Chemical characterization of a novel polysaccharide ASKP-1 from Artemisia sphaerocephala Krasch seed and its macrophage activation via MAPK, PI3k/Akt and NF-κB signaling pathways in RAW264.7 cells

The aim of this study was to investigate the molecular mechanism underlying the immunomodulatory effect of the purified Artemisia sphaerocephala Krasch seed polysaccharide (ASKP-1) in RAW264.7 macrophages. Chemical characteristic analysis revealed that ASKP-1 consisted of 14.1% mannose, 56.9% glucose and 19.6% galactose with the average molecular weight of 9.08 x 10(5) Da and the mixed glycan backbone structure containing 1 -> 4)-Glcp (39.8%), 1 -> 6)-Galp (18.8%), 1 -> 3,6)-Manp (19.6%), 1 ->)-Glcp (10.8%), 2 -> 6)-Manp (4.0%) and 2 -> 3,5)-Araf (7.0%). In vitro studies showed that ASKP-1 markedly induced the release of cytotoxic molecules (NO and ROS) and secretion of the cytokines (TNF-alpha, INF-beta, and IL-6) and significantly enhanced the phagocytosis of RAW264.7 macrophages. Furthermore, TLR4 was found to be a recognized target of ASKP-1 and its related mitogen-activated protein (MAPK) and phosphoinositide 3-kinase (PI3K)/Akt, including phosphorylated ERK, JNK, p38 and Akt, were rapidly activated by ASKP-1 in RAW264.7 macrophages. Moreover, ASKP-1 was found to cause the nuclear translocation of the nuclear factor NF-kappa B subunit p65 and the degradation of I kappa B-alpha in RAW264.7 macrophages. All these findings suggest that MAPK, PI3K/Akt and NF-kappa B pathways are involved in ASKP-1-induced macrophage activation, and ASKP-1 is a potential immunomodulating function food.