The Effect And Mechanism Of Astragalus Polysaccharides On The Inflammatory Factors Secretion From Macrophage Stimulated By Lipopolysaccharide

Astragalus Polydsaccharide(APS) is one of the most effective and important ingredient isolated from the roots of Astragalus membranaceus, which signifycantly enhances immunity organ's function, promotes production of antibodies, improves macrophage activity, and strengthens of T,B cells and other immune cells such as NK cells.Lipopolysaccharide is a large molecular structure ingredient in the outer membrane of gram negative bacteria, it can induce macrophage to synthesis and release inflammatory mediators to result in many inflammatory reaction.So restraining the inflammatory mediators from macrophage becomes the investigative and therapeutical target of anti-inflammotory drugs. Human mononuclear macrophage U937 cells were induced to be mature by phorbol 12-myristate 13-acetate (PMA),and then stimulated by LPS to be inflammatory cell model to research the effect and mechanism of APS on the inflammatory factors secretion from macrophage stimulated by LPS. The main work as follow:1. Human mononuclear macrophage U937 cells were cultured, and indu-ced to be mature by PMA. The reasonable concentration of APS for the following experiments was studied by MTT assay.The result displayed that compared with the controls, the growth of U937 cells treated with 37.5～150μg/ml concentration of APS had no significant difference.2. After establishing inflammatory cell model,37.5～150μg/ml concentration of APS were treated for 48h. TNF-αand IL-1βreleased from the U937 cells were detected by ELISA assay. NO released from the U937 cells was detected by Griess assay. Expression of TNF-α, IL-1βand iNOS mRNA in U937 cells were detected by real-time PCR. The result displayed that the concentration of TNF-α, IL-1βand NO released from LPS stimulated U937 cells increased significantly than those in the controls(P<0.01). After treatment with APS, the levels were significantly lower than those in LPS group(P<0.05). TNF-α, IL-1βand iNOS mRNA expression in LPS group significantly increased as compared with control group(P<0.01). TNF-α, IL-1βand iNOS mRNA expression in each APS group significantly decreased as compared with LPS group(P<0.05).The content of endonuclear NF-κB was detected by western blot. The result showed that NF-κB P65 expression in LPS group significantly increased as compared with control group(P<0.01). NF-κB P65 expression in every APS group significantly decreased as compared with LPS group(P<0.05).In addition to macrophage, lymphocytes and dendritic cell are also the important immunocytes in the body. Lymphocytes are from central lymphoid organ and peripheral lymphoid organ, and distribute in peripheral blood, spleen, lymphaden and Peyer's patches, they migrate uninterruptedly between immunological induction and effector position in the eneral immune system and mucosal immune system, with themselves' differentiation and maturation, and participate in body's immune response. Dendritic cell is the most effective antigen presenting cells in the body, it could absorb,process and transmit antigen. APS impact cell multiplication and phenotype to reflect its immunoregulatory function.This thesis has done the following experiments based on theview:1. Peripheral blood mononuclear cell, lymph node cell, spleen lymphocytes and Peyer's patches lymphocyte were separated, and induced to proliferation by ConA. The effect of different concentration of APS on the growth of the cells was studied by MTT assay. The result displayed that compared with the controls, the growth of lymphocytes cells treated with 37.5～300μg/ml concentration of APS had significant difference (P<0.01)2. The mononuclear cells (MNCs) isolated from murine bone marrow were cultured in RPMI 1640 medium containing recombinant mouse granulocyte macrophage colony-stimulating factor(rmGM-CSF) and interleukin-4(IL-4). Six days later, add in 150μg/ml concentration of APS to affect 48h. The result displayed that Morphologically typical DCs were observed after the culturing for 8 days, with high expression of CD80, CD86 and murine major histocompatibility complexⅡI-A/I-E.