| BackgroundPostmenopausal Osteoporosis(PMO),as a common and high-incidence systemic and metabolic bone disease,has attracted more and more researchers.It is well known that postmenopausal women are prone to osteoporosis,which has been proved to be related to the lack of estrogen in vivo.Estrogen deficiency leads to the imbalance between bone resorption and formation.Previous studies have clearly shown that osteoclasts are the direct target of estrogen.Estrogen has many direct and indirect effects on the formation,activity and life span of osteoclasts.However,the exact mechanism of estrogen regulating bone formation remains unclear.Periostin is mainly expressed in periosteum and periodontal tissue,which is one of the most important extracellular matrix proteins to maintain the integrity of periodontal tissue structure and function.Periostin also plays a key role in regulating bone metabolism.17β-E2 and POSTN can promote bone formation,and may regulate osteogenesis through similar mechanisms.ObjectivesStarting from in vivo studies of bone growth and in vitro studies of proliferation and differentiation of bone marrow stromal cells(BMSCs),this study explores the relationship between 17β-E2 and POSTN,and the mechanism of 17β-E2 participating in regulating differentiation of BMSCs.Prevention and treatment of postmenopausal osteoporos is provides a new molecular target.Methods1.In vivo effects of 17β-E2 on bone formation and POSTN expression in OVX-ratsEight-week-old rats were randomly divided into two groups.The ovariectomy(OVX)and sham-operation(Sham)were performed.At 6 weeks after operation,Ct.Th,BV/TV and BMD of femur were measured by micro-CT to identify the success of postmenopausal osteoporosis animal model.The effects of 17β-E2 on osteogenesis in OVX-rats were observed by micro-CT at 2and 6 weeks after treatment.The distal femoral tissues of Sham,OVX and OVX+E2groups were detected by HE staining,dynamic analysis and Safranin-O stained(SO).The cartilage and bone formation,cortical thickness,bone mass and bone mass were analyzed and compared.The expression of POSTN in femoral tissue was detected by immunohistochemical method to verify the effect of 17β-E2 on cortical bone formation and the effect of estradiol on POSTN in vivo.2.Study on Osteogenic Differentiation and POSTN Expression of OVX-BMSCs by 17β-E2 in VitroBMSCs were isolated by whole bone marrow adherence separation method and subcultured to observe the difference of general growth state of the three groups of cells.Flow cytometry was used to identify the difference of molecular phenotypes on the surface of the three groups.Proliferation activity of CCK-8 cells was measured to detect the proliferation ability of the three groups of BMSCs.RT-PCR was used to detect the expression of ALP and Runx2,alkaline phosphatase(ALP)staining and activity test of BMSCs in each group.The osteogenic capacity of cells in each group was measured.The expression of POSTN in Sham-BMSCs,OVX-BMSCs and OVX+E2-BMSCs was identified by q RT-PCR,immunofluorescence and Western blot,and the effect of 17β-E2 on bone formation and POSTN expression in OVX-BMSCs in vitro was verified.3.Mechanisms of 17β-E2 in osteogenic differentiation of OVX-BMSCsOVX-BMSCs were stably transfected with a lentivirus carrying the GFP(Lev-GFP)and POSTN-GFP(Lev-POSTN)genes.Transfected OVX-BMSCs were pretreated with17β-E2 and Dkk1 respectively,the expressions of POSTN,β-catenin,RUNX2 and ALP were identified by q RT-PCR and Western blot.After transfection of OVX-BMSCs with si-Postn,the cells were pretreated with Wnt3 a and 17β-E2 respectively.The expressions of POSTN,β-catenin,RUNX2 and ALP were identified by q RT-PCR and Western blot,and the mechanism of POSTN-mediated17β-E2 regulating the osteogenic differentiation of OVX-BMSCs was discussed.Results1.In vivo study of 17β-E2 on bone formation and POSTN expression in OVX-ratsMicro-CT was used to detect bone formation 2 and 6 weeks after17β-E2 treatment.It was found that 17β-E2 promoted bone formation in OVX rats as early as 2 weeks after treatment.Dynamic analysis of the femur 6 weeks after treatment showed that bone formation mainly occurred in the periosteum.The expression of POSTN on the surface of the periosteum in OVX+E2 group was higher than that in OVX group by immunohistochemical method.It was found that the location of POSTN coincided with that of bone formation.2.Study on Osteogenic Differentiation and POSTN Expression of OVX-BMSCs by 17β-E2 in VitroThe ALP activity of OVX+E2-BMSCs was significantly higher than that of OVX-BMSCs.The results of osteogenesis-related gene expression were consistent with result of ALP activity.17β-E2 significantly increased the expression of POSTN in OVX-BMSCs.It is located in the cytoplasm.Therefore,17β-E2 increased the bone formation of OVX-BMSCs and the expression of POSTN.3.Mechanisms of 17β-E2 in osteogenic differentiation of OVX-BMSCsIn the transfected Lev-Postn group,17β-E2 +Dkk1 significantly decreased the expression of POSTN,β-catenin,RUNX2 and ALP,and inhibited osteogenic differentiation.These results suggest that Wnt pathway plays an important role in the osteogenic differentiation of 17β-E2 and POSTN.After transfection of OVX-BMSCs with si-Postn,OVX-BMSCs were treated with Wnt3 a and 17β-E2.The results showed that17β-E2 +Wnt3a slightly increased the expression of β-catenin and RUNX2 and the capability of osteogenic differentiation,but it’s not statistically significant(P >0.05).These results indicate that POSTN mediated17β-E2 to regulate the osteogenic differentiation of OVX-BMSCs.Conclusion1.17β-E2 promotes the expression of POSTN and regulates the proliferation and differentiation of BMSCs.2.17β-E2 reduces osteoporosis and promotes osteogenesis through the POSTN-Wnt/β-catenin pathway. |
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