The immunostimulatory activity of Epimedium flavonoids involves toll-like receptor 7/8

<i>Epimedium</i> flavonoids, primarily composed of icaritin, icariin I and icariin II, were observed to significantly enhance the expression of surface co-stimulatory molecules (CD40, CD80, CD86) and major histocompatibility complex (MHC-I, MHC-II) in bone marrow-derived dendritic cells (BMDCs) and RAW 264.7 cells. Additionally, the production of chemokines and pro-inflammatory cytokines was significantly increased in RAW 264.7 cells. <i>In vivo</i>, the findings demonstrated that the vaccine adjuvant containing <i>Epimedium</i> flavonoids significantly increased the serum concentration of total OVA-specific IgG compared to the control group. SPR analysis revealed that icariin II exhibited the highest binding response to TLR7, while icariin I and icariin II showed the strongest interactions with TLR8 protein, even surpassing the positive control drug, Resiquimod. The NF-κB/SEAP assay further confirmed that icaritin, icariin I, and icariin II enhanced NF-κB activity and stimulated SEAP secretion through TLR7/8 activation.