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

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