Discovery And Mechanism Investigation Of The Anti-osteoporosis Candidate Compounds Through Screening Based On Potential Molecular Targets In Bone Metabolism

With the increase of aging population,the number of osteoporosis patients in China is increasing year by year.Currently,most of anti-osteoporosis drugs have many side-effects while exerting the curative effect.Thus,to find safer and more effective drugs is still required.Osteoporosis is caused by abnormal bone metabolism due to the imbalance between bone formation and bone resorption.Promoting osteoblast differentiation and/or inhibiting bone resorption has become valid strategy for the treatment of osteoporosis.RUNX2,as a key transcription factor of osteoblast differentiation,is a gathering point of multiple signaling pathways involved in osteoblast differentiation and bone formation.RUNX2 transcriptional activators are speculated to have potential effects on osteoblast differentiation.NF-?B is one of the most important transcription factors regulating osteoclast differentiation,and inhibiting the activity of NF-?B may inhibit osteoclast formation.In this study,we intend to screen the small molecule compounds library and the natural products library using the cellular screening model based on RUNX2 and NF-?B transcriptional activity,to identify the candidate compounds that have potentials to promote osteoblast differentiation and to inhibit bone resorption,respectively.In order to achieve this goal,this study is divided into two parts.Part One:High throughput screening based on transcriptional activity of RUNX2 and study on the anti-osteoporosis effect of Compound T63Objective:To screen about 20,000 small molecule compounds using the cellular screening model based on RUNX2 transcriptional activity and to investigate the effect and molecular mechanism of T63 on bone metabolism both in vitro and in vivo.Methods:The luciferase reporter assay system was used to detect the effects of the tested compounds on the transcriptional activity of RUNX2.The effects of T63 on the cell proliferation,ALPL activity,mineralized nodules and genes expressions were measured by MTT method,p-NPP method,Alizarin red staining and PCR,respectively,in the mouse multipotent mesenchymal stem cell-like fibroblasts(C3H10T1/2)and mouse precursor osteoblasts(MC3T3-E1)cells.Western blot,ChIP and RNAi were applied to observe the effects of T63 on related signaling pathways involved in the regulation of osteoblast differentiation.ROS level and activity of ERE were analyzed by flow cytometry(FACS)and dual luciferase reporter assay system.The effects of T63 on the formation of lipid droplets and adipogenesis-related genes expression were measured by Oil Red staining and real-time PCR,respectively.TRAP staining,F-actin staining,PCR and Western blot were used to evaluate the effects of T63 on osteoclast formation,cell fusion,osteoclast differentiation related genes expressions and signaling pathways,respectively,in the osteoblast/osteoclast co-culture system and RANKL induced osteoclasts.Combining ovariectomy with dexamethasone treatment(OVX-D)rats was used to investigate the effects of T63 in vivo on bone mineral density,bone volume fraction and serum biomarkers.Results:Compound T63 was identified to be able to increase RUNX2 transcriptional activity from the screening of 20,760 compounds in the cellular screening model.T63 significantly increased ALPL activity,cell mineralization and Alpl,Bglap and Sppl,Runx2 and Bmp2 mRNA expressions without apparent cytotoxicity.Effect of T63's osteogenic effect on the human osteoblast cell lines MG63 and hFOB1.19 was consistently confirmed.Besides,the formation of adipocytes and mRNA expressions of Ppary2,Srebfl and Fabp4 in C3H10T1/2 cells was decreased after T63 treatment.T63 increased both mRNA and protein level of RUNX2 as well as the enrichment of RUNX2 protein in the Alpl promoter,whereas had little effect on the expression of transcription factor OSX.Knockdown of RUNX2 by shRNAs reduced RUNX2 transcriptional acitivity and T63-induced ALPL activity,indicating the osteogenic role of T63 is dependent on RUNX2 expression.Mechanistically,T63 increased both BMP/Smadl/5/8 and Wnt/?