Immune regulation mechanism of polysacchride extracted from Acanthopanax senticosus on RAW 264.7 cells through activating the TLR/MAPK/NF-?B signaling pathway

Acanthopanax senticosus is a unique wild resource in Northeast China. Its main active ingredient is polysaccharide, which has 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, FT-IR. Results showed that ASPS-A1 is mainly composed of a-1,4-D-GalA, a-1,5-L-Ara, and ß-1,4-D-Gal. RT-qPCR experiments and RNA-seq analysis was used to study the immunoregulatory mechanism of ASPS-A1. The results showed that ASPS-A1 could significantly up-regulated the levels of cytokines iNOS, IL-1ß, IL-6 and TNF-a activated macrophages through MAPK, NF-?B, and Toll-like receptor signaling pathways. Inhibitory experiments were further confirmed that ASPS-A1 promote the expression of iNOS, TNF-a, and IL-6 via TLR4 receptor, and TNF-a and IL-1ß via 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. Overall design: The RNA-seq analysis was performed on RAW 264.7 cells. RAW 264.7 cells were cultured in 6-well plates at a concentration of 2×105 cells/mL for 24 h, then the supernatant was discarded, and the medium containing ASPS-A1 (200 µg/mL) was added. The cells were collected for 24 h, treated with 1 mL Trizol, and immediately placed in a refrigerator at -80 ?. Fresh Acanthopanax senticosus leaves were taken and extracted with ultrasonic-assisted extraction at a material-liquid ratio of 1:20, rotary evaporation, alcohol precipitation, and lyophilization to obtain Acanthopanax senticosus leaves crude polysaccharide (ASPS). DEAE cellulose-52 column was used to separate and purify ASPS, and the acidic polysaccharide (ASPS-A) and neutral polysaccharide (ASPS-N) were obtained. ASPS-A was further isolated and purified using a Superdex-G100 gel column and eluted by NaCl at a flow rate of 0.15 mL/min. The liquid was collected, dialyzed, and freeze-dried to obtain ASPS-A1.