Icariside II suppresses Enterovirus 71 replication via blockade of NF-κB p65 activation.

Enterovirus 71 (EV71) infection remains a serious public health threat without a specific antiviral drug. Icariside II (ICS II), a natural active compound, has shown various pharmacological effects. This study aims to evaluate the anti-EV71 activity of ICS II and to decipher whether the NF-κB pathway, a key signaling mediator in viral pathogenesis, is involved in its mechanism of action. Firstly, the cytotoxicity of ICS II was assessed by CCK-8 assay to determine its maximal non-cytotoxic concentration. The antiviral efficacy of ICS II was determined by assessing cytopathic effect (CPE), viral titer (Reed-Muench method), and viral VP1 protein expression (Western blot and qRT-PCR) in EV71-infected human rhabdomyosarcoma (RD) cells. Network pharmacology prediction, molecular docking, and drug affinity responsive target stability (DARTS) assays were integrated to identify the key interacting target. Immunofluorescence and Western blot were performed to evaluate NF-κB p65 nuclear translocation. ELISA was performed to determine the concentrations of inflammatory cytokines. The results showed that ICS II exhibited significant anti-EV71 activity at a non-cytotoxic concentration (10 μM), markedly reducing CPE, viral titer, and VP1 expression. We identified NF-κB p65 as a key interacting target of ICS II, and demonstrated that ICS II effectively impeded EV71-induced NF-κB p65 nuclear translocation. Accordingly, treatment with ICS II markedly reduced the levels of the inflammatory cytokines IL-1β, IL-6, and TNF-α. Our study reveals that the anti-EV71 activity of ICS II is potentially associated with modulation of NF-κB p65, providing mechanistic insights into the antiviral effects of this natural product.