Polysaccharide extracted from Polygonatum kingianum Coll. Et Hemsl. Activates macrophages via the TLR4/NF-κB/MAPK pathway and exhibits vaccine adjuvant effect

Abstract

Polygonatum kingianum Coll. et Hemsl. (Huang Jing), a traditional Chinese medicinal herb, has long been used as a functional food and immune-enhancing remedy. Its polysaccharide component (PKP) is believed to drive immunomodulatory effects, though the molecular mechanisms behind its adjuvant potential remain unclear. PKP was isolated using water extraction, ethanol precipitation, enzymatic digestion, and purification. The immunostimulatory activity was assessed in vitro using RAW264.7 macrophages and primary macrophages from TLR4^+/+ and TLR4^−/− mice. Cytokine secretion (TNF-α, IL-6), nitric oxide production, and gene/protein expression were evaluated through RT-PCR, Western blotting, and immunofluorescence to analyze the activation of the TLR4/NF-κB/MAPK pathway. In vivo, the adjuvant activity of PKP was tested in BALB/c mice immunized with ovalbumin (OVA), measuring antigen-specific immune responses. The results revealed that PKP robustly activated macrophages in a TLR4-dependent manner, significantly enhancing TNF-α (3.8-fold) and IL-6 (2.5-fold) secretion, and upregulating MyD88, TRIF, p65-NF-κB, and MAPK phosphorylation. TLR4^−/− macrophages showed abolished responses, confirming TLR4 as the critical receptor. PKP also enhanced antigen-specific IgG titers and Th1/Th2 cytokine production (IFN-γ, IL-4) in OVA-immunized mice, demonstrating its adjuvant efficacy. Notably, while PKP exhibited activity at higher in-vitro concentrations than LPS, its low toxicity and plant origin constitute key advantages over bacterial endotoxins; accordingly, PKP should be framed not as a more potent LPS alternative, but as a safer adjuvant candidate for vaccine development. In conclusion, PKP activates macrophages through the TLR4/MyD88/TRIF/NF-κB/MAPK axis and functions as a potent vaccine adjuvant, providing mechanistic validation for its traditional use and positioning it as a promising low-toxicity candidate for improving vaccine-mediated immunity.