The Research Of Salidroside On The Fracture Regeneration And Angiogenesis And The Corresponding Mechanism

Objectives: With a greater incidence of fractures in military training. In recent years, studies have shown that hypoxia can cause fracture delayed union or nonunion, reduce bone mineral density, and increase the incidence of the disease such as osteoporosis, fracture. Our country is the largest country in the plateau people living in the world,people are also affected by the hypoxia environment increasingly large,and the amed police troops in plateau are charged with various tasks all year round, it is facing serious altitude hypoxia injury issue. Therefore, the study of hypoxia bone metabolism and bone regeneration has a very important military and social significance. Researches have confirmed that salidroside has significant effect on anti-hypoxia anti-osteoporosis and angiogenesis. However, studies on the regulation of salidroside on bone regeneration and angiogenesis after fracture has not been reported,In this experiment, we choose mice with unstable tibial fracture and human umbilical vein cell line, EA.hy926 and human umbilical vein endothelial cell(HUVEC) and fetal mouse metatarsal as the research models to study the regulation of salidroside on bone regeneration and angiogenesis after fracture, and also explore possible molecular mechanism of salidroside.Methods:1 6-week-old male Kunming mice were randomly divided into two time points of 17 and 31 days,and each time point were randomly divided into five groups: ①Sham group ②FM group ③SAL-L group(200 μM) ④SAL-H group(800 μM) ⑤DFO group(200 μM). Mice in the Sham group were subject to sham operation. Tibial fracture surgery was performed in the other groups. Administration of drugs local injection every other day was carried out in 7 to 17 days after operation. The Sham and FM group received vehicle only.2 The fracture healing was evaluated by X-ray observation. The histomorphological analyses were performed by H.E stain. The hypoxia and HIF-1α signal pathway in Bone tissue were detected with immunohistochemistry. The parameters of bone biomechanics were determined with Instron materials testing machine.3 The proliferation activity assay with MTS method.4 The migration ability assay by Transwell method.5 Regulating effect of salidroside on tube formation of vascular endothelial cell by matrigel tube formation assay.6 The effect of salidroside on angiogenesis of fetal mouse metatarsal by fetal mouse metatarsal angiogenesis.7 Application of anti-VEGF antibody blocking experiments.8 The m RNA expression levels of HIF-1α signal pathway of vascular endothelial cell were detected by reverse transcription polymerase chain reaction(RT-PCR).Results:1 X-ray and HE staining showed salidroside has a good role in promoting fracture healing. Histomorphological change in mice tibial: salidroside could improve the trabecular area percentage, trabecular width and trabecular number in callus tissue, and reduce the trabecular separation of callus tissue. The change of parameters of bone biomechanics: materials and structural mechanics performance was obviously improved after salidroside therapy, and bone toughness and strength were enhanced obviously. Salidroside could also improve the fracture anoxic condition, increase the microvascular density of bone tissue, its mechanism might be related regulation HIF-1α expression, which up-regulated expression of downstream target gene VEGF.2 Salidroside could markedly promote vascular endothelial cell proliferation and migration. And salidroside could significantly promote tube formation of HUVEC and angiogenesis of fetal mouse metatarsal. However, anti-VEGF antibody(Avastin) can block the above phenomenon caused by salidroside.3 In normoxic state,salidroside could significantly promote m RNA HIF-1α and downstream target gene VEGF expression,and inhibit m RNA expression of downstream control gene p VHL in vascular endothelial cell. In hypoxic environment,salidroside could up-regulate HIF-1α, VEGF and p VHL m RNA expression levels.Conclusions:1 Salidroside could improve the fracture anoxic condition, increase the microvascular density of bone tissue, promote the formation of new bone, and accelerate fracture healing. It could significantly promote parameters of bone biomechanic and bone toughness and strength. This role may be by adjusting the mechanism of HIF-1α signaling pathways.2 Under hypoxic and normoxic conditions, salidroside could significantly promote proliferation, migration and tube formation of vascular endothelial cell, and angiogenesis of fetal mouse metatarsal through the regulation of HIF-1α signal pathway to promote expression of target genes VEGF.