Investigation On The Mechanism And Prevention Of Osteoporosis By A New Model Of Hypoxia Mimicking Agent

Currently,the therapy for osteoporosis has tended to clarify the regulatory mechanism of osteoblast differentiation and bone formation.Activating hypoxia /HIF-1? pathway promotes the beneficial effect of bone formation,and guides the development of medical hypoxia mimicking agents and prevention and treatment of bone lesions.As a chelating agent,Desferrioxamine can reduce the content of iron in bone tissue,and increase the expression of HIF-1? in tissue cells,and stimulate the activity of osteoblasts and improve the blood supply of bone tissue,in order to prevent osteoporosis.However,many systemic toxicities related to DFO have been reported in the literature.To achieve the goal of increasing the concentration of DFO in bone tissue and reducing the systemic adverse effects of drugs,and achieving the optimal effect of DFO in bone,we designed and synthesized a new type of hypoxia mimicking agents' SF-DF0.Objective:This study was to evaluate the ability of SF-DFO to promote hypoxia /HIF-1? pathway in vitro,and to evaluate the effect on osteoporosis in vivo,and tried to explore the related mechanism.Methods:1)We designed and synthesized a new type of hypoxia mimicking agents' SF-DF0 through the patent technology.We used cell culture,CCK-8 detection,Real-time PCR detection and luciferase reporter gene detection to evaluate the toxicity of SF-DFO and the role of hypoxia /HIF? pathway in vitro.2)This research used a single intravenous injection of SF-DFO in mice at four-time points to evaluate the distribution of SF-DFO in vivo;We used intraperitoneal injection of SF-DFO in ovariectomy mice osteoporosis model for five-week continuity,and used biomechanical and Micro-CT detection,histological staining and pathological sections,to evaluate SF-DFO side effects in treatment of osteoporosis and the adverse effects of the important organs.3)We used the irradiated rat osteoporosis model and hip specimens from patients,ovariectomized mice osteoporosis model and Vhl knockout primary osteoblasts as the research objects,and used cell culture,tissue morphology,Real-time PCR detection,Westernblot detection and immunohistochemical detection,to evaluate the correlation between hypoxia /HIF-1? pathway and EZH2.Results:1)The SF-DFO has lower toxicity of bone marrow mesenchymal stem cells and primary mice osteoblasts than DFO,and the ability to activate HIF signaling pathway is similar to DFO,and SF-DFO can promote the expression of VEGF and HO-1.2)The binding rate of SF-DFO to serum protein in mice is low,however the binding rate to bone is higher than DFO.It can significantly improve bone mineral density,bone microstructure and mechanical properties of femur in ovariectomized mice,and SF-DFO has little side effects on tissue damage.SF-DFO promote the expression of HIF-1 ?,Runx2,and Osterix of bone tissue.3)The bone mineral density and osteoblast activity decreased in the irradiation of rat,and the expression of BMP2 and Runx2 decreased and the expression of EZH2 increased in bone marrow mesenchymal stem cells in the irradiation of rat.The expression of EZH2 levels increased in human osteoporosis hip tissue.The expression of EZH2 decreased in the SF-DFO treatment of osteoporosis in ovariectomized mice.The expression of EZH2 decreased in the Vhl knockout primary osteoblast cells.Conclusions:1)The new type of hypoxia mimicking agents' SF-DF0 has the characteristics of low toxicity and the ability to activate the hypoxia /HIF-1? pathway.2)SF-DFO has a stable bone targeting effect in vivo,which can improve bone mineral density and bone microstructure in ovariectomized mice,and no obvious side effects are observed,and promote the expression of HIF-1 ? of bone tissue3)SF-DFO may regulate osteoblast differentiation by activating the hypoxia /HIF-1? signaling pathway and inhibit the expression of EZH2.