Study On The Mechanism Of Active Components Of Er-Zhi-Wan In Regulating Bone Remodeling

Bone remodeling is a key pathological process of osteoporosis.Osteogenesis mediated by osteoblasts and bone resorption mediated by osteoclasts are dynamically coupled.Regulating the proliferation and differentiation of osteoblasts and osteoclasts is an important direction in the treatment of osteoporosis.This study evaluated the effect and molecular mechanism of the compatibility of effective components of Er-Zhi-Wan on osteoblasts and osteoclasts.Previous experiments screened potential targets for anti-osteoporosis,and completed the construction of biological regulatory network model.The first part of this study aims to investigate the role of other Semaphorins in wedelolactone-enhanced osteoblastogenesis and-inhibited osteoclastogenesis.Wedelolactone inhibited RANKL-induced Sema4 D and Sema7 A production,but had no effect on RANKL-reduced Sema6 D expression in osteoclastic RAW264.7 cells.In mouse bone marrow mesenchymal stem cells(BMSC),wedelolactone reversed osteogenic medium(OS)-reduced Sema7 A expression and OS-enhanced Sema3 E mRNA expression,but no effect on OS-reduced Sema3 B mRNA expression.Addition of Sema4 D antibody promoted wedelolactone-reduced TRAP activity and bone resorption pit formation.Wedelolactone combined with Sema4 D antibody inhibited the formation of Sema4D-Plexin B1 complex.In co-culture of BMSC with RAW264.7 cells,Sema7 A antibody,similar with Sema 3A antibody,reversed wedelolactone-enhanced ALP activity and mineralization level,but promotedwedelolactone-inhibited TRAP activity.However,Sema3 E and Sema3 B antibodies had no effect.Further,wedelolactone enhanced the binding of Sema7 A with PlexinC1 and Beta1,but addition of Sema7 A antibody partially blocked this binding.Our data demonstrated that wedelolactone inhibited Sema4 D production and Sema4D-PlexinB1 complex formation in RAW264.7 cells,thereafter inhibiting osteoclastogenesis.At the same time,wedelolactone enhanced osteoblastogenesis through promoting Sema7 A production and Sema7A-PlexinC1-Beta1 complex formation in BMSC.The second part of the present study aimed to evaluate the combined effect of wedelolactone and oleonuezhenide,two compounds from Chinese formula Er-Zhi-Wan,on osteoblastogenesis and the underlying molecular mechanisms based on signal network.Wedelolactone combined with oleonuezhenide enhanced osteoblast differentiation and bone mineralization.Osteoblastogenesis-related marker genes including osteocalcin,Runx2,and osteorix were upregulated in the presence of wedelolactone and oleonuezhenide.At the molecular level,oleonuezhenide did not affect GSK-3? phosphorylation induced by wedelolactone,but elevated casein kinase2-alpha(CK2?)expression,resulting in ?-catenin and Runx2 nuclear translocation.In addition,30 ?M wedelolactone-induced cytotoxicity in bone marrow mesenchymal stem cells was relieved by 9 ?M oleonuezhenide.These cells were protected by oleonuezhenide and maintained osteoblastic activity.Oleonuezhenide increased Wnt5 a and CK2? expression.Wedelolactone-reduced extracellular signal-regulated kinase(ERK)phosphorylation was reversed by oleonuezhenide.These results suggested that oleonuezhenide enhanced the effects of wedelolactone on osteoblastogenesis.The results of this paper will provide a scientific basis for Er-Zhi-Wan clinical anti-osteoporosis treatment,and promote the development and clinical application of innovative traditional Chinese medicine for the ingredients of Er-Zhi-Wan for osteoporosis.