Study On The Effect Of Pine Pollen Sulfated Polysaccharide On RAW264.7 Cell Surface Receptors

It is unknown if the pine pollen polysaccharides function through the cell surface or enter cells.In the previous laboratory,the fluorescence labeling of Pinus massoniana pollen polysaccharide showed that polysaccharides could enter the cell.After treated with cell surface receptor TLR4 inhibitor,it could significantly inhibit the entry of polysaccharides into cells,suggesting that polysaccharide may effect cell function through cell surface receptors.Besides that the fluorescent tagged polysaccharides provide a technical basis for tracer polysaccharides.In our laboratory it has also shown that sulfated pine pollen polysaccharides have more functions than unesterified polysaccharides,but it is unclear whether sulfated pine pollen polysaccharides function in cells or through cell surface receptors.Therefore,the pollen polysaccharides of Pinus massoniana were esterified and labeled with fluorescence,and the effects of polysaccharides and cell surface receptors were investigated.The main contents of this paper are as follows:1.The extraction,separation,purification and detection of pine pollen polysaccharides.The pine pollen polysaccharides were extracted by hot water extraction.Trichloroacetic acid was used to remove protein and ethanol was fractionally precipitated to obtain 60% ethanol-precipitated polysaccharide,named PPM60.And then separated and purified by medium pressure preparative chromatography.Five polysaccharide peaks were separated and the purity of each polysaccharide peak was measured.The better-purified peak III was selected and named PPM60-III.2.The sulfation and fluorescence labeling of PPM60-III were carried out.The PPM60-III was sulfated by chlorosulfonic acid pyridine method and the sulfate substitution degree was 1.21,which was named SPPM60-III.SPPM60-III was labeled with tyrosine reduction method,and the fluorescence substitution degree was 0.43%,and it was named FSPPM60-III.The results of infrared spectrometer and fluorescence spectrometer showed that the PPM60-III was successfully sulphated and fluorescently labeled.The content of polysaccharide in FSPPM60-III was measured,which was 71.74%.We also used FDSS(FITC-dextran sulfate sodium)for controlled experiments.3.To explore the effect of FSPPM60-III on the cell activity,phagocytosis and migration of RAW264.7 cells.The results showed that both FSPPM60-III and FDSS significantly promoted cell proliferation at a concentration of 200?g/mL.Follow-up experiment was selected as 200?g/m L for polysaccharide concentration.The results showed that PPM60-III and FSPPM60-III can significantly promote phagocytosis;FSPPM60 can significantly promote cell adhesion;PPM60-III,FDSS and FSPPM60-III have no significant effect on cell migration.4.To investigate the role of three receptors TLR4,TLR2 and Dectin-1 on the surface of macrophages with polysaccharides.CLSM results show colocalization of the three receptors with FSPPM60-III and FDSS.After the cells were treated with inhibitors of the three receptors,polysaccharides were added for FCM detection.The results showed that all three receptor inhibitors significantly inhibited the entry of polysaccharides into the cells,indicating that the polysaccharides may interact with the three receptors.5.The effects of three receptors on the intracellular calcium and cytokine secretion of IL-1?,IL-6,TNF-? in RAW264.7 were investigated.The results showed that the increase of intracellular calcium concentration induced by FSPPM60-III and FDSS was related to TLR4,TLR2 and Dectin-1 receptors on the macrophage surface.Three receptor inhibitors can also or partially inhibit the increase of cytokine secretion by polysaccharides;and the results again indicate that FSPPM60-III and FDSS act through three receptors on the surface of macrophages.Conclusion: FSPPM60-III can significantly promote cell proliferation,cell adhesion and cell phagocytosis to enhance the immune regulation ability of the body.And the co localization of FSPPM60-III and cell surface receptors can significantly inhibit the entry of polysaccharides into cells.Addition of receptor inhibitors can significantly inhibit the increase of intracellular calcium and cytokines induced by FSPPM60-III.It is suggested that the cell surface receptor TLR4,TLR2 and Dectin-1 are the targets of FSPPM60-III,and the FSPPM60-III may also combine with other receptors on the cell surface to play polysaccharide effects.