Daidzein Couples Osteogenesis-angiogenesis By Targeting Caveolin-1/EGFR Signaling

BackgroundPhytoestrogens are polyphenols similar to human estrogens found in plants or extracted from plant precursors.In recent years,studies have found that it has antioxidant,anti-inflammatory and anti-osteoporosis activities,and also can prevent aging-related cardiac-cerebral vascular and other chronic diseases.Daidzein is one of the most important phytoestrogens,and the research on its anti-osteoporosis bone protective effect mainly focuses on osteoblasts and osteoclasts,meawhile,the research on its endothelial cells is relatively few.Caveolae are 50-100 nm sized cystic invaginations on the plasma membrane,and in vertebrates contain three Caveolae proteins,encoded as Caveolin-1(Cav-1),Caveolin-2(Cav-2),Caveolin-3(Cav-3).Caveolins are mainly found on the cytoplasmic membrane,but also in the Golgi apparatus,endoplasmic reticulum,vesicles and cytoplasm.Caveolin-1 is widely expressed in adipocytes,endothelial cells,osteoblasts,fibroblasts,smooth muscle cells and various epidermal cells.Recent studies found that inhibiting Caveolin-1 or knocking out its gene can enhance osteoblast proliferation and matrix mineralization,and play a role in increasing bone mass.In addition,several important studies have found a close relationship between osteogenesis and angiogenesis.Therefore,whether Daidzein can promote physiological functions of endothelial such as cell migration and proliferation,thereby promoting angiogenesis and increasing boneMethodsFirst,bilateral ovariectomy(OVX)was performed on 3-month-old female C57BL/6 mice to establish an osteoporotic animal model,and then 25 mg Daidzein/KG bodyweight was administered to the OVX mice by intragastrical gavage for 8 weeks.Then,it was determined whether the change in bone mass was caused by changes in osteoblasts or osteoclasts by HE staining,OCN staining and TRAP staining.Then,immunofluorescence co-staining of CD31,EMCN and P-EGFR was performed to explain the changes in the number of blood vessels in the distal femoral medullary cavity of each group and the possible reasons for the changes.Secondly,in vitro cell culture experiments,the effect of Daidzein on the migration and proliferation of rat bone marrow endothelial cells(BMECs)was studied.Finally,Western Blot and QPCR were used to detect the expression of related proteins and genes,and to clarify the related molecular mechanism of bone mass change.In addition,we also explored its effect in aged male C57BL/6 mice,gavage 25mg Daidzein/KG bodyweight for 8 weeks,took the femur for Micro-CT scan,and confirmed the change of bone mass in each group.Then,it was determined whether the change of bone mass was caused by the change of osteoblast or osteoclast by HE staining,OCN staining and TRAP staining.Results1.Daidzein promotes the increase of bone mass in OVX mice by inhibiting caveolin-1.Compared with the Sham group,the bone mass in the OVX group was significantly reduced,and after the intervention of Daidzein,the bone mass in the Dai group was significantly increased compared with the OVX group.The results of HE staining,OCN staining and TRAP staining showed that the number of osteoblasts in the OVX group was significantly reduced and the number of osteoclasts was significantly increased,while the number of osteoblasts in the Dai group was significantly increased and the number of osteoclasts was significantly reduced.Daidzein increases bone mass by promoting osteoblasts and inhibiting osteoclasts functions.2.The results of immunofluorescence co-staining of distal femur sections confirmed that,compared with the Sham group,the positive fluorescence intensity of CD31 and EMCN in the OVX group was significantly weakened,and after the intervention of Daidzein,the positive fluorescence intensity of CD31 and EMCN in the Dai group was significantly increased compared with the OVX group,and the expression of phosphorylated EGFR protein(P-EGFR)increased in Dai group compared with OVX group.It is proved that OVX can reduce the number of blood vessels and Daidzein may increase angiogenesis and improve bone mass by promoting the expression of P-EGFR protein.3.Western Blot results:10μM Daidzein can inhibit the expression of phosphorylated caveolin-1 protein(P-caveolin-1),promote phosphorylated EGFR protein(P-EGFR),phosphorylated PI3K protein(P-PI3K)and phosphorylated AKT protein(P-AKT)protein expression,indicating that Daidzein may promote angiogenesis through the Caveolin-1/EGFR/PI3K-AKT signaling pathway,thereby increasing bone mass.QPCR results:The expressions of vascular endothelial growth factor(VEGF)and angiopoietin-1(ANGPT1)in the 10 μM Daidzein group were higher than those in the control group,which proved that Daidzein could promote the expression of VEGF and ANGPT1 in vascular endothelial cells.4.Micro-CT analysis of distal femoral cancellous bone in aged male mice showed three items:the ratio of bone volume to tissue volume(BV/TV),bone mineral density(BMD),and trabecular bone number(Tb.N),which have increased significantly.The results of Micro-CT analysis of distal femoral cortical bone showed:cortical bone area(Ct.Ar),cortical bone inner diameter(endosteal perimeter),cortical bone outer diameter(periosteal perimeter),cortical bone thickness(cortical thickness)four indicators were significantly increased.It was confirmed that Daidzein can also increase bone mass in aged male mice.Histological staining,OCN staining and TRAP staining of femoral sections could observe an increase in the number of cancellous bone trabeculae near the growth plate of the distal femoral medullary cavity after Daidzein intervention,and an increase in the number of osteoblasts and a decrease in osteoclasts around the trabeculae.ConclusionsDaidzein can increase bone mass in ovariectomized mice(OVX),the possible mechanism is that it can promote endothelial cell angiogenesis by targeting the Caveolin-1/EGFR/PI3K-AKT signaling pathway to achieve angio-osteogenesis coupling,thereby increasing bone mass.Caveolin-1/EGFR signaling pathway may become a new target for anti-osteoporosis treatment.