Areca nut polysaccharide induces M1 macrophage polarization through the NF-κB and MAPK pathways independent of TLR2 and TLR4 signaling

In this study, the structure of Areca nut polysaccharide (ANP) was characterized, and its effects on macrophage activation and the underlying molecular mechanisms were investigated. ANP was identified as a glucan with a molecular weight of 24.5 kDa, and its structure was analyzed using XRD, SEM, FT-IR, methylation, and NMR techniques. The main chain of ANP is composed of →4)-α-D-Glcp-(1 → and →4,6)-α-D-Glcp-(1→, with a branched α-D-Glcp-(1 → chain. Furthermore, the activation of macrophages by ANP was explored. Stimulation of RAW264.7 cells with ANP in vitro increased the expression of inflammatory cytokines (TNF-α and IL-6) and NO levels. Flow cytometry showed that ANP induced M1 macrophage polarization. RNA-seq and Western blot analyses revealed that ANP activated the NF-κB and MAPK pathways. Importantly, TLR2- and TLR4- specific antibodies did not affect ANP-induced M1 polarization, whereas endocytosis inhibitors reduced the production of inflammatory cytokines in ANP-treated macrophages. In conclusion, ANP engages macrophages without interacting with TLR2 and TLR4 receptors, inducing M1 polarization through the NF-κB and MAPK signaling pathways.