Evaluation Of Immune Enhancement Function Of Polysaccharides From Angelica Dahurica And Blackberry Seeds Based On RAW264.7 Cells

The immune system has the important function of maintaining the normal life activities of the body.The body judges the "non-self" component according to the immune system,and destroys the damaged cells and tumor cells produced by the foreign bacteria and the human body,so as to maintain the balance of the human body function.Many studies have confirmed that plant polysaccharides can safely improve human immune function.Our group have isolated and purified nine polysaccharides from A.dahurica and blackberry seed,among which ADPs-1a,ADPs-3a and BSP-2 in high contents.All the above polysaccharides had certain inhibitory effects against tyrosinase and anticoagulant activity.Since no immunological studies on pure polysaccharides from A.dahurica and blackberry seed have been reported,,ADPs-1a,ADPs-3a and BSP-2 were used to explore the immune activity in this study.Macrophages play an important role in immune system by phagocytosis and release cytokines to participate in various stages of the immune reaction.There are two types of polarization on macrophages: M1 and M2.M1 cells exhibited pro-inflammatory reaction,maintained Th1-dependent immune response,and produced inflammatory mediators(such as IL-1,IL-6,IL-12,NO,TNF-?)to engulf microorganisms or infect / mutating cells.M2 cells showed anti-inflammatory response,enhanced Th2 response,released anti-inflammatory factor(such as IL-4,IL-10,TGF-?),secreted arginase 1,and promoted the production of Th2 cytochemical attractant.At present,most of the bioactive polysaccharides reported in the literaturet could promote the polarization of macrophage M1,stimulate the expression of IL-1,IL-6,IL-12 and TNF-?,promote Th1-directed adaptive immune responses,and inhibit the Th2 response.In this paper,the immunomodulatory function and potential mechanism of ADPs-1a,ADPs-3a and BSP-2 were detected by RAW264.7 cell with LPS(1 ?g/mL)as positive control group.First,RAW264.7 cells were stimulated with different concentrations of ADPs-1a(100-1600 ?g/mL),ADPs-3a(37.5-600 ?g/mL)and BSP-2(37.5-600 ?g/mL).Cell viability was detected by MTT assay.Phagocytic performance of cells was detected by neutral red method.The content of TNF-?,IL-6 and NO was determined by ELISA kit and Nitric oxide kit.The expression levels of iNOS,TNF-? and IL-6 mRNA were measured by qRT-PCR.The expression levels of key proteins in MAPK and NF-?B signals before and after the use of pathway inhibitors were determined by Western blot,and the mechanisms of ADPs-1a,ADPs-3a and BSP-2 on macrophage immune regulation were discussed.ADPs-1a,ADPs-3a and BSP-2 could significantly increase the phagocytic activity of RAW264.7 cells,and the ability of RAW264.7 cells to release NO and secrete TNF-? and IL-6.On this basis,ADPs-1a,ADPs-3a and BSP-2 could also significantly up-regulate the gene expression levels of iNOS,IL-6 and TNF-?,as well as the phosphorylation of p65 protein in the nucleus.It indicated that ADPs-1a,ADPs-3a and BSP-2 could promote the expression of NF-?B from cytoplasm into nucleus.Similarly,it was found that the phosphorylation level of p38 MAPK,ERK1/2,SAPK/JNK protein in MAPK pathway were significantly increased,and the level of proteins is significantly reduced after pretreatment with pathway inhibitor.These results suggested that the immunomodulatory effects of ADPs-1a,ADPs-3a and BSP-2 on macrophages may be related with activation of MAPK and NF-?B signaling pathways.The results provided a scientific basis for the further research and development of ADPs-1a,ADPs-3a and BSP-2.