and antiviral drugs assumes critical importance. Despite numerous efforts employing diverse strategies, only the poliovirus vaccine and the EV-A71 vaccine have received clinical approval to date [5, 6]. The considerable diversity of enterovirus serotypes, substantial evolutionary divergence, and ongoing mutations present formidable challenges to vaccine development. The virus’s rapid mutation rate and the complexity associated with developing vaccines for multiple serotypes underscore the urgent need for broad-spectrum antiviral therapies. In contrast to the immediate and visible impact of SARS-CoV-2 on human health, research into enterovirus infections, which predominantly affect children and individuals with compromised immune systems, has often been neglected. This disparity underscores the significant challenges in this field of study. To date, there are no approved drugs specifically targeting enterovirus infections; treatment primarily focuses on alleviating symptoms through symptomatic methods such as antipyretics and antidiarrheal medications.

The multi-target effects of natural medicines offer a promising approach to treating diverse diseases from a holistic perspective [7, 8]. By acting on multiple targets within the human body, natural medicines can simultaneously address various pathological processes, thereby achieving comprehensive therapeutic outcomes. This characteristic positions natural medicines as a valuable resource in modern medicine, particularly for complex diseases that traditional drugs struggle to manage effectively. In this study, we focused on punicalagin, a natural compound known for its anti-inflammatory properties. Through a series of in vitro and in vivo experiments, we investigated the therapeutic potential of punicalagin against major serotypes of enteroviruses that cause severe diseases, specifically coxsackievirus B3 (CVB3) and enterovirus A71 (EV-A71), which are representative of the A and B species of enteroviruses [9, 10]. Utilizing systems pharmacology approaches and corroborating our findings with experimental validation, we thoroughly explored the mechanisms by which punicalagin exerts its antiviral effects against CVB3 and EV-A71.

Materials and methods

Compound and reagents

Punicalagin (HY-N006, MedChemExpress); CCK-8 Cell Counting (A311-01, Vazyme Biotech Co., LTD, Nanjing, China); DMEM (10566016, GIBCO,); FBS (10091155,GIBCO); PBS (10010023,GIBCO); Typsin-EDTA (25200072,GIBCO); F-12 K (21127022, GIBCO); TUNEL BrightRed Apoptosis Detection Kit (A113, Vazyme Biotech Co., LTD, Nanjing, China); AceQ^®UniversalSYBRqPCRMasterMix (Q511, Vazyme Biotech Co., LTD, Nanjing, China); FreeZol Reagent (R711, Vazyme Biotech Co., LTD, Nanjing, China); DMSO (60313ES60, YEASEN, Shanghai, China); Hifair^® II 1st Strand cDNA Synthesis SuperMix (11120ES, YEASEN, Shanghai, China).

Network pharmacology study of punicalagin against enterovirus infection and its subsequent complications

The targets of the punicalagin were retrieved through the SwissTarget database (http://swisstargetprediction.ch/), PharmMapper database (https://www.lilab-ecust.cn/pharmmapper/), CTD database (https://ctdbase.org/) and ETCM database (http://www.tcmip.cn/ETCM/index.php/Home/). A comprehensive search for enterovirus infection and its associated complications was conducted in the Genecards database (https://www.genecards.org/) to identify relevant targets. The search keywords for complications were as follows: Aseptic meningitis; Pulmonary edema; Encephalomyelitis; Myocarditis; Encephalitis; Pneumonia. The targets associated with complications of enterovirus infection were mapped against the drug action targets to identify intersection targets, which were then visualized using a Venn diagram. Subsequently, the overlapping genes were subjected to Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) enrichment analysis using R Studio version 4.4.2. Finally, the “Overlap Genes-GO/KEGG Pathway” network was constructed using Cytoscape version 3.9.1.

