Mechanism Study Of The Role Of Neobavaisoflavone In Inhibiting Osteoclastogenesis And Alleviating Bone Loss Induced By Ovariectomy

Objective: With the rapid increase of our aging population group,our society faces huge challeges to prevent and control osteoporosis.Moreover,drawbacks of classic anti-resorptive drugs such as bisphosphonates have been exposed in clinical application.Hence,there is an urgent need to explore the next generation of anti-resorptive or anti-osteoporosis drugs to ensure safe and effective clinical usage.Traditional Chinese Medicine like seeds from Psoralea corylifolia(Buguzhi)has beed used for hundreds of years in Chinese history and now are widely applied in accelerating bone healing and osteoporosis treatment.However,as a result of the complexity of the mixtures of Psoralea corylifolia,as well as the lack of powerful tools to isolate and identify bioactive components for bone metabolism treatment,the potential bioactive monomers have not yet been fully exploited.The object of this study is to explore high-affinity monomers from the seeds of Psoralea corylifolia using two-dimensionalcell membrane chromatographic/C18column/time-of-flight mass spectrometry,and to examine the inhibitory effects and molecular mechanisms on osteoclastogenesis and osteoclast functions from one of the discovered potential bioactive component-neobavaisoflavone.It will lay scientific foundations for developing new drugs for bone metabolism manipulation.Method: Firstly,this study took advantage of two-dimensional RAW264.7 cell membrane chromatographic/C18 column/time-of-flight mass spectrometry to screen,isolate,and to identify the potential bioactive components from the ethanol extracts from Psoralea corylifolia.Neobavaisoflavone(NBIF)was discovered and validiated by affinity to RAW264.7 membrane.We set different NBIF concentration and treatment time courses to explore the inhibitory effects on osteoclast differentiation by TRAP staining using RAW264.7 cell line and bone marrow monocytes with the stimulation of NBIF.Osteoclast function was examined by Actin-ring assay and bone resorption assay.Western Blot was applied to demonstrate the specific markers’ levels for osteoclast differentiation and function.Immunoflorence was used to determine the nuclear translocation of NFATc1 upon RANKL stimulation.Calcium oscillation was detected after RANKL was added.Furthermore,Co-IP was used to elucidate whether NBIF affects the recruitment of TRAF6 and c-Src to RANK upon RANKL stimulation.μCT and HE staining were used to clarify the protective role of NBIF to the bone volume for ovariectomy-induced postmenopausal osteoporosis mice.The inhibitory effect on osteoclast differentiation in vivo was further determined by TRAP staining.Lastly,the in vivo bone resorption inhibitory effect was examined by ELISA experiment.Result: NBIF was identified as a potential bioactive component through two-dimensional cell membrane chromatographic/C18 column/time-of-flight mass spectrometry,as it showed high affinity to certain proteins on RAW264.7 cell membrane.TRAP staining,actin-ring assay,bone resorption assay collectively indicated that NBIF exhibited strong inhibitory effects on osteoclast differentiation and function in a concentration-and time-dependent manner.Western Blot results revealed that NBIF interrupted the activation of key pathways(NF-κB、MAPK、Akt)and the expression of key osteoclast marker genes(MMP9 、 Cathepsin K 、 CTR 、 TRAP)that are required for osteoclastogenesis.Immunoflorence showed that the nuclear translocation of NFATc1 was blocked by NBIF treatment.Calcium oscillation fluctuation was also suppressed by the treatment of NBIF treatment.Co-IP showed that NBIF disrupted the recruitment of TRAF6 and c-Src to RANK,which is located in the cellular membrane.In vivo,μCT、HE staning showed that NBIF significantly preserved trabecular bone volume from ovariectomy-induced bone loss.TRAP staining of paraffin sections and ELISA experiment revealed that NBIF inhibited osteoclastogenesis and bone resorption markers in the serum.Conclusion: In this study,two-dimensional cell membrane chromatographic/C18column/time-of-flight mass spectrometry was proved to be a powerful tool to screen and identify potential powerful bioactive monomers from traditional Chinese medicine.NBIF was further proved to potently inhibit osteoclastogenesis and osteoclast functions in vitro and in vivo.As for the molecular mechanism,we showed that NBIFcould possibly exert it effects by disrupting the recruitment of TRAF and c-Src to RANK,blocking NFATc1 nuclear translocation and calcium oscillation,as well as inhibiting the activation of NF-κB、MAPK、Akt signaling pathway and key osteoclastogenesis marker genes(MMP9、Cathepsin K、CTR、TRAP).NBIF is a promising agent to serve as an osteoclastogenesis inhibitor to be used in treating high bone turnover disease.Further exploration of the molecular targets of NBIF in the membrane can pave way to discover new generation of anti-resorptive and anti-osteoporosis drug.