GSK-3β/β-catenin信号通路介导的淫羊藿素预防绝经后骨质疏松的机制研究

Osteoporosis is a major bone metabolic disorder to threat people's health in our aging society. Determinating the fate of bone marrow mechencymal stem cells (BMSCs), i.e. promoting osteogenic differentiation and inhibiting adipogenic differentiation, becomes an important strategy to prevent osteoporosis. Our previous study found that icaritin was effective to prevent bone loss in ovariectomized (OVX) mice, and further it promoted the bone marrow mechencymal stem cells ostegenic differentiation and inhibited their adipogenic differention in vitro. However, the molecular mechanism for icaritin on preventiong osteoporosis remains unclear. Our recent study found that icaritin could inhibit the activity of GSK-3β by phosphoralyation of its serine 9 site, and promoted the translocation of β-catenin into nuclei. It was reported that inhibiting GSK-3β and promoting nuclear translocation of β-catenin, was key for promoting BMSCs osteogenesis and inhibiting their adipogenesis. Thus we hypothesize that icaritin may promote osteogenesis and inhibit adipogensis of BMSC to prevent osteoporosis via GSK-3β/β-catenin signaling pathway. For testing the hypothesis, we are going to construct steady cell lines with constitutively active GSK-3β and with down-regulated expression of β-catenin in vitro and establish ovariectomized osteoporotic mice model in vivo. Further we will use various techonologies, such as bone histomorphometery, imaging, MRI, immunohistochemistry and ex vivo BMSC culture , ect. , to elucidate the mechanism of GSK-3β/β-catenin signaling pathway mediating the effects of icaritin on preventing osteoporosis. Our project will not only provide a scientific basis for exploring the role of icaritin's target, but also provide basis for developing drug development.

调控骨髓基质干细胞（BMSC）的成骨、成脂分化是治疗骨质疏松的重要策略。淫羊藿素能预防去势小鼠骨丢失，促进BMSC成骨分化、抑制其成脂分化，然而分子机制尚不清楚。我们的最新研究发现淫羊藿素可抑制GSK-3β的活性，并促进β-catenin在细胞质聚集、转核。而抑制GSK-3β的活性、促进β-catenin转核，是促进BMSC成骨分化、抑制其成脂分化的关键。由此形成研究假设：淫羊藿素可能通过GSK-3β/β-catenin信号通路调控BMSC的定向分化发挥抗骨质疏松作用。本项目拟在体外建立持续活化的GSK-3β和β-catenin表达沉默的稳定细胞株，在体内建立去势小鼠骨质疏松模型，通过组织形态学、影像学、磁共振技术、免疫组化，及BMSC离体诱导分化等方法，阐明GSK-3β/β-catenin信号通路介导淫羊藿素预防骨质疏松的作用机制，为探索淫羊藿素的作用靶点及进一步的新药开发提供科学基础。

调控骨髓基质干细胞(BMSC)的成骨、成脂分化是治疗骨质疏松的重要策略。淫羊藿素能预防去势小鼠骨丢失，促进BMSC成骨分化、抑制其成脂分化，然而分子机制尚不清楚。本项目重点研究淫羊藿素通过对GSK-3β/β-catenin信号通路调控预防骨质疏松的作用机制。首先，我们采用小鼠前成脂细胞系发现淫羊藿素通过对蛋白激酶（PKB/Akt）在473丝氨酸位点的磷酸化在上游调控GSK-3β/β-catenin信号通路；并进一步证明淫羊藿素的抑成脂与促成骨分化与雌激素受体（ER）细胞核信号通路作用无关，可能与雌激素膜受体相关。接着发现，淫羊藿素快速且显著增加GSK-3β在9位丝氨酸位点的磷酸化，从而调控下游的β-catenin信号通路，促进β-catenin的入核，发挥转录调控作用；此外，淫羊藿素还可促进成骨和成脂分化时β-catenin的基因表达，利用β-catenin的小干扰RNA研究发现β-catenin部分参与淫羊藿素的促成骨和抑成脂作用。另一方面，采用去卵巢小鼠骨质疏松模型，确认了淫羊藿素可通过促进骨形成并抑制骨吸收预防骨丢失的作用；同样在大鼠模型中，淫羊藿素抑制OVX导致的体重增加、不促进子宫增殖、减缓腰椎松质骨的骨丢失、并且维持皮质骨的力学性能。对比上游Akt信号通路抑制剂LY294002和下游的GSK-3β的调控因子LiCl对去卵巢大鼠骨质疏松的调控作用；并发现LY294002对抗淫羊藿素的促成骨作用，但增加其抑制破骨功能。通过项目开展，发表相关期刊论文2篇；会议论文5篇，并获得1次壁报奖；申请发明专利2项，其中1项获得授权；培养研究生2名。