Investigation of the interaction between punicalagin and key proteins which play a pivotal role in antiviral signaling pathways via molecular docking

Initially, we retrieved the target protein structures from the PDB database (https://www.rcsb.org/) and the structural formula of punicalagin from the PubChem database (https://pubchem.ncbi.nlm.nih.gov/). Subsequently, we utilized Chem3D 18.1 to convert the two-dimensional structure of punicalagin into a three-dimensional structure. Following this, we employed AutoDock software version 1.5.6 to individually dock punicalagin with the respective target proteins and calculated their binding energies. Ultimately, we performed a visual analysis of the docking results using PyMOL software version 4.6.0. Additionally, the root mean square deviation (RMSD) values were predicted using PyMOL.

Cells and virus

Vero, A549 cell lines were purchased from the National collection of authenticated cell cultures of Chinese Academy of Sciences and cultured in DMEM, or F-12 K medium (Gibco) containing penicillin-streptomycin (1% v/v) and fetal bovine serum (FBS, 10%; Gibco) at 37 °C in an atmosphere of 5% CO2. Wild-type EV-A71 strain (Enterovirus A71 isolate 71/HuN/CHN/2016, GenBank: ON502278.1) and CVB3 strain (strain Nancy, GenBank: JN048468.1) were kept in our laboratory and produced in Vero cells and preserved at − 80 °C. Virus titer (TCID₅₀) was calculated by the Reed-Muench method as described previously.

Cytotoxicity assays

The cells were seeded overnight in 96-well plates. Punicalagin (200–6.25 µM) was added to the cells at various concentrations. Cell viability after 48 h of incubation was measured using Cell Counting Kit-8 (CCK-8), following the manufacturer’s instructions. The cytotoxic concentration (CC₅₀) of compounds was determined through linear regression analysis.

Cytopathic effect (CPE) inhibition

In the preliminary experiments designed to investigate whether punicalagin exhibits antiviral activity, interferon-deficient and virus-susceptible Vero cells were utilized. To assess CPE inhibitory activity of punicalagin, Vero cells were seeded in 96-well plates and cultured overnight. One hour post-viral infection (MOI = 0.1), the supernatant containing the virus was removed, and the cells were washed thoroughly with PBS. Subsequently, varying concentrations of punicalagin (10–0.03125 µM) were added to the wells. Subsequently, Cell viability was determined using the CCK-8 assay at 48 h post-infection to calculate the rate of CPE inhibition. The EC₅₀ values for the compounds were calculated through linear regression analysis of the CPE inhibition curves.

Time of drug addition assay

A549 cells were seeded into 6-well plates and subsequently infected with EV-A71 and CVB3 (MOI = 1). Treatment with 5 µM Punicalagin was administered at three distinct time points: prior to infection (Pre: 6–0 h), post-infection (Post: 1–16 h), and both pre- and post-infection (Pre: 6–0 h + Post: 1–16 h). This experimental design aimed to elucidate the specific stages of the viral life cycle where Punicalagin exerts its inhibitory effects. Following a 16-hour incubation period post-infection, RNA was extracted from the treated cells and quantified via qPCR to determine the VP1 levels of EV-A71 and CVB3.

Quantitative real-time PCR (qRT-PCR)

The RNA was extracted from cells using TRIzol reagent (Sangon, China). The transcripts of various genes were quantified using SYBR Green master mix (vazyme, China) according to the manufacturer’s instructions. Gene transcript levels were determined using the ΔΔCT method. The information of the primers was presented in Table S1.

Apoptosis assay

Apoptosis was evaluated using the TUNEL assay kit (A113, Vazyme, Nanjing, China). Cells were gently washed with phosphate-buffered saline (PBS) (G101, Vazyme, Nanjing, China) and subsequently stained with 4′,6-diamidino-2-phenylindole (DAPI; Catalog No. 40728ES03, YEASON, Shanghai, China). The stained cells were examined under a fluorescence microscope (Model Ti2-U, Nikon, Tokyo, Japan) to quantify the number of apoptotic cells. The mean count of TUNEL-positive cells was determined from three randomly selected microscopic fields.

