Structural Characterization of Polysaccharide Derived from and Its Immunostimulatory Effect on RAW264.7 Cells.

A polysaccharide from Gastrodia elata (named GEP-1) was isolated with a DEAE-52 column and Sephadex G-100 column. The structural characteristics showed that GEP-1 was mainly composed of glucose (92.04%), galactose (4.79%) and arabinose (2.19%) with a molecular weight of 76.444 kDa. The polydispersity (Mw/Mn) of GEP-1 was 1.25, indicating that the distribution of molar mass (Mw) was relatively narrow, which suggested that GEP-1 was a homogeneous polysaccharide. Moreover, the molecular conformation plot of the root mean square (RMS) radius ( 1/2) versus Mw yielded a line with a slope less than 0.33 (0.15 ± 0.02), displaying that GEP-1 is a compact and curly spherical molecule in NaNO3 aqueous solution. NMR and methylation analyses revealed that the main chain structure of GEP-1 was α-(1→4)-glucans. Furthermore, it was proven that GEP-1 possessed cytoproliferative and enhancing phagocytic activities and induced cytokine (TNF-α, IL1-β) and nitric oxide (NO) release in macrophages by upregulating the related gene expression. In addition, the RNA-seq results suggested that the GEP-1-induced immunomodulatory effect was mainly caused by activation of the NF-κB signaling pathway, which was further verified by NF-κB ELISA and pathway inhibition assays. As a result, GEP-1 exhibits the potential to be developed as a novel cheap immunostimulant without obvious toxicity.