Exploring the Vaccine Adjuvant Effect and Mechanism of Epimedium Using Network Pharmacology, Molecular Docking, and...

Background:Epimedium is a natural herb with immunomodulatory potential, but its vaccine adjuvant properties remain poorly understood. Objective: The aim of this study was to elucidate the adjuvant effects of Epimedium and the underlying molecular mechanisms. Methods: Network pharmacology was used to identify bioactive compounds and targets of Epimedium from the TCMSP database, and immunomodulation-related targets from GeneCards and OMIM. PPI networks, KEGG/GO enrichment, molecular docking, and molecular dynamics (MD) simulations were performed. In vivo, female BALB/c mice were immunized with the Staphylococcus aureus (S. aureus) vaccine subunit HI antigen, either alone or with low- or high-dose icariin (ICA). Serum antibody responses (IgG, IgG1, IgG2a, IgG2b) were measured by ELISA. Survival against lethal S. aureus USA300 challenge was monitored. Results: Network pharmacology predicted 488 targets and 13 pathways. Core targets included IL6, TP53, EGFR, CTNNB1, HIF1A, HSP90AA1, JUN, MTOR, SRC, and AKT1. KEGG/GO analysis indicated involvement of T cell receptor and NOD-like receptor signaling pathways in inflammatory responses. Molecular docking and MD simulations confirmed stable ligand-target binding. Experimental validation showed that ICA significantly enhanced HI-specific antibody responses and induced a Th2-biased humoral immune response (IgG1/IgG2a ratio > 1), which is particularly relevant for vaccines targeting extracellular pathogens such as S. aureus. ICA also improved survival after lethal bacterial challenge. Conclusions: This study identifies potential bioactive compounds, core targets, and key pathways of Epimedium as a vaccine adjuvant. Experimentally, ICA, as a representative component, enhanced HI-specific antibody responses and conferred protection against lethal S. aureus challenge. Together, these findings offer a computational-experimental basis that may guide further mechanistic investigation.