Mice

The newborn ICR mice (one-day old) were intraperitoneally injected with 106 TCID50 or 105 TCID50 of EV-A71 and CVB3 respectively. The mice were divided into 3 groups (10 mice per group): the Negative Control (NC) group, the Viral Infected group (CVB3/EV-A71), and the Treatment group. After a 2-hour interval, the pups received oral administration of punicalagin (5 mg/kg) in PBS or saline for two consecutive days. The control group received an injection of an equal volume of the solvent. The mice were monitored daily for clinical syndromes and mortality. The study protocol was approved by the Institutional Animal Care and Use Committee of the Institute of Laboratory Animal Science, Jiangsu university with approval number (permit number: IACUC-AP-2023032101).

Histopathological examinations

Histopathological analyses were conducted on brain and heart tissues (Randomly selected surviving mice from the normal control group, the virus-infected group (2 dpi), and the virus-infected group with oral administration (2 dpi), n = 3.) perfused with 10% neutral buffered formalin. The tissues were subsequently paraffin-embedded, sectioned at a thickness of 10 μm, and stained with hematoxylin and eosin (H&E). The resulting slides were digitally scanned and analyzed using a NIKON DS-U3 slide scanner.

Statistical analysis

The data were presented as the mean ± standard deviation (SD). Statistical differences were evaluated using Student’s t-test or analysis of variance (ANOVA). A p-value less than 0.05 was considered statistically significant. The log-rank test was employed for analyzing the Kaplan-Meier survival curves.

Results

The potential of punicalagin as a drug candidate for treating enterovirus infections in immunocompromised individuals was predicted using network pharmacology

Punicalagin, as an anti-inflammatory drug, has been widely reported for its anti-inflammatory activity [11, 12], and its structure is shown in Fig. 1A. By integrating the SwissTarget database, PharmMapper database, and ETCM database, we predicted its action targets, identifying a total of 229 targets, as shown in Fig. 1B. This study focused on exploring the potential of Punicalagin in treating different clinical symptoms caused by various serotypes of viral infections. Therefore, severe cases of various inflammatory types were included in the analysis of severe cases of enterovirus infection in immunocompromised populations. Through the GeneCards database, we integrated disease-related targets for severe cases of enterovirus, especially EV-A71 and CVB3, which are common representative viruses of types A and B, using keywords such as aseptic meningitis, pulmonary edema, encephalomyelitis, myocarditis, encephalitis, and pneumonia. A total of 9,911 targets were identified, as shown in Fig. 1C. By using a Venn diagram to analyze the correlation between candidate drugs and disease targets, we found that more than 80% of the predicted targets of Punicalagin were related to the targets of severe cases after enterovirus infection, suggesting its potential for treating different diseases with the same drug, as shown in Fig. 1D. Further, we conducted GO and KEGG analysis on the intersecting targets to predict the molecular mechanisms of their actions. Through GO enrichment analysis, we found that the drug’s effects were highly related to cell proliferation regulation, inflammatory factor regulation, and apoptosis factor regulation, as shown in Fig. 1E. KEGG signaling pathway analysis further suggested that its mechanism of action was related to multiple molecular pathways such as PI3K-Akt, MAPK, TNF, and IL-17, which are associated with inflammation and apoptosis, as shown in Fig. 1F. Therefore, through network pharmacology, we preliminarily predicted the potential of Punicalagin as a drug for treating different diseases with the same drug. It may address different clinical symptoms caused by viral infections through its anti-inflammatory and anti-apoptotic activities.

Fig. 1.

Network pharmacology-based analyses of the active ingredients of Punicalagin against different serotypes of enterovirus infection. (A) Chemical structure of Punicalagin. (B) Predicted targets of Punicalagin. (C) Genes associated with enterovirus infection syndromes. (D) Venn diagram illustrated the shared targets between Punicalagin and enterovirus infection. (E) Gene Ontology (GO) analysis of the overlapping targets shown in panel (D). (F) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of the overlapping targets shown in panel (D)

Investigation of the antiviral efficacy of Punicalgin against CVB3 and EV-A71 Viruses in both in vitro and in vivo

