Mechanistic Investigations Of The Effective Components Of Spatholobus Suberectus For The Prevention And Treatment Of Osteonecrosis Of Femoral Head Based On The Theory Of Huo Xue Hua Yu

Objective:This investigation were performed to investigate morphological changes of Formononetin,Daidzein and Calycosin,which are three effective components from Spatholobus suberectus,on osteoclast with bone marrow macrophages(BMMs)into osteoclasts.To-investigate the effect and the underlying molecular mechanism of Formononetin on osteoclast differentiation.To investigate the effect and the underlying molecular mechanism of Formononetin,Daidzein and calycosin on osteoblast with bone marrow mesenchymal stem cells(BMSCs)into osteoblasts.Combining the above cell experiments to investigate the mechanisms of the effective components of Spatholobus suberectus for the prevention and treatment of bone destruction in ONFH.Methods:(1)BMMs from C57/BL6 mice(Both male and female are included)were isolated and cultured from the bilateral femur and tibia.The cell viability assay was performed with cell counting kit(CCK8)and the safety dosages of Formononetin,Daidzein and Calycosin for in vitro investigation were measured.BMMs were were cultured with the stimulation of M-CSF and RANKL.DMSO were added and served as the Control group.Different concentrations of Formononetin,Daidzein and Calycosin were added and served as the targeted intervention group The cell culture was fixed at 4-6 days and Tartrate-resistant acid phosphatase(TRAP)staining was performed.The TRAP-positive osteoclasts under each view were counted and statistical analyses were performed.(2)Proteins from with 10 ?M Formononetin cells were collected and the expression of osteoclastic transcriptional factors of NFATcl and c-Fos was detected by Western blot assays.The expression of phosphorylated protein of ERK in osteoclastogenesis was also detected.RNA was extracted after 96-hour's treatment of cell culture,and the expression of osteoclastic-associated genes of CTSK,TRAP,MMP9 and Car 2 were detected by Real-Time PCR.(3)BMSCs also from the bilateral femur and tibia were plated on 24-well plates and were cultured with the stimulation of osteogenic medium and different concentrations of Formononetin,Daidzein and Calycosin were added and served as the targeted intervention group The cell culture was fixed at 5-7 days and Alkaline phosphatase(ALP)staining was performed.After 21 days alizarin red staining was performed.RNA was extracted and the expression of osteoblastic-associated genes of OCN,Col-1,ALP and Runx2 were detected by Real-Time PCRResults:(1)The cell viability assays identified that the safe and effective dosages,of Formononetin,Daidzein and Calycosin are less than 20?M(p>0.05).(2)Formononetin inhibits osteoclastogenesis in a dose-dependent manner and completely inhibits osteoclastogenesis at 10?M.At the protein level,Formononetin significantly suppressed the expression of NFATc1 and c-Fos in osteoclastogenesis,but exerted no inhibitory effects on the expression of phosphorylated expression of ERX.Formononetin significantly suppresses the expression of functional osteoclast genetic expressions of CTSK,TRAP,MMP9 and Car 2(P=0.000,P<0.05).(3)After 7 days of BMSCs culture,ALP staining showed that three effective components could promote the differentiation of osteoblasts,and the number of alizarin red stained mineralized nodules increased significantly after 21 days of culture.Three effective components significantly promote the expression of functional osteoclast genetic expressions of OCN,Col-1,ALP,Osterix and Runx2(P=0.000,P<0.05).Conclusion:The component of traditional Chinese medicine Spatholobus suberectus could control the bone formation and bone resorption to prevent and treat bone destruction of ONFH.The mechanism may be related to the promotion of osteoblast production and activity and inhibition of osteoclastogenesis and activity.