Immunostimulatory effects mechanism of polysaccharide extracted from Acanthopanax senticosus on RAW 264.7 cells through activating the TLR/MAPK/NF-κB signaling pathway

Acanthopanax senticosus is a unique wild resource in Northeast China. Its main active ingredient is a polysaccharide, which has a prominent immunomodulatory effect. In this study, the purified polysaccharide component of Acanthopanax senticosus leaves(ASPS-A1) with strong immunomodulatory activity was isolated by column chromatography, and its structure and properties were characterized by HPGPC, and FT-IR. Results showed that ASPS-A1 is mainly composed of α- 1,4-_D-GalA, α- 1,5-_L-Ara, and β- 1,4-_D-Gal. RT-qPCR experiments and RNA-seq analysis were used to study the immunoregulatory mechanism of ASPS-A1. The results showed that ASPS-A1 could significantly up-regulate the levels of cytokines iNOS, IL- 1β, IL- 6, and TNF-α activated macrophages through MAPK, NF-κB, and Toll-like receptor signaling pathways. Inhibitory experiments further confirmed that ASPS-A1 promotes the expression of iNOS, TNF-α, and IL- 6 via the TLR4 receptor, and TNF-α and IL- 1β via the TLR2 receptor. In order to identify the target of ASPS-A1, molecular docking experiments were conducted. The results demonstrated that ASPS-A1 could bind to both TLR4 and TLR2, forming stable complexes with the cavities on the protein surface through hydrogen bonding and hydrophobic interaction. The docking scores indicated that ASPS-A1 could regulate the immune response through TLR2 and TLR4 signaling pathways, with a particularly strong interaction with TLR4. In summary, this study screened and characterized the most immunoreactive components of Acanthopanax senticosus polysaccharide, disclosed the immunomodulatory mechanism of ASPS-A1, and furnished a research basis for its potential application as a natural immune enhancer.