To verify the application effect of network pharmacology in the treatment of severe models caused by enterovirus A and B, this study selected CVB3 as the representative of Enterovirus B species and EV-A71 as the representative of Enterovirus A species. By establishing virus infection models in susceptible cells Vero and neonatal mice, the alleviating effect of Punicalagin on the pathogenesis symptoms of virus-infected cells and neonatal mice was evaluated. Figure 2A showed the inhibitory effect of Punicalagin on the cytopathic effect (CPE) of CVB3 virus infection, with a half-maximal effective concentration (EC₅₀) of 0.77 µM at 48 h post infection (hpi); for EV-A71 virus infection, the EC₅₀ of Punicalagin was 1.01 µM, as shown in Fig. 2B at 48hpi. Cell morphology observation further visually demonstrated the significant improvement of Punicalagin on the CPE of virus-infected cells, as shown in Fig. 2D. In addition, the 48-hour half-maximal cytotoxic concentration (CC₅₀) of Punicalagin on Vero cells was 128 µM. According to the calculation of the selectivity index (SI), the SI value of this drug for Vero cells infected with the above two viruses was greater than 100, indicating the potential of Punicalagin to be low-toxic and highly effective. Given that this study aimed to focus specifically on the treatment of immunocompromised individuals, particularly children, following enterovirus infection, neonatal mice were selected based on literature references to establish an animal model for viral infection. It was observed that neonatal mice showed clinical symptoms such as cyanosis, paralysis, and even severe death after virus infection. However, after treatment with Punicalagin, the clinical condition of neonatal mice was significantly improved, as shown in Fig. 2E. The survival rate after 7 days post infection (dpi) of administration was calculated by statistical methods, and it was found that Punicalagin significantly increased the survival rate of both virus infections after 7dpi, as shown in Fig. 2F and G.

Fig. 2.

In vitro and in vivo experiments verified the heterologous therapeutic effect of Punicalagin on CVB3 and EV-A71 infections. (A) The inhibitory effect of Punicalagin on the CPE of CVB3-infected Vero cells was detected by the CCK-8 method, and its EC₅₀ was calculated. (B) The inhibitory effect of Punicalagin on the CPE of EV-A71-infected Vero cells was detected by the CCK-8 method, and its EC₅₀ was calculated. (C) The cytotoxicity of Punicalagin on Vero cells was detected by the CCK-8 method, and its CC₅₀ was calculated. (D) Morphological observation of the rescue effect of Punicalagin on Vero cells infected with CVB3 and EV-A71. (E) Morphological observation of the rescue effect of Punicalagin on neonatal mice infected with CVB3 and EV-A71. (F, G) The survival rate of neonatal mice (n = 10) infected with lethal doses of CVB3 and EV-A71 was evaluated after treatment with Punicalagin. (H) Hematoxylin and eosin (H&E) staining was performed to evaluate brain and heart pathology in representative sections from various treated mouse groups. The sample size for each group was (n = 3). Data are expressed as mean ± standard deviation (SD), and comparisons between groups are indicated by asterisks in the figure, *p < 0.05, **p < 0.01

In addition, we randomly selected three surviving mice from the groups that were administered or not administered the drug two days after EV-A71 and CVB3 infection for dissection to examine the improvement of viral symptoms by the drug. The results are shown in Fig. 2H. In the brain slices, the CA1 and CA3 regions of the hippocampus could be observed. In the EV-A71 virus-infected group, obvious neuronal morphological abnormalities such as cell shrinkage and nuclear pyknosis were seen, indicating neuronal damage (indicated by purple arrows in the figure), while these conditions were significantly improved after the addition of the drug. In the ventroposteromedial nucleus of the thalamus (VPN) region, these phenomena were even more pronounced. For the analysis of cardiac pathological slices, it could be seen that after CVB3 infection, myocardial cells also showed obvious cell shrinkage and nuclear pyknosis (indicated by purple arrows in the figure), and in addition, there was strong eosinophilia (indicated by red arrows in the figure). After the addition of the drug, these conditions were also significantly improved.

