The Study Of Biotin Labeling Of Icaritin And Fishing Icaritin Promote Skeleton Formation Receptor

Icaritin,the most effective metabolites of icariin isolated from traditional Chinese medicinal plants,which has a strong promotion of bone formation activity.Epimedin belongs to flavonoids and has phytoestrogenic activity.This may be one of the mechanisms that promote the activity of bone formation.Secondly,it has been found that icaritin has the activity of activating non-estrogen signaling pathway in promoting bone formation.Capability is such as BMP(Bone morphogenetic protein),MAPK(Mitogen-activated protein kinase),WNT,etc.It is not clear how icaritin activates these signaling cascades,the initial target of icaritin to activate these signaling pathways is unknown.In order to elucidate the complete signal transduction pathway,the following studies are to be carried out in this study: 1.Biotinylated icaritin for biotinylated icaritin;2.Osteoblasts for detection of synthetic drug activity and immunofluorescence tracer;3.Fish the icaritin receptor using biotin/streptavidin affinity purification method;4.Mass spectrometry identification results of icaritin receptor and its composite protein and literature reports and ours combination of previous studies,infers a complete signal transduction pathway;5.Use blocking method to verify and finally establish at least one complete signal transduction pathway.Methods : This subject first synthesized biotinylated icaritin by biotin and icariin,verify the activity of synthetic drugs and the ability to promote osteoblast maturation and mineralization,and the distribution of immunofluorescent tracer drugs in osteoblasts.Then using Bio-molecule interaction analysis core-technology(Biacore),fishing icaritin receptor,mass spectrometry detection,immunoprecipitation and immunofluorescence colocalization techniques to verify the fishing protein;subsequent blocking method to detect fishing protein’s influence in the process of the drug’s action,determine the drug’s transduction pathway in its process of promoting osteogenic activity.The results of the study indicate that biotinylated icaritin can be successfullysynthesized and that the ability of osteoblasts to verify the synthesis of drugs to promote cell maturation and mineralization is not affected;therefore,follow-up studies can be carried out,using the Biacore fishing method to fishing the affinity protein with biotinylation icaritin,analyzed by mass spectrometry to obtain the chemokine receptor type 4(CXCR4).The blocking method have shown that blocking CXCR4 has a significant effect on the maturation and mineralization of osteoblasts,and has a significant effect on the expression of BMP2-Smad1/5/9 signaling pathway related proteins.Epimedin may promote the maturation and mineralization of osteoblasts maybe through the CXCR4-BMP2-Smad1/5/9 signaling pathway.The conclusions are as follows:1.Biotin labeling can successfully synthesize biotinylated icaritin and ensure that its drug activity is not lost.2.Biacore fishing method was used to capture the protein related to biotinylated icaritin and G protein-coupled receptor-related protein(CXCR4)was detected by mass spectrometry.Immunofluorescence staining successfully colocalized the biotinylated icaritin and CXCR4.3.Co-immunoprecipitation verified the results of Biacore fishing.The expression of CXCR4 protein in different time was significantly higher than other time at 15 min and 30 min.BMP2 also showed the same trend.4.Icaritin promotes bone formation dependent on the CXCR4-BMP-Smad signaling pathway.Blocking CXCR4 significantly inhibited the protein expression of BMP2 and p-Smad1/5/9 in osteoblasts.