The Study On Sea Urchins Polysaccharide Regulated BMP-2 Relate For Inducing Osteogenic Activity

Due to the particularity of marine life and environment,the synthesis process of polysaccharide in marine organisms is different from that of terrestrial faunas,which produces bioactive substance with novel structure and special physiological functions.Marine polysaccharide could be alternatived as an active ingredient for drugs to prevent or cure some diseases due to its various biological activity,such as immunomodulatory,antitumor,antivirus,anti-aging,reducing blood pressureand anti-obesity.With the increasing research on the structure of sea urchin polysaccharide,it raises concerns about the biological functions and pharmacological activities of sea urchin polysaccharides and has made great progress in anti-tumor and immunoregulation activity.The study aimed to investigate the effects of the sea urchin polysaccharide(SUP)combined with recombinant human bone morphogenetic protein-2(BMP-2)on the osteogenic differentiation of mouse osteoblast(MC3T3-E1 cells)in vitro.SUP was extracted by complex enzyme method,ethanol precipitation and ultrafiltration.The monosaccharide composition was analyzed by 1-phenyl-3-methyl-5-pyrazolone(PMP)derivatization.MC3T3-E1 cells model treated with different concentrations of SUP were used to test the osteogenic activity of SUP.MTT method was adopted to detect the proliferation rate of MC3T3-E1 cells and the activity of alkaline phosphatase(ALP)was detected by BCIP/NBT staining.The staining of mineralized nodules and content of osteocalcin were detected by alizarin red staining and Elisa.Immunofluorescence was used to detect the binding ability of BMP-2 to cells surface and Western Blot assay was used to detect the expression of BMP-2 and Runx-2 related to osteogenic differentiation in MC3T3-E1 cells.Finally,the effect of SUP on the proliferation and differentiation of MC3T3-E1 cells was analyzed by SPSS software.The results indicate that SUP contained mannose(Man),glucosamine(GlcN),glucose(Glc)with molecular weight of 3.5-5kD.MC3T3-E1 induced with SUP shows morphological changes,such as,distinct cell plasm salience,compact cell link.MTT result shows that SUP with the concentration of 1μg/mL,5μg/mL,10μg/mL,25μg/mL,50μg/mL and 100μg/mL accelerated the proliferation of MC3T3-E1 cells during 24-120 h.SUP modulated BMP-2 led to a significant increase in the activity of alkaline phosphatase(ALP)at concentrations of SUP for 10 μg/mL and BMP-2 for 100 ng/mL compared with BMP-2 alone.The result is also verified by alizarin red staining and detecting the contents of osteocalcin at the period of mineralization.BMP-2 has the strongest binding capacity with cell under the concentration of SUP for 10 μg/mL and BMP-2 for 100 ng/ml by immunofluorescence analysis.Differentiation of osteogenesis cells related to the structure and function sites of BMP-2 and cell surface receptor.In addition,western Blot result indicates that SUP promotes the expression of BMP-2 and Runx-2 protein.It was demonstrated in the experiments that SUP and BMP-2 have synergistical effects on promoting the proliferation,differentiation and calcification of MC3T3-E1 cells by regulating transcription factor of BMP-2 signal path.This finding indicates that SUP could promote the differentiation into mature bone cells and has the potential as a new type of functional food development and utilization,which provides a new idea for the prevention or mitigation of osteoporosis and fracture disease.