Preparation Of Fermented Astragalus Polysaccharides And Its Effect On The Signaling Pathway Of Murine Bone Marrow-derived Dendritic Cells Maturation In Vitro

Probiotics FGM,Streptococcus alactolyticus,was isolated from chicken caecum.Previous study showed that FGM fermentation could be used to improve the yield of polysaccharides of astragalus membranaceus.Polysaccharides from astragalus membranaceus fermented with FGM Strain had the preventive and therapeutic effect of hepatic fibrosis in rat,promote production performance of broiler chicken,improve immune organ index,and enhance the immunity.This study investigated that the effects of the fermented astragalus membranaceus(FAPS)on the signaling pathway of bone-morrow-derived dendritic cells maturation using the method of ELISA,RT-PCR and western blot in mice.The methods of FAPS extraction and purification were optimized.The main reults and conclusions as follows:1.Response surface method was used to optimize main process parameters for the extractions of polysaccharides from astragalus membranaceus fermented with probiotic FGM strain.The results showed that extraction time,extraction temperature and solid-liquid ratio were significant factors on polysaccharide content from the fermented astragalus membranaceus.An interactive effect was observed between every two factors.The optimum extraction conditions as follow: time was 65 min,temperature was 80 ℃,solid-liquid ratio was 1∶9,and the polysaccharide content was 6.72 mg / m L under this condition.2.The yield and purity of polysaccharides were detected using phonel-sulfate method,as well as the level of endotoxin using tachypleus amebocyte lysate(LAL)method.The results showed that the endotoxin of the fermented astragalus polysaccharides and the astragalus polysaccharides extracted with the optimization method were less than 0.1 EU / m L.The yield of fermented astragalus polysaccharides was 3.31% and higher than 1.91% of astragalus polysaccharides.The purity of fermented astragalus polysaccharides and astragalus polysaccharides were 91.3% and 88.5% respectively.3.After the dendritic cells was treated with different concentrations of fermented astragalus polysaccharides,the DCs volume was obviously increased and the surface convex was longer and more obvious.ELISA and RT-PCR results showed that the concentration of cytokine(included IL-12,TNF-α,IL-1β,and IL-6)and their m RNA were increased significantly.The dendritic cell treated with fermented astragalus polysaccharides could induce T cell proliferation.4.RT-PCR and Western blot techniques results indicated that the fermented astragalus polysaccharides could induce the maturation of dendritic cell through TLR2 or TLR4 signaling pathway.FAPS could significantly increase the protein expression of phosphorylated JNK,P38 and NF-k Bp65.These results suggested that MAPK and NF-k B signaling pathway were activated with the maturation process of dendritic cells treated by FAPS.In conclusion,extraction time,extraction temperature and solid-liquid ratio were significant factors on polysaccharide content fermented from astragalus membranaceus and there was interactive effects between these factors.Using the optimized extraction and purification method,the higher purity of polysaccharide could be obtained from the fermented astragalus and the non-fermented astragalus.Fermented astragalus polysaccharide could induce the maturation of dendritic cells through JNK MAPK signaling pathway,P38 MAPK signaling pathway and NF-κB signaling pathway.