In conclusion, the experimental results at both the cellular and animal levels fully confirmed the effective inhibitory effect of Punicalagin on different serotype enterovirus infections.

Punicalagin might exhibit antiviral efficacy by targeting critical factors that induce severe intestinal viral infections

Based on the GO and KEGG analysis results showed in Fig. 1, we subsequently utilized Cytoscape software to conduct an in-depth investigation of the genes associated with the top 10 signaling pathways, ranking them according to node degree and betweenness centrality. The analysis revealed that the common targets of severe enterovirus infection overlaped with Punicalagin were positioned within the top ten KEGG signaling pathways, as illustrated in Fig. 3A. The top 20 targets with the highest degree values were PRKCA, AKT1, STAT3, BCL2, PRKCB, IL6, EGFR, MAPK10, IL1B, MAPK14, TNF, PRKCG, BAD, CASP3, RAC1, GRB2, MAP2K1, JAK1, RXRA, and HSP90AA1, respectively. In the top 10 GO enrichment analysis of cellular processes, as depicted in Fig. 3B, the top 20 targets with the highest degree values were TNF, IL6, PRKCA, AKT1, IL1B, ITGB3, TGFB2, ADAM17, MAPK14, BAD, SYK, PRKCE, ALOX5, LCK, PPARD, CASP3, PRCKD, PRCKQ, GSTP1, and HGF, respectively. Therefore, PRKCA, AKT1, MAPK14, IL-6, TNF, IL1B, BAD, and CASP3, which had common degree values in KEGG and GO analyses, were considered as the key targets for Punicalagin in treating co-morbidities of severe enterovirus infections of different serotypes and were further subjected to molecular docking. The docking results indicated that Punicalagin had good binding activity with the above-mentioned key targets as shown in Fig. 3C-H; Table 1 (all binding energies are less than 0, and all root mean square deviations (RMSD) are less than 2) [13, 14].

Fig. 3.

Molecular docking technology was used to explore the key genes in the top 10 signaling pathways of co-morbidities after Enterovirus infection in Punicalagin. (A) Gene and pathway analysis diagram of drug-disease overlapping genes and top 10 KEGG pathways. (B) Gene and pathway analysis diagram of drug-disease overlapping genes and top 10 GO pathways. (C) Docking results of Punicalagin with MAPK14 protein. (D) Docking results of Punicalagin with PRCKA protein. (E) Docking results of Punicalagin with AKT1 protein. (F) Docking results of Punicalagin with BAD protein. (G) Docking results of Punicalagin with CAPS3 protein. (H) Docking results of Punicalagin with IL1B protein

Table 1.

Molecular Docking analysis results of Punicalagin with target proteins

Target Protein	PDB ID	RMSD	Binding energy
MAPK14	6SFO	0.000	−3.59
PRKCA	2ELI	0.871	−2.11
AKT1	3O96	0.002	−2.77
CASP3	1RE1	0.000	−4.43
BAD	1G5J	NA	−5.45
IL1B	68YI	0.598	−2.78
TNF	2AZ5	0.011	−1.43
IL6	1ALU	0.000	−1.28

NA: The relevant protein structure in the PDB database do not contain the original ligands

The binding affinities were in the order of target BAD > CASP3 > MAPK14 > IL1B > AKT1 > PRKCA > TNF > IL6. These binding results suggested that Punicalagin might treat co-morbidities of severe enterovirus infections of different serotypes by targeting BAD, CASP3, MAPK14, IL1B, AKT1, PRKCA, TNF, and IL6.

