Pine Pollen Sulfated Polysaccharides Affect The Cytoplasmic Signaling Pathway Of RAW264.7 Cells

There were some researches focused on the polysaccharide from pine pollen and its effects on immune cells about metabonomics in our lab.We also studied the effects of polysaccharides with different molecular weight on the immune cells and the influence of FITC labeled polysaccharide from pine pollen on the cells.We have obtained the skilled technique of labeling FITC to the polysaccharide from pine pollen.In this study,we wonder whether the sulfonic polysaccharide from pine pollen could be labeled the FITC.It was reported that FITC labeled polysaccharide from pine pollen could enter cells,involved in the toll-like receptor 4and most percentage of pinocytosis,including the clathrin.It means that there are some other ways existing involved in the entering of the polysaccharide,apart from the receptor-mediated endocytosis.According to the results in our lab,the FITC labeled polysaccharide could enter the cells very quickly.Hence,we guess that the entered polysaccharide may mediated the downstream immune response through the intracellular receptors.In this study,the interaction of NOD2 and NLRP3 in the cytoplasm was investigated to induce the downstream immune response after the polysaccharide was entered into the RAW264.7 cells.The main contents of this paper are as follows:1.Extraction of pollen polysaccharide by the method of water boiling and ethanol precipitation used in the laboratory.First,the pollen was extracted with hot water and the three chloroacetic acid method was used in addition to the precipitation of 20%,40%,60%and 80%ethanol.The precipitated polysaccharide by 60%ethanol was worked as the object and named it PPM60,with 47.32%of polysaccharide.The components of crude polysaccharide PPM60 were purified by medium pressure preparation chromatography.It was found that the third peak of PPM60 had the best separation.Hence,the third peak of PPM60 was acted as the experiment object,named it PPM60-III2.The labeling of sulfonic and FITC to the PPM60-III.PPM60-III was sulfated with pyridine-chlorosulfonic acid,and the product was named SPPM60-III after freeze-drying,with 1.21 of sulfonic degree.SPPM60-III was reacted with tyrosine and named SPPM60-Tyr-III after freeze-drying.The reaction of SPPM60-Tyr-III to fluorescein isothiocyanate?Fluorescein isothiocyanate,FITC?was named FSPPM60-III after freeze-drying.The fluorescence degree of FSPPM60-III was0.43%with 71.74%of polysaccharide.3.Using MTT method to detect the FSPPM60-III activity of RAW264.7 cells in mouse macrophages,we found that the best concentration FSPPM60-III on the activity of RAW264.7 cell was 200?g/mL.The best concentration of FDSS,as the positive control,was 200?g/mL on the activity of RAW264.7 cell.For PPM60,the concentration was 400?g/m L.In the subsequent experiment,these mentioned concentrations were acted as the experimental concentration.The effects of FSPPM60-III on the adhesion,migration and phagocytosis of RAW264.7 cells were detected by adhesion test,scratch healing experiment and neutral red test.It was found that FSPPM60-III significantly enhanced the ability of cell adhesion,migration and phagocytosis on the RAW264.7 cell,compared with other components.4.Using laser scanning confocal microscope,we can observe that the signaling of fluorescent labeled polysaccharides can enter into cells.The content of FSPPM60-III and FDSS in cells was 5%and 24.3%,respectively,tested by flow cytometry.5.The detection of fluorescent labeled polysaccharides by immunofluorescence markers can co-localize with NOD receptors.After adding NOD2 receptor inhibitor Ponatinib and NLRP3 receptor inhibitor MCC950 to RAW264.7 cell,the co-localizations of FSPPM60-III and FDSS and receptors were decreased.By adding Ponatinib and/or MCC950,it was found that the amount of RAW264.7 cells entered cells was reduced.6.By detecting intracellular calcium concentration,it was found that both FSPPM60-III and FDSS could increase the intracellular calcium concentration.The increase of intracellular calcium ions was reduced by adding Ponatinib and/or MCC950 to the cells treated with polysaccharides.Through the secretion of ELISA kit for detection of cell of four cytokines IL-6,TNF-?,IL-1?,IL-18,FSPPM60-III and FDSS are able to increase the cultivation of four cytokines concentration in medium.The increase of cytokines in the medium was reduced by adding Ponatinib and/or MCC950 to the cells treated with polysaccharides.In this study,the pollen polysaccharide of Pine massoniana was successfully sulfated and fluorescently labelled,named FSPPM60-III.It is confirmed that FSPPM60-III could enter the RAW264.7 cells and localize with the intracellular receptors NOD2 and NLRP3.These two receptors might be involved in mediating the effects of FSPPM60-III on the immune activity of RAW264.7 cells,including the changes in the concentration of[Ca²⁺]_I and the secretion of a variety of cytokines.