项目以体内外两个层面为视角，沿着Hedgehog通路平衡调控成骨和成脂分化的思路，朝着阐述复叶耳蕨黄酮促进骨质疏松骨再生机制的目标开展工作。体外以MSCs成骨和成脂定向分化细胞模型为对象，运用骨质疏松大鼠血浆药理学方法，以含药血浆干预成骨和成脂分化过程，采用特殊表型染色、qPCR、Western Blot、FIA等技术，评价MSCs成骨和成脂分化能力，检测Hedgehog通路关键分子表达水平，进一步采取外源性抑制和激动Hedgehog通路的方法，证实含药血浆借助Hedgehog通路增强MSCs成骨分化同时抑制成脂分化过程，系统阐述复叶耳蕨黄酮作用机制。体内以去卵巢大鼠模型为对象，运用骨形态计量、显微CT和成骨分化表型特征分析等方法，定性（量）评价骨再生效能，证实复叶耳蕨黄酮借助Hedgehog通路以增强成骨同时抑制成脂方式，促进骨质疏松病损区骨再生，为中西医结合治疗骨质疏松提供理论依据。

Abstract: The effects of Arachniodes flavonoids on promoting bone regeneration in osteoporosis have attracted extensive attentions; however, the cellular and molecular mechanisms remain unclear. We propose to employ a combination of in vivo and in vitro methods to understand how Arachniodes flavonoids promote osteoporotic bone regeneration through regulating Hedgehog signaling pathway. By using the cell models of directed osteogenic and adipogenic differentiation of mesenchymal stem cells (MSCs), we will first analyze the samples obtained from Medicated Plasma of Ovariectomized rat model to explore the molecular mechanisms leading to theosteogenic and adipogenic differentiation of MSCs by a series of integral　in vitro assays including qPCR, Western Blot, ELISA and immunofluorescence. These results will be further verified independently by up- or down- regulating Hedgehog signal pathways using specific agonists or inhibitors. In vivo, we will generate osteoporosis-ovariectomy rat model, then give various dosage of Arachniodes flavonoids to different experimental groups. After 8 and 12 weeks, the bone histomorphometry, bone ultrastructure, bone mineral density and specific gene expression　of each experimental group will be analyzed to evaluate the effects of Arachniodes flavonoids on promoting osteogenic differentiation and inhibiting adipogenic differentiation via hedgehog pathway in osteoporosis animal. This study will provide mechanism insights and theoretical basis of Chinese herbal medicine in the treatment of osteoporosis.