Quercetin Stimulates Bone Marrow Mesenchymal Stem Cell(BMSC) Differentiation Via Estrogen Receptor And BMP Signal Pathway

Objective.Quercetin is one of the principal flavonoids and widely used in traditional Chinese medicine.A variety of studies have begun amassing evidence regarding the anti-inflammatory,anti-carcinogenic,neuroprotective,and cardioprotective effects of quercetin.Moreover several recent studies demonstrated that quercetin inhibits ovariectomy induced osteopenia in female rats.However,the mechanism by which quercetin induces osteogenic differentiation remains unclear.The present study aimed to investigate the overall effect of quercetin on mouse bone marrow mesenchymal stem cell(BMSC)proliferation and osteogenic differentiation in vitro.Materials and Methods.A frozen vial of mouse BMSCs isolated from C57BL/6 mice was purchased from Cyagen Biosciences Inc.The cells were thawed and then expanded in the complete medium(CM).The cells treated with quercetin at the indicated concentrations before being cultured for an additional 6 days.Then cell activity was determined by CCK-8 method at predetermined time.The cells were treated with quercetin or estrogen for 7 or 21 days.The effects of quercetin on BMSC osteogenic differentiation were analyzed by an alkaline phosphatase(ALP)assay kit,and Alizarin Red S staining(ARS).The cells were treated with quercetin or estrogen for 7 days,then the transcription level of osterix(OSX),runt-related transcription factor 2(RUNX2),and osteopontin(OPN)was determined with quantitative real-time PCR(qPCR).The cells were treated with quercetin or estrogen for 7 days with the estrogen signaling pathway blocked by estrogen receptor antagonist,and then the transcription level of OSX,RUNX2,and OPN was determined with qPCR.The cells were treated with quercetin for 7 days with the estrogen signaling pathway blocked by estrogen receptor antagonist,and then the expression level of bone morphogenetic protein 2(BMP2),Smad1,Smad4,OSX,RUNX2,and OPN,and the Smad1 phosphorylation was determined with western blot.Results.The CCK-8 and ALP assays and ARS staining showed that quercetin significantly enhanced BMSC proliferation,ALP activity,and extracellular matrix production and mineralization,respectively.The qPCR results indicated that OSX,RUNX2,and OPN transcription in the presence of osteoinduction medium,and the western blotting results indicated that quercetin enhanced bone morphogenetic protein 2(BMP2),Smad1,Smad4,RUNX2,OSX,and OPN expression and Smad1 phosphorylation.Treatment with the ER inhibitor ICI182780 blocked the effects of quercetin.Conclusions.Our data demonstrated that quercetin promotes BMSC proliferation and osteogenic differentiation.Activating or enhancing the BMP/smad signaling pathway via estrogen receptor pathway is the possible mechanism by which quercetin plays its pharmacological role.