Punicalagin inhibited the infection of different serotypes of enteroviruses via its anti-apoptotic mechanism

Based on the results of molecular docking, Punicalagin exhibited significant high affinity for molecules such as BAD and CASP3. Previous studies have established that these molecules are integral components of the mitochondrial apoptotic pathway [15, 16]. Through GO and KEGG analyses (Fig. 1), we observed that the molecular functions associated with Punicalagin in relation to viral infection complications were predominantly enriched in anti-apoptotic and inflammatory mechanisms. Based on these findings, we hypothesized that the anti-apoptotic effect of Punicalagin might represent a primary molecular mechanism underlying its response to severe complications caused by different serotypes of enterovirus infection. To test this hypothesis, we employed the TUNEL assay to meticulously evaluate the extent of apoptosis in cells infected with CVB3 and EV-A71 (Virus infection MOI = 0.1, adsorption time = 1 h, drug treatment initiated at 48 hpi), both before and after treatment. As illustrated in Fig. 4A, following CVB3 infection, the red fluorescence indicative of apoptotic signals was markedly intensified, signifying a pronounced increase in apoptosis. However, upon treatment with Punicalagin, the red fluorescence was significantly attenuated, suggesting that Punicalagin effectively mitigated the extent of apoptosis. This trend was corroborated in the EV-A71 infection experiment depicted in Fig. 4B. Collectively, these experimental findings consistently demonstrated that Punicalagin might efficaciously alleviate apoptosis induced by both CVB3 and EV-A71, thereby diminishing the severity of cytopathic effects.

Fig. 4.

Analysis of apoptosis levels following Punicalagin treatment in Vero cells infected with CVB3 or EV-A71 using the TUNEL Assay. (A). Vero cells infected with CVB3. (B). Vero cells infected with EV-A71. (Scale bar = 100 μm, n = 3)

The anti-inflammatory activity of punicalagin might effectively alleviate the inflammation caused by different serotypes of enterovirus infection

Through an in-depth analysis of the mechanism of action of Punicalagin and its associated protein interaction network, we have identified its ability to interact with multiple inflammatory factors, indicating its significant influence on inflammation regulation. Previous studies have highlighted Punicalagin as a promising anti-inflammatory agent, garnering considerable attention and research interest [11, 17]. Given that severe cases of enterovirus co-infections are often closely linked to inflammatory responses, we further investigated the impact of Punicalagin on the expression of inflammation-related factors following viral infection. Considering the inherent interferon deficiency in the Vero cell line and the fact that pulmonary infections are a major concern in severe enterovirus cases [18, 19], we selected A549 cells, which possess functional interferon pathways and are derived from lung tissue, as our research model to examine the effects of Punicalagin on the inflammatory response post-enterovirus infection. We systematically evaluated the CPE improvement of Punicalagin on A549 cells infected with CVB3 and EV-A71 viruses, and determined its EC₅₀. The results indicated that after 48 h of treatment, the EC₅₀ values for Punicalagin were 1.7 µM against CVB3 and 1.59 µM against EV-A71 (Fig. 5A, B). Morphologically, Punicalagin demonstrated a significant amelioration of CPE in virus-infected cells, as illustrated in Fig. 5C. Subsequently, we performed comprehensive analyses of inflammatory markers including IL6, IL1B, CXCL8, TNF, IFNA, IFNB, and IFNG. The findings, presented in Fig. 5D, E, revealed that infection with CVB3 or EV-A71 led to elevated expression levels of these inflammatory factors. However, upon administration of Punicalagin, the expression levels of these inflammatory markers were markedly reduced in a dose-dependent manner. This evidence suggested that Punicalagin’s anti-inflammatory properties significantly mitigate the inflammatory response induced by different serotypes of enterovirus infection, thereby enhancing its antiviral efficacy.

Fig. 5.

Analysis of the anti-inflammatory effects of punicalagin during CVB3 and EV-A71 Infections in A549 Cells. (A) Punicalagin demonstrated a dose-dependent inhibitory effect on CVB3 infection in A549 cells. (B) Punicalagin exhibited a dose-dependent inhibitory effect on EV-A71 infection in A549 cells. (C) Morphological changes in A549 cells were observed 48 h post-infection with CVB3 or EV-A71, both in the presence and absence of punicalagin treatment, as assessed by the CPE inhibition assay. (D) qRT-PCR analysis revealed that punicalagin treatment significantly reduced the levels of cytokines/chemokines in CVB3-infected A549 cells, and (E) Likewise, punicalagin treatment significantly decreased the levels of cytokines/chemokines in EV-A71-infected A549 cells (n = 3). Data are presented as mean ± SD, with statistical significance indicated by asterisks or hashtag: ####/**** p < 0.0001, ###/*** p < 0.001, ##/** p < 0.01, #/* p < 0.05. (*) denote comparisons between virus-infected groups and control groups, while (#) indicate comparisons between treated and untreated virus-infected groups

