Preparation Of Polysaccharide From Astragalus Radix And Primary Study On Its Activating Mechanisms On Macrophages And Dendritic Cell

Astragalus polysaccharide (APS) are important bioactive components extracted from Astragalus membrabaceus, which have multiple functions including immunomodulation, antitumor, antioxidant and so on. Dendritic cells (DC) are the most important professional antigen-presenting cells which play vital role in initiating T-cell responses against microbial pathogens and tumors. Macrophages are one of the highly efficient phagocytes, with beneficial in host defense and anti-tumor immunity. DC, as well as macrophages, play noticeable roles in both innate and adaptive immunity.Concretely this study included three parts. In the first part, three fractions of APS were obtained by hot water extraction, alcohol precipitation, gel-permeation chromatography and ultra filtration. Their weight-average molecular weights (MW) were detected by Gel Permeation Chromatog-Size Exclusion Chromatography (GPC-SEC). The69KD fraction was choosed to be further studied the effect on macrophages function because of its high homogenicity, production and purity.In the second part, APS (2-AMAC-APS) labeled by fluorescence material2-aminoacridone (2-AMAC) was applied to investigate the binding activity with a macrophage cell line--RAW264.7, and then the conjugation between RAW264.7and APS was observed by laser-scanning microscope. Griess method and ELISA were performed to investigate the production of NO, TNF-α or GM-CSF secreted from macrophage cell line RAW264.7. PMB, which is a specific inhibitor for LPS, was used to exclude the contamination of APS.The influence on p65level of NF-κB heterodimer p50/p65in the nuclear extract from activated macrophage cell line RAW264.7induced by APS, as well as NO, TNF-α secretion from activated RAW264.7further blocked by NF-κB inhibitor, were examined by Western blot. Anti-TLR4was used to evaluate the NO production of macrophage induced by APS, so that the possible mechanism of APS via TLR4/NF-κB signal pathway were preliminary discussed. In the third part, we performed basic research to explore the effects of ASP on the phenotypic and functional maturation of DC derived from murine bone-marrow cells. DC surface molecules including CD11c and MHC class Ⅱ molecules and the endocytosis capacity of DC were determined by immunofluorescence staining and flow cytometry. The protein level of mouse IL-12in supematants was detected by ELISA. Furthermore, the scanning electromicroscope was used to explore the ultra-structure of DC which was activated by APS..The results showed that the polysaccharide with the average MW of69KD was successfully obtained, weighting0.952g and containing92%suger. APS1abled by2-AMAC could specifically bind to macrophage in a time-dependent manner, and the binding effect could be significantly blocked by unlabled APS, which demonstrated that the inhibition could possibily due to the saturation of specific receptor.50or100μg/ml APS could remarkably boost the production of NO (P<0.05), and25,50or100μg/ml APS could siginificantly increase the level of TNF-a, GM-CSF secreted by macrophage. Macrophage treated by LPS was used as the positive control. To test the possible contamination of bacterial LPS within APS, we examined the effect of PMB on APS-induced production of NO. The data demonstrated that the activation of macrophages by APS was not a result of the contamination of LPS. Moreover, anti-TLR4could totally block the production of NO from RAW264.7induced by APS. The western blot results demonstrated that NF-κB was involved in the course of the activation induced by APS. The peak of protein level of NF-κB from nucleus was detected6h after stimulating by APS. In addition, PDTC, an inhibitor of NF-κB, could significantly block NO production from RAW264.7induced by APS.APS (10,50,100or250μg/ml) could significantly increase the expression of CD11c and MHC class Ⅱ molecules on DC. The ability of non-stimulated DC to uptake FITC-dextran was much higher than that of APS-or LPS-treated DC. The concentration of IL-12secreted by DC was futher analyzed by ELISA. The results showed that APS-treated DC secreted much higher level of IL-12secretion, and the morphology of APS-or LPS-treated DC bearing with long protrusions was also more mature than untreated DC.Conclusions:1. We have successfully established a novel method with full intellectual property for separation, purification and identification of APS and using this method we are able to obtain desired APS with different molecule weights.2. APS can efficiently induce NO, TNF-α and GM-CSF secretion from RAW264.7through binding with TLR4.3. APS can significantly up-regulate the expression of MHC class Ⅱ molecules and CDllc on DC, boost the secretion of IL-12, suppress the ability of phagocytosis and induce the maturation of the phenotype and functions of DC.