破骨细胞自噬在糖皮质激素诱导的骨丢失过程中的作用及PI3K/Akt/mTOR信号通路的调节机制

Glucocorticoid-induced bone loss is closely related with the increased bone resorption caused by osteoclast. Autophagy related proteins play critical roles in the formation of ruffle border of osteoclast. PI3K/Akt/mTOR signal pathway is important for the regulation of cellular autophagy. Our preliminary study found that dexamethasone could up-regulate the expression of Beclin 1, which was reversed by Akt inhibitor. However, the effect of osteoclast autophagy on glucocorticoid-induced bone loss and the regulatory mechanisms are not well elucidated. Therefore, the purpose of this project is to investigate the effect of glucocorticoid on osteoclast autophagy and its structural and functional changes, and the regulatory mechanisms of PI3K/Akt/mTOR signal pathway in mice model and primary cultured osteoclasts by Real-time PCR, Western blot, immunohistochemistry, eletromicroscopy and co-immunoprecipitation methods. This project helps to better understand the mechanisms of glucocorticoid-induced osteoclast autophagy, and provides new insights and new solutions for the treatment of glucocorticoid-induced osteoporosis.

糖皮质激素（GC）诱导的骨量丢失与破骨细胞介导的骨吸收增强密切相关。自噬相关蛋白在破骨细胞皱褶缘形成所介导的骨吸收过程中发挥关键作用。PI3K/Akt/mTOR是调节细胞自噬的一个重要信号转导通路。我们前期工作发现地塞米松可诱导破骨细胞自噬相关蛋白Beclin 1表达增加，而预先孵育Akt抑制剂则下调地塞米松诱导的Beclin 1表达增加。目前，破骨细胞自噬在GC诱导的骨丢失过程中的作用及其信号调节机制尚不清楚。本课题拟采用GC诱导的小