The capacity of punicalagin to bind with viral capsid proteins indicated its potential direct antiviral efficacy against enteroviruses

Next, we specifically investigated the binding affinity of the drug to the capsid proteins of various enterovirus serotypes. We selected the VP1 protein as the target for our research and performed molecular docking with Punicalagin. Previous studies have established that the VP1 protein is a critical component of enterovirus particles and plays an essential role in viral infection [20, 21]. The results of our molecular docking revealed that Punicalagin exhibited high binding affinity to the VP1 proteins of CVB3 and EV-A71, as illustrated in Fig. 6A and B; Table 2. For the experimental validation, we utilized interferon-deficient Vero cells as the model system to examine the direct inhibitory effect of the compound on the viral VP1 protein (Virus infection MOI = 0.1, adsorption time = 1 h, drug treatment initiated at 48 hpi). The results demonstrated that, as illustrated in Fig. 6C and D, the expression of the viral VP1 protein was significantly suppressed following drug treatment.

Fig. 6.

Investigation of the inhibitory wffect of Punicalagin on viral VP1 proteins. (A) Docking result of Punicalagin and CVB3 VP1 protein. (B) Docking result of Punicalagin and EV-A71 VP1 protein. (C) Evaluation of the inhibitory effect of Punicalagin on EV-A71 VP1 using qRT-PCR. (D) Evaluation of the Inhibitory Effect of Punicalagin on CVB3 VP1 Protein Using qRT-PCR (n = 3). (E) Scheme for the time of drug addition assays in A549 cell. (F) The effects of punicalagin on EV-A71 replication at 5 µM were evaluated (n = 3). (G) The effects of punicalagin on CVB3 replication at 5 µM were evaluated (n = 3). Data are expressed as mean ± standard deviation, with statistical significance indicated by asterisks or hashtags: ****/#### P < 0.0001, ***/### P < 0.001, **P < 0.01, *P < 0.05

Table 2.

Molecular Docking analysis results of Punicalagin with VP1 proteins of CVB3 and EV-A71

Target Protein	PDB ID	RMSD	Binding energy
VP1 (EV-A71)	6DIZ	0.000	−2.12
VP1 (CVB3)	3JD7	0.000	−2.82

In addition, to explore whether Punicalagin can prevent enterovirus infection, the experiment used EV-A71 and CVB3 to infect non-interferon-deficient A549 cells as host cells. The experiment was divided into three groups: the pretreatment group (Pre), where A549 cells were pretreated with Punicalagin 6 h before virus infection; the post-treatment group, where Punicalagin was administered 1 h after virus infection (Post); and the pretreatment + post-treatment group (Pre + Post), where Punicalagin was given both before and after virus infection. The results showed that all three groups could inhibit viral replication compared between the test groups and the virus infection group. Notably, compared with the post-treatment group, the pretreatment group and the pretreatment + post-treatment group could more significantly inhibit the expression of viral VP1 (Fig. 6E -G).

These findings provided robust evidence supporting the compound’s potential as a broad-spectrum antienteroviral agent. Consequently, it is imperative to further investigate the structural optimization of the compound, its delivery methods, and underlying molecular mechanisms to fully realize its antiviral potential.

