Structural characterization and macrophage activation of a hetero-galactan isolated from Flammulina velutipes

We isolated and purified a new polysaccharide (WFVP-N-b1) with a molecular weight of 20 kDa from Flammulina velutipes. Results showed that WFVP-N-b1 is composed of an α (1 → 6)-linked D-galactan backbone and branched at the O-2 of its Galp residues by an α-D-(1 → 6)-linked Manp attached to t-β-D-Glcp or t-α-D-Fucp side chains. WFVP-N-b1 can significantly induce cytokines secretion and release of toxic molecules. On a cellular level, WFVP-N-b1 is recognized by Toll-like receptor 4 (TLR4). Thereby, the hetero-galactan increased the phosphorylation of mitogen-activated protein kinases (MAPKs) and Akt, promoted degradation of IκB-α and the nuclear translocation of the NF-κB p65 subunit. Importantly, our results indicate that WFVP-N-b1 activated macrophage is mediated by autophagy, as blockade of WFVP-N-b1-induced autophagy by Baf-A1 significantly decreases macrophage activation. This is the first report that hetero-galactan-induced macrophage activation is mediated by autophagy. Collectively, WFVP-N-b1 activated RAW264.7 cells through MAPKs, autophagy, and Akt/NF-κB signaling pathways via TLR4 receptor.