To explore the immunomodulatory activity of polysaccharides from Ampelopsis grossedentata, two polysaccharides named AGP1 and AGP2 were isolated and purified by DEAE-cellulose 52 column and Sephacryl S-300HR chromatography. AGP1 and AGP2 were composed of fucose, arabinose, rhamnose, galactose, glucose, mannose, galacturonic acid, and glucuronic acid, with a ratio of 0.5: 10.2: 0.9: 31.8: 7.4: 3.4: 21.6: 24.2 and 0.4: 6.0: 0.5: 23.3: 3.3: 6.2: 33.5: 26.8, respectively. The average molecular weights of AGP1 and AGP2 were found to be 6.60 × 10 Da and 7.24 × 10 Da, respectively. AGP1 contained →4,6)-Galp-(1 → glycosidic linkages, while AGP2 contained →2)-Galp-(1 → and →2,3,4)-Glcp-(1 → glycosidic linkages. The structures of AGPs were characterized by Fourier transform infrared spectroscopy, nuclear magnetic resonance, and scanning electron microscope. The immunomodulatory activity of AGPs was investigated in RAW264.7 cells, and the results indicated that AGPs significantly activated macrophages, promoted cells differentiation and NO secretion, increased the expression of IL-6 and TNF-α, and induced macrophage M1 polarization. Transcriptomic analysis indicated that AGP1 and AGP2 regulated a total of 1043 and 970 differentially expressed genes respectively, which were identified in different immune related signaling pathways. Moreover, the immunoblot demonstrated that AGPs exerted immune-promoting effects through the TLR4, MAPK and NF-κB signaling pathways in macrophages. Consequently, AGPs have potent immunomodulatory activity and can be considered as immunomodulators in medical and food industries.
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