Discussion

This study comprehensively employed system pharmacology methods and experimental validation approaches to deeply analyze the mechanism of Punicalagin in treating representative strains of enterovirus A and B. Enterovirus A and B can cause serious diseases including hand-foot-mouth disease, meningitis, and myocarditis, posing a significant threat to infants, young children, and individuals with weakened immune systems [22, 23]. Currently, there are no specific antiviral drugs for these viruses, making the exploration of Punicalagin’s antiviral mechanism as a potential therapeutic agent of great scientific significance. Enteroviruses are diverse, with significant serotype differences and rapid viral mutations, which pose significant challenges for vaccine development and antiviral drug research [24, 25]. Moreover, current treatments for enteroviruses mainly rely on symptomatic treatment, lacking targeted antiviral drugs. Therefore, the study of Punicalagin’s antiviral activity, especially its potential therapeutic mechanism within the framework of system pharmacology [26, 27], provides a new perspective for future research on antiviral natural products.

This study employed network pharmacology to predict and analyze the correlation between the targets of Punicalagin and the severe complications caused by Enterovirus A and B. Through GO and KEGG pathway analysis, the study revealed that Punicalagin demonstrated significant antiviral activity against EV-A71 and CVB3 in Vero and A549 cells without causing cytotoxicity and with a low selectivity index. The in vivo antiviral effect of Punicalagin was evaluated in a neonatal mouse model by intraperitoneal injection of the virus. In the neonatal mouse model, Punicalagin significantly increased the survival rate of mice after viral infection and alleviated clinical symptoms. Through network pharmacology, the study found that the predicted targets of Punicalagin were highly correlated with the targets of severe complications after enterovirus infection. GO and KEGG analysis revealed its potential anti-inflammatory and anti-apoptotic mechanisms. Molecular docking technology was used to analyze the interaction between Punicalagin and key molecules to verify its affinity with these molecular targets. The study found that Punicalagin had varying degrees of affinity for key molecules such as CASP3, BAD, MAPK14, PRKCA, AKT1, IL1B, TNF, and IL6, revealing its potential impact on the inflammatory response and apoptosis of host cells after viral infection, which was verified by experiments. Additionally, molecular docking analysis revealed that Punicalagin had a significant binding affinity with the VP1 protein, suggesting its potential to inhibit viral replication. Experiments confirmed that Punicalagin effectively inhibited the expression of the viral capsid protein VP1 in EV-A71 and CVB3-infected host cells. In conclusion, we believe that Punicalagin has significant potential to enhance the effectiveness of current clinical antiviral methods and immunotherapies in treating severe co-morbidities caused by enterovirus infections in immunocompromised individuals.

Conclusion

This study systematically evaluated the therapeutic effects of Punicalagin on different serotypes of enteroviruses by integrating systems pharmacology and experimental validation. The research findings demonstrated that Punicalagin exerted anti-inflammatory and anti-apoptotic activities, effectively combating severe diseases caused by enterovirus A and B. These results highlighted its potential as a safe and efficacious drug for treating enterovirus infections. Furthermore, this study provided preliminary insights into the molecular mechanisms underlying Punicalagin’s efficacy in treating diseases with similar symptoms, offering valuable guidance for future research on antiviral natural products.

Electronic Supplementary Material

Below is the link to the electronic supplementary material.

Author contributions

Yuwei Liu: Writing– review & editing, Writing– original draft, Visualization, Validation, Software, Methodology, Investigation, Formal analysis, Data curation, Conceptualization; Jing Cheng: Writing– original draft, Visualization, Methodology, Investigation, Data curation; Nana Du: Writing– original draft, Visualization, Methodology, Investigation; Ping Wu: Methodology, Investigation; Shixing Yang: Methodology, Investigation; Likai Ji: Methodology, Investigation; Quan Shen: Methodology, Investigation; Xiaochun Wang: Methodology, Investigation; Songyi Ning: Methodology, Investigation; Xiaodan Zhang: Validation, Supervision. Hongfeng: Validation, Supervision. Wen Zhang: Writing– review & editing, Validation, Conceptualization and Supervision,

Funding

The project was financially supported by the National Natural Science Foundation of China 82101630 To Yuwei Liu.

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethics approval and consent to participate

Ethics approval and consent to participate: All animal experiments in this study were approved by the Institutional Animal Care and Use Committee of the Institute of Laboratory Animal Science, Jiangsu university with approval number (permit number: IACUC-AP-2023032101).

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.