Data Sheet 1_The immunostimulatory activity of Epimedium flavonoids involves toll-like receptor 7/8.docx

BackgroundThe flavonoids found in Epimedium exhibit a wide range of pharmacological activities, with their immunostimulatory effects emerging as a significant area of research in recent years. However, the underlying mechanism of their immunostimulatory activity remains unclear.
PurposeTo investigate the immunostimulatory effects and elucidate the specific mechanisms of Epimedium flavonoids both in vitro and in vivo.
MethodsThe immunostimulatory effects and underlying mechanisms of flavonoids from Epimedium were evaluated in vitro using a variety of techniques, including cell viability assays, flow cytometry, real-time reverse transcription-quantitative polymerase chain reaction (qRT-PCR), enzyme-linked immunosorbent assay (ELISA), molecular docking, plasmid recombination and transformation, recombinant protein expression, surface plasmon resonance (SPR), and NF-κB/SEAP assays. To investigate the immune response in animal experiments, Epimedium flavonoids were compared with traditional adjuvants, utilizing biochemical analysis and flow cytometry.
ResultsEpimedium 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. In vivo, the findings demonstrated that the vaccine adjuvant containing Epimedium 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.