| Background:Osteoporosis,characterized by low bone mass,increased bone fragility and a higher risk of bone fractures[3],is one of common bone matabolic diseases.The incidence of osteoporosis has gradually risen in recent years,seriously affecting the health and normal life of the elderly.When osteoporosis happens on the jaw,bone mineral density would be reduced and biomechanical properties would be decreased,which would lead to the accelerating of the height and width of the alveolar ridge bone so as to make a big difficulty for prosthodontics.Thus there is a great need to discover novel anabolic agents for osteoporosis treatment,slowing down the absorption rate of the alveolar bone and maintaining its height to ensure the denture smoothly and aesthetically.Ginkgo biloba L,naturally occurring plant estrogens,has been extensively reported on their ability to affect bone metabolism.Ritu et al[1]proved that GBE(Ginkgo Biloba Extract)have positive effects on bone mineral density,bone microstructure,and osteoblast function in OVx(ovariectomized)rats.Wu Z et al[2]proved that GBE can promote the osteogenic differentiation and inhibit adipogenic differentiation of theBMSCs(bone marrow stem cells)in vitro,which provides a new direction for the treatment of osteoporosis at the cellular level.However,at the present time few studies have been concerned with the effect of GBE on cultured osteoblasts and the precise molecular mechanisms underlying the observed anabolic effects of GBE on osteoblasts are still under investigation.Over the past few years,the Wnt/?-catenin signaling pathway has been shown to be an important component of bone mass accrual,regulation,and maintenance[4,5].Growing evidence suggests that activation of this pathway increases osteoblastic differentiation and subsequent bone formation,while suppressing osteoclastogenesis[6].And the Wnt/?-catenin signaling pathway is of interest as a novel therapeutic target for development of osteoporosis treatment.However,the link between GBE and the Wnt/?-catenin signaling pathway in the case of osteogenic process has not been clarified.Thus in the present study,to settle this issue,we examine whether GBE activates Wnt/?-catenin signaling pathway in osteoblasts and if Wnt/?-catenin signaling pathway is required during GBE-induced osteoblastic differentiation.Objective:To investigate the effect of GBE on the differentiation of osteoblast and its possible mechanism of action in vitro.Methods:1.Cell cultureThe MG63 human osteoblasts were incubated in complete medium composed of DMEM,10% fetal bovine serum(FBS),and 100 IU/m L penicillin/streptomycin in a humidified 5% CO2 incubator at 37?.The culture medium was refreshed every 1-2 days before the cells reached80% confluence.And then the cells were subcultured by treatment with0.25% trypsin/0.02% EDTA and seeded into fresh flasks with the same medium for subsequent experimental procedures.2.The effect of GBE on the differentation of MG63Applying the different concentrations of GBE on the MG63.To estimate the effects of osteogenic ability of GBE by Alkaline phosphatase(ALP)activity at days 4 and 7,Alizarin red staining at 2 week.Quantitative real-Time PCR(q RT-PCR)was taken to test the expression of OC(Osteocalcin),Runx2,COL1A1(Collagen type 1,Alpha 1)m RNA at 1 week.3.The effect of GBE on the Wnt/?-catenin signaling pathway of MG63Applying the 75?g/m L GBE on the MG63 cells,and the inhibitor group underwent a pretreatment of DKK1,which is the specific inhibitor of Wnt/?-catenin signaling pathway.The results were evaluated by ALP activity at day 7,Alizarin red staining at 2 week.q RT-PCR was taken to test the m RNA expression of OC,Runx2,COL1A1,?-catenin and cyclin D1 at 1 week.Western Blot was adopted to inspected the protein level of ?-catenin,cyclin D1 and subcellular localization of ?-catenin.Results:1.Incubation of MG63 cell in GBE-supplemented culture medium,especially at the concentration of 75?g/m L,significantly increased ALP activity at days 4,7 as well as the calcium nodule formation and matrix mineralization by Alizarin red staining after 14 days incubation of GBE.2.The cultures treated with 75?g/m L GBE demonstrated that OC,Runx2,COL1A1,?-catenin and cyclin D1 m RNA expression were higher compared to the control culture.Western Blot analysis showed that75?g/m L GBE increased the ?-catenin,cyclin D1 protein level and the protein levels of ?-catenin in the nuclear fractions at day 7.However,the positive effects of GBE above were abolished by DKK1,a blocker of the Wnt/?-catenin recepetor.Conclusion:GBE enhances the osteogenic differentiation of the MG63 via activation of the Wnt/?-catenin pathway and the 75?g/m L was the most effective concentration. |
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