Modulation Of T Cell Responses By Fucoidan To Regulate The Osteogenesis

Objective:The influence of sterilized fucoidan on T cell proliferation and differentiation is investigated.We are also looking into how fucoidan affects osteogenic differentiation by affecting T cells and thus osteogenic differentiation.Methods:In this study,fucoidan was sterilized by high-temperature autoclave and characterized by UV-visible spectroscopy,X-ray diffraction,nuclear magnetic resonance and Fourier transform infrared（FT-IR）spectroscopy before and after autoclaving.In addition,the monosaccharide composition and molecular weight of fucoidan before and after autoclaving were determined by high-performance liquid chromatography.Mouse primary T lymphocytes were extracted by immunomagnetic bead negative selection,and the autoclaved fucoidan was co-cultured with T cells to determine the effect of fucoidan on T cells.We set up five groups:0 mg/m L（control group）,0.4 mg/m L fucoidan group,2mg/m L fucoidan group,10 mg/m L fucoidan group,and 50 mg/m L fucoidan group,respectively.The effect of fucoidan on T cell proliferation was determined by CCK-8 and CFSE staining,and the effect of fucoidan on T cell apoptosis was determined by Annexin V/propidium iodide staining.The effect of fucoidan on T-cell differentiation was determined using flow cytometry.To investigate the effect of fucoidan on osteogenesis by affecting T cells,we co-cultured fucoidan with T cells and then took the supernatant with MC3T3-E1 cells and determined its effect on osteogenesis by detecting alkaline phosphatase expression and staining for calcium nodules.Graph Pad Prism 8 or Origin 9was used to evaluate all statistical analyses.Results:（1）The fucoidan before and after autoclaving showed typical absorbance peaks at 260 nm in UV-visible spectroscopy,and the overall crystallinity of fucoidan before and after autoclaving was low at 23°in X-ray diffraction analysis.The NMR analysis of fucoidan before and after autoclaving showed signal peaks at 4.66,4.32,4.00 and 3.70 ppm,representing 3-linkedα-L-fucose,α-L-fucose,3-linkedβ-D-galactose andα-L-rhamnopyranose residues,respectively.The FT-IR analysis of fucoidan before and after autoclaving showed characteristic peaks at 3403,2936,1632,1214,1074 and 830 cm^-1,representing O-H,C-H,C=O,S=O,C-O-C and C-O-S,respectively.The total sugar content of fucoidan before and after autoclaving was 78.61%and 70.91%,the sulfate content was28.42%and 30.23%,and the glyoxylate content was 0.9%and 10.7%,respectively.The above results proved that high-temperature autoclave treatment led to the depolymerization of fucoidan,but its active groups did not change.（2）The proliferation and apoptosis experiments of sterilized fucoidan on T cells proved that fucoian could reduce the apoptosis of T cells and promote the proliferation of T cells,and the proliferation effect was better with the increase of fucoian concentration in a certain range（0.4-50 mg/m L）.（3）The results of flow cytometry proved that the addition of fucoidan reduced the proportion of differentiated T cells,and the higher the concentration of fucoidan,the lower the proportion of differentiated T cells.Besides,the addition of fucoidan had no effect on the ratio of CD4+T and CD8+T cells.（4）The supernatant of fucoidan co-cultured with T cells had no significant effect on the proliferation of MC3T3-E1 cells.（5）The supernatant of T cells promoted the expression of alkaline phosphatase,while the supernatant of fucoidan co-cultured with T cells was able to inhibit the expression of alkaline phosphatase.Alizarin red staining similarly demonstrated that the supernatant of fucoidan co-cultured with T cells could inhibit the formation of calcium nodules.Conclusion:High-temperature autoclave sterilization caused depolymerization of fucoidan,but had no effect on its bioactive moieties.In a certain range（0.4-50 mg/m L）,the proliferative effect of fucoidan on T cells was enhanced with increasing concentration.On the contrary,fucoidan had an inhibitory effect on the differentiation of T cells,and the higher the concentration,the more pronounced the inhibitory effect.Fucoidan could inhibit osteogenesis by affecting T cells.