Mycelial polysaccharides of Lentinus edodes (shiitake mushroom) in submerged culture exert immunoenhancing effect on macrophage cells via MAPK pathway

A crude polysaccharide fraction (termed cLEP) and two derived fractions (termed LEP1 and LEP2) from Lentinus edodes mycelia were purified and characterized. LEP1/-2 were classified as α-type heteropolysaccharides with 1 → 2, 1 → 3, 1 → 4, 1 → 6 linkages. Their monosaccharide components were respectively Gal, Glc, Man, Ara, Fuc, and Rha (molar ratio 10.17:9.75:9.01:1.61:1.26:1), and Glc, Man, Fuc, Rha, and Gal (molar ratio 5.18:4.69:2.85:1.43:1). In vitro culture experiments with macrophage RAW264.7 cells showed no cytotoxic effects of the polysaccharides. Phagocytosis (neutral red uptake) was significantly enhanced by LEP1/-2. Levels of NO, TNF-α and IL-6 were higher in LEP1/-2-treated groups than in cLEP-treated group. qRT-PCR analysis showed that LEP1/-2 had greater enhancing effect on mRNA transcription of iNOS, TNF-α, and IL-6 genes. Western blotting analysis revealed that LEP1/-2 strongly promoted phosphorylation of kinases ERK and JNK, and suggested that they exert immunoenhancing effects via MAPK signaling pathway.