| The polysaccharides extracted from the pollen of Pinus massoniana has a variety of biological activities,such as the inhibition of Hep G2,the regulation of T,B lymphocyte immunity in the spleen and Ca2+ level in smooth muscle cells.Based on the previous work of the laboratory,the single component of polysaccharides was isolated and purified by a new chromatography technique called MPLC(medium pressure liquid chromatography).We used the methods of chemical modification and degradation on polysaccharide to explore the effects of structure on the immune regulation of macrophages,and analysis the corresponding relationship between them.1.Polysaccharide from 200 g Piuns massoniana pollen was extracted with hot water and precipitated by 20%,40%,60% and 80% alcohol.Protein which may interfere the study was removed by trichloroacetic acid method.We obtained 0.98 g PPM20,0.54 g PPM40,2.98 g PPM60 and 2.74 g PPM80.PPM60 and PPM80 were used as the material for separating the single component.2.The polysaccharides of pine pollen were isolated and purified by medium pressure preparative chromatography.The four standard dextrans were mixed and then separated with MPLC in order to explore the appropriate chromatographic conditions.The final conditions were determined as the elution flow rate of 2.5 ml/min with distilled water,the sample volume was 1ml and the concentration was 80 mg/ml.The column pressure was controlled at 2-4 bar,delayed6 minutes to collect the component and collect a tube every 40 s.The polysaccharide PPM80 was separated by MPLC.According to the experimental requirement,the second peak which purified and collected was named PPM80-B.We measured its molecular weight was 317.76 K.PPM80-B was degradated using hydrochloric acid into small molecule polysaccharide which named DPPM80-B and its molecular weight was 3.13 K.The two polysaccharides were sulfated with chlorosulfonic acid-pyridine method.The new components named SPPM80-B and SDPPM80-B,their degree of the substitution is 1.08 and 1.34 respectively.3.MTT assay was used to detect the effects of four polysaccharides on the relative activityof mouse macrophages RAW264.7 at different concentrations.The results showed that the pine pollen polysaccharide could promote the cell growth in low concentration and inhibition of cell growth in high concentration,the optimal concentration of proliferation was 400 ?g/ml.The effect of chemical modification on the activity of polysaccharides was showed that sulfated polysaccharides significantly enhanced the proliferation of RAW264.7 cells,which was 46.2%higher than that of non-sulfated polysaccharides.In terms of molecular weight,the proliferation ability of non-degradable polysaccharides on macrophages was 24.3% higher than that after degradation.4.The macrophages were stimulated by four polysaccharide components.The effects of polysaccharides on immunoregulation were studied by measuring the adhesion,migration,phagocytosis and secretion of cytokines and inflammatory mediators.We observed the role of polysaccharides in the cell functions by adding the inhibitor of polysaccharide receptor TLR4 and explore the molecular mechanism of cell surface receptors to identify polysaccharide components and trigger signaling pathways.The results showed that the sulfated polysaccharide significantly enhanced the adhesion,migration and phagocytosis of macrophages,induced the secretion of cytokines IL-1,IL-6,TNF-? and the production of NO and NOS compared with control group.In the respect of cell migration and secretion of cytokines,the biological activity of undegraded polysaccharides is higher than that after degradation.TAK-242 treatment group effectively inhibited the effect of sulfated polysaccharide on macrophage immunomodulatory ability,and the inhibition rate was about 45%.It is verified that the process of activating macrophages by polysaccharide components is partly affected by the recognition of surface receptor TLR4. |
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