-catenin signaling pathways.Both Noggin(inhibitor for BMPs signaling)and DKK1(inhibitor for WNT signaling)markedly suppressed T63-induced RUNX2 expression and osteogenic phenotypes,suggesting BMPs and WNT/?-catenin signaling mediated T63-induced osteoblast differentiation through regulating RUNX2 expression.Moreover,T63 decreased the level of ROS and enhanced the transcriptional activity of estrogen response element(ERE)in MC3T3-El cells.With regard to the role of T63 in osteoclastogenesis,T63 inhibited Rankl expression and the number of TRAP-positive cells in the osteoblasts and osteoclasts co-culture system.The compound also directly suppressed RANKL-induced osteoclastogensis as well as the fusion of osteoclasts in a dose-dependent manner without significant cytotoxicity.T63 also inhibited osteoclastogenesis-related gene expression as well as MAPKs and Akt activation.T63 was also found to markedly increase the bone mineral density(BMD)and bone mineral content(BMC)in femurs,tibia and lumbar vertebrae of the OVX-D osteoporosis rat model in vivo.Moreover,T63 increased the number of osteoblasts and serum ALPL level,while reduced TRAP-positive staining area in the femurs and serum NTX-1 level as compared with OVX-D model group.Conclusion:A small molecule T63 is identifed as a RUNX2 transcriptional activator,which has potent anti-osteoporosis effect both in vitro and in vivo.T63 could increase RUNX2 transriptional activity through regulating both BMPs and canonical Wnt/?-catenin pathway to promote osteoblast differentiation.The compound also suppresses RANKL-induced osteoclast differentiation through affecting the signaling pathways involved in osteoclastogenesis regulation.T63 merits further study for potential application in osteoporosis treatment.Part Two:Screening of NF-?B transcriptional activity inhibitors and study on the effect of Aesculin on osteoclast differentiationObjective:We aim to establish a screening model based on NF-?B transcriptional activity,and to identify natural compounds with potentials to inhibit osteoclast differentiation.The effect of AES on bone metabolism both in vitro and in vivo and the underlying mechanism will be investigated.Methods:Liposome transfection method was used to transfect the RAW264.7 cells with NF-?B plasmid containing luciferase reporter gene and luciferase reporter assay system was used to detect the effects of natural compounds on luciferase activity.The cytotoxicity of AES in the RAW264.7 cells was measured by MTT method.TRAP staining,F-actin staining and PCR assay were used to investigate the effects of AES on RANKL-induced osteoclast formation,fusion and genes expressions,respectively.Western blot,dual luciferase reporter assay system and gene overexpression method were used to discuss AES's molecular mechanism.The effect of AES on the osteoblast differentiation in the MC3T3-El cells was investigated by MTT,p-NPP method and Alizarin Red staining.In addition,OVX-D rats was used to observe the anti-osteoporosis effect of AES in ivivo.Results:The screening model based on NF-?B transcriptional activity was established,and Aesculin(AES)was identified from 132 compounds.AES(5-20 ?M)inhibited RANKL-induced osteoclast differentiation and F-actin rings formation as well as the genes expressions dose-dependently without significant cytotoxicity.Also RANKL-induced MAPKs activation was inhibited after AES treatment.In addition,AES inhibited Rank gene expression,and overexpressed RANK level significantly blocked AES's effect on the osteoclast differentiation as well as the NF-?B activity upon RANKL induction,suggesting RANK might be involved in the regulation of AES during osteoclastogenesis.However,AES had no significant effect on the osteoblast differentiation.In the in vivo experiments,AES treatment in the OVX-D models increased BMD,BMC and the BV/TV%,while decreased NTX-1 level at both doses(20 mg/kg and 100 mg/kg),as compared with OVX-D control group.Conclusion:AES is identified to decrease NF-?B transcriptional activity in te screening model.It protects against bone mass loss by suppressing RANKL-induced osteoclastogenesis and transduction signals.AES therefore could be a promising agent for the treatment of osteoporosis.