Effects Of Hedysarum Polysaccharides And Flavonoids On The Osteogenic Differentiation Of RBMCSs And ROBs

With the aging of the population,the incidence of osteoporosis is increasing year by year,posing a great threat to human health.So far,the use of traditional Chinese medicine to treat osteoporosis has attracted the attention of more and more researchers.In this paper,Hedysarum polysaccharides and flavonoids in Hedysari Radix were administered to rat bone marrow marrow stromal cells?rBMSCs?and rat calvarial osteoblasts?ROBs?cultured in vitro,in order to screen out the component that has better osteogenic effects of both cells and explore the mechanism for promoting osteogenesis.The specific research contents carried out in this paper are as follows:The first part measured the changes of rBMSCs and ROBs activity and osteogenesis-related factors after treatment with different concentrations of three kinds of Hedysarum polysaccharides?HPS-1,HPS-2,HPS-3?.The results of MTT assay found 20 and 50?g/mL HPS-1 significantly promoted cells proliferation,inhanced Alkaline phosphatase?ALP?activity,and increased the areas and numbers of calcified nodules of rBMSCs and ROBs?P<0.05 or P<0.01?,while HPS-2 and HPS-3 did not significantly promote the osteogenesis of the two types of cells.These results show that only HPS-1 of the three Hedysarum polysaccharides can significantly promote osteogenic differentiation and mineralization of rBMSCs and ROBs.The second part measured the changes of rBMSCs and ROBs activity and osteogenesis-related factors after treatment with different concentrations of five kinds of flavonoids?calycosin,formononetin,ononin,isoliquiritigenin and medicarpin?from Hedysari Radix.It was found that five components could promote proliferation,inhance ALP activity,increase calcium contents and the areas and numbers of calcified nodules of rBMSCs and ROBs?P<0.05 or P<0.01?.Among them,10^-6mol/L calycosin has better effects on promoting osteogenic differentiation of both cells,and is superior to HPS-1.In the third part,HPLC method was used to determine calycosin concentrations in intracellular,extracellular and on the cell membrane of ROBs after different treatment time to understand the dynamic effects of calycosin in ROBs.The results showed that the linear range of extracellular calycosin was 1.300⁷.800?g/mL?R²=0.9997?.The linear range of calycosin of the cell membrane?dissociation solution?and intracellular fluid?lysate?was 0.052⁰.650?g/mL?R²=0.9995?.The average recoveries were 100.75%?RSD=1.22%?,99.10%?RSD=1.92%?,and100.34%?RSD=1.79%?for calycosin in extracellular fluid,dissociation fluid,and lysate.And each sample solution was stable for at least 24 h.The results show that a reliable HPLC method is established for the determination of calycosin content that specifically binds to cells.In the fourth part,we investigated whether IGF-1R/PI3K/Akt signaling pathway was involved in effect of calycosin promoting bone formation.It was found that the levels of phosphorylation of IGF-1R and Akt in ROBs were increased after treatment with 10^-6 mol/L calycosin.When using GSK1904529A and LY294002,the inhibitor of IGF-1R and PI3K,respectively,the rise of ALP activity,the expression of osteogenic related genes and proteins,the area and number of calcified nodules were significantly weakened after calycosin treatment?P<0.05 or P<0.01?,and the ability of calycosin to promote the differentiation and mineralization of ROBs was significantly inhibited.These suggest that IGF-1R/PI3K/Akt signaling pathway is necessary for the bone formation advanced by calycosin.