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

Jingyu Wu ^1,2 Jingwei Jin ^1,2* Yi Ou ^1,2 Min Yao ^1,2 Jiaquan Liu ^1,2 Hengxing Ran ^1,2 Zhengrong Wu ^1,2 Rihui Wu ^1,2 Li-She Gan ^1,2,3 Dongli Li ^1,2*

• ¹ School of Pharmacy and Food Engineering, Wuyi University, Jiangmen, China
• ² International Healthcare Innovation Institute, Wuyi University, Jiangmen, Guangdong Province, China
• ³ School of Pharmaceutical Science, Zhejiang Chinese Medical University, Hangzhou, Jiangsu Province, China

The 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 2 their immunostimulatory activity remains unclear. Purpose: To investigate the immunostimulatory effects and elucidate the specific mechanisms of Epimedium flavonoids both in vitro and in vivo. Methods: The 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. Results: Epimedium 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 3 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.