Study On The Influence Of Astragtlus And Polyporus Umbellate Polysacchaide On The Intestinal Epithelial Cells And The Phagocytosis Of Macrophage Via The Intestinal Epithelial Cell

Polysaccharide or multi-polysaccharide was a kind of polar natural macromolecules having higher polymerization degree over 10 with Molecular mass from ten thousands to millions,which was basically consisted of aldoses linked by indicant bond,and was almost found in all organisms including animal,plant,microbe and seaweed.Polysaccharide is the most important biomacromolecule in organism and one of the basic materials in keep normal life activity.Besides biological functions,Polysaccharide also has important medicine value.As great part of protect people from diseases, the Chinese medicine is naturally occurring drugs,Polysaccharide,as one of the effective ingredients in TCM,is widely used in TCM as a treatment for immuno-regulation,tumor, inflammatory,viral,etc.Recently,most study focusedon the function of herbal polysaccharide on immunomodulation,anti-tumor with low toxicity,it has great potentiality in clinical application.With development of modem science and technology as well as molecular biology, the research on Polysaccharide had been further studied to cellular and molecular levels, but the effective pathway after Polysaccharide taken orally is not very clear.It is seemed that the Studies of polysaccharides has been confined to the traditional immune cells,and incomprehension on the function of interaction between Polysaccharide and intestinal epithelial cells,which Polysaccharide first contacted after oral administration.Intestinal epithelial cells are the first line of defense,participating in innate immune response and can play the role of antigen presenting;besides,it can have"cross-talk"with mucosal immune cells.The effection of polysaccharides on intestinal epithelial cells plays its pharmacological role through the regulation of antigen presenting ability as well as the secretion of Cytokines,chemokines,defensins and other mediators of intestinal epithelial cells,killing the pathological target directly or influencing immune system by indirect effect by improving or reducig the cross-talk between intestinal epithelial cells and other immune cells.It is a good ideas to be studied on.Exploring on the mode of action of oral administration polysaccharides means make people well understandingthe role of polysaccharides,it will settle a bsae for Changing dosage form of polysaccharides as well as modifying the molecular molecular constitution of polysaccharides.We chose astragalus polysaccharides and polyporus umbellatus polysaccharose as our research object,in oder to study the effect of astragalus polysaccharides and polyporus umbellatus polysaccharose on the expression of HBD-1(humanβ-defensins-1),HBD-2 (humanβ-defensins-2)mRNA,IL-8 and TLR4,seting one pole layer of the intestinal epithelial cells with the cells SW480,which was stimulated by LPS to imitate the internal environment of the distance of intestinces.And the influnence of astragalus polysaccharides and polyporus umbellatus polysaccharose on the phagocytosis of macrophage-like cells THP-1 has been also studied via the intestinal epithelial cells.1.Experimental research1.1 Study on biological characteristics of SW480 cellsSW480 growth curve drawning,screening against a number of the best.The sensitivity of SW480 cells to lipopolysaccharide was assayed by CCK-8,in accordance with experimental results and reference materials select the appropriate stimulation concentration so as to stimulated intestinal environment of the intestinal inferior segment. Select the appropriate concentration of astragalus polysaccharide(APS)and polyporus umbellate polysaccharide(PUPS)by toxicity test.1.2 Effect on the expression of APS and PUPS on SW480 cells HBD-1,HBD-2 mRNAUsing RT-PCR detect the HBD-1,HBD-2 mRNA expression of APS,PUPS acting on Single-layer model of intestinal epithelial cells of SW480.1.3 Effect on the expression of APS and PUPS on SW480 cells IL-8Using RT-PCR and ELISA detect the IL-8 mRNA expression and secretion of APS, PUPS acting on Single-layer model of intestinal epithelial cells of SW480.1.4 Effect on the expression of APS and PUPS on SW480 cells TLR4Using RT-PCR and FCM detect the TLR4 mRNA and cell surface marker TLR4 expression of APS and PUPS acting on Single-layer model of intestinal epithelial cells of SW480 and evaluate the single-layer model of intestinal epithelial cells possible receptor of SW480 though APS and PUPS.1.5 The influence of APS and PUPS acting on the cross-talk between intestinal epithelial cells and lamina propria macrophageEstablish two models of APS and PUPS acting on the single-layer of intestinal epithelial cells SW480 with its conditioned medium co culture with macrophage-like cells THP-1. The fluorescent microsphere which is fluorescence Labeled latex beads was Phagocytosis by macrophage THP-1 on the principle of Macrophage phagocytosis of inert material,and the phagocytosis were detected by flow cytometry method and Calculate the phagocytic rate and phagocytic index.2.Conclusion2.1 The expressed HBD-2,IL-8 of SW480 cells by APS,PUPS may through TLR4The proximal end of small intestine is bacteria-free,while there is a great quantity of bacterias and high level LPS in the distal end of small intestine and colon of normal organism.In order to explore the effect of APS,PUPS on the expression ofβ-defensins HBD-1,HBD-2 and IL-8 and the possible receptor,SW480 cells was cultured in vitro,and two cell model including the unstimulated SW480 cells and the stimulated SW480 cells by LPS were built to simulate the epithelial cell in physiological condition of upper and lower intestine.First,the effect of APS,PUPS on the expression ofβ-defensins HBD-1,HBD-2 was detected by PT-PCR,the results indicated that APS,PUPS had no effect on expression of HBD-1 mRNA in the intestinal tract,but could induce intestine epithelia cells expressing HBD-2 mRNA in upper intestine,while the induced expression did't exist in the intestine epithelia cells of lower intestine.IEC is the major source IL-8 of human body,so the expression of IL-8 was detected in the APS and PUPS stimulated SW480 cells by RT-PCR,ELISA,the results suggested that APS,PUPS could promote the secretion of IL-8 through inducing the transcription of IL-8 RNA.Research had indicated that the activation of TLR4 signal transduction pathway promotes the transcription and translation of HBD-2,IL-8,so the expression of TLR4 was detected in the APS and PUPS stimulated SW480 cells by RT-PCR,FCM,the result implied that TLR4 may be one receptor of SW480 cells recognizing APS and PUPS.2.2 APS,PUPS increased the phagocytosis ofmacrophage via intestine epithelia cellsIEC is located to the macrophage of lamina propria,and there is a"cross talk"between them,IEC affects the phagocytosis of macrophage via paracrine secretion.In order to explore the effect of APS,PUPS on the phagocytosis of macrophage via IEC,two cell model including the unstimulated SW480 cells and the stimulated SW480 cells by LPS were built to simulate the epithelial cell in physiological condition of upper and lower intestine,and PMA induced the macrophage-like THP-1 cells was adopted to simulate the macrophages of lamina propria.The results suggested that the cytokines of IEC induced by APS and PUPS increased the Phagocytic rate of macrophage,and the Phagocytic index of macrophage in lower intestine could also be increased by APS,PUPS.conclusively,APS,PUPS increased the phagocytosis of macrophage via IEC.3.The meaning of researchThrough the establishment of single-layer model of intestinal epithelial cells and the stimulation with LPS of SW480 so as to simulated the intestinal segment on the internal environment of the single-layer intestinal epithelial cells,investigate the expression of APS and PUPS on the intestinal epithelial cellβ-defensins HBD-1 and HBD-2,chemokine IL-8 and the effect of the"cross-talk"between intestinal epithelial cells and lamina propria macrophages.The study also tests the possible receptor TLR-4 on the intestinal epithelial cells of SW480.The paper makes clear that oral administration of APS,PUPS play its role in immune regulation by intestinal epithelial cells,and laid the foundation of a comprehensive study of the role of polysaccharide in the gastro-intestinal mechanisms and a new target of the polysaccharide.