红景天苷经氧感应信号通路抗骨质疏松分子机制的研究

Hypoxia-inducible factor-1α (HIF-1α) is a key molecule that couples angiogenesis to bone formation during bone development and regeneration and suggest the feasibility of molecular targeting of the HIF pathway to enhance bone formation. As a traditional Tibetan medicine, Rhodiola has extensive pharmacological effects including anti-hypoxia, angiogenesis and anti-osteoporosis. Previously, we found that salidroside (SAL), the main effective component of Rhodiola, was able to promote proliferation, early differentiation as well as expression of HIF-1α and vascular endothelial growth factor (VEGF) in human osteoblastic MG-63 cells. Meanwhile, we also found that hypoxia (1% oxygen) increased the expression of interleukin-8(IL-8) in osteoblast-derived cells. These data suggest that SAL may play an important role in the regulation of bone development and regeneration via activation of the HIF pathway coupling angiogenesis and bone formation. In this project, we will first determine the role of HIF-1α in hypoxia-induced IL-8 expression of osteoblast. Using neonatal rat calvaria osteoblasts, human osteoblast-like MG-63 cells, human umbilical vein endothelial cells (EAHY926), fetal mouse metatarsals and two animal models of osteoporosis, we will investigate whether SAL affects bone development and regeneration via activation of oxygen sensing signal pathway coupling angiogenesis and bone formation from the tissular, cellular and molecular level. Moreover, we will further explored the mechanisms underlying its actions.

低氧诱导因子(HIF)-1α是骨代谢和骨再生过程中调节骨形成耦联血管生成的重要介导因子，可能成为促进骨生成药物研究的新靶点。藏药红景天具有抗缺氧、促血管新生及抗骨质疏松等多种药理作用，本课题组前期工作证实，其主要活性成分红景天苷(SAL)可促进成骨细胞增殖、早期分化及HIF-1α和VEGF表达；同时还发现低氧可促进成骨细胞表达另一种重要促血管生成因子IL-8。提示SAL可能通过激活HIF-1通路介导血管生成和骨生成耦联而在调节骨代谢与再生中发挥重要作用。本项目拟在原工作基础上，首先确定HIF-1α在低氧诱导的成骨细胞IL-8表达中的作用，同时利用新生大鼠颅骨成骨细胞、人成骨样MG-63细胞、人脐静脉内皮细胞EAHY926、胎鼠跖骨及两种骨质疏松症动物模型，从组织、细胞及分子水平研究SAL调控氧感应信号通路介导血管生成和骨生成耦联，进而调节骨代谢与骨再生过程，同时揭示其抗骨质疏松的相关机制。

HIF-1α是骨代谢与再生中调节骨形成耦联血管生成的重要介导因子，可能成为促进骨生成药物研究的新靶点。藏药红景天具有抗缺氧、促血管新生及抗骨质疏松等多种药理作用，红景天苷(SAL)是其主要活性成分。本项目首先证实低氧或内源性过表HIF-1α均可显著上调成骨细胞IL-6、IL-8的表达水平；并首次证明HIF-1α能促进IL-6、IL-8的转录活性，二者亦是 HIF-1α的下游靶基因。同时证实：1）常氧下SAL除上调成骨细胞HIF-1α、VEGF和下调pVHL表达外，还增强其HIF-1α的转录活性；低氧可促进成骨细胞HIF-1α核转位、转录活性增强，并使其表达水平升高，低氧还可上调VEGF表达及下调pVHL表达；SAL预处理后可下调HIF-1α表达、减少HIF-1α核转位，但增强HIF-1α转录活性及上调VEGF和pVHL表达。2）常氧和低氧下SAL可促进成骨细胞增殖、分化及矿化，低氧可导致成骨细胞凋亡率增加，SAL能抑制低氧诱导的成骨细胞凋亡；SAL可通过上调Osterix、Runx2表达促进成骨细胞的分化和成熟。3）常氧下SAL可促进血管内皮细胞增殖、迁移和管腔形成以及胎鼠跖骨血管生成；低氧下血管内皮细胞的增殖能力减弱而其迁移、管腔形成能力以及胎鼠跖骨血管生成能力增强，SAL预处理后可促进血管内皮细胞增殖、迁移和管腔形成以及胎鼠跖骨血管生成。应用抗VEGF抗体Avastin证实，常氧和低氧下SAL促血管内皮细胞增殖、迁移和管腔形成以及胎鼠跖骨血管生成功能是VEGF部分依赖的。4）SAL可显著增加去势大鼠股骨骨密度、湿重、干重及灰重，降低去势大鼠血清Ca、BGP及ALP水平而增加E2水平；SAL能显著改善骨质疏松部位材料力学和结构力学的性能，增强骨的韧性和强度，并能显著抑制去势大鼠骨的高转换状态及活跃的骨矿化与骨改建；SAL还可改善去势大鼠骨组织中的缺氧状况，增加骨组织的微血管密度，其机制可能与其活化HIF-1α/VEGF通路有关。本成果在国际上率先证明SAL通过调控成骨细胞HIF-1/VEGF信号通路介导血管生成骨形成耦联，从而促进骨代谢和骨再生。该成果为全面深入阐明SAL促进骨生成的作用机制奠定基础，同时也为今后临床应用SAL治疗和预防骨质疏松症提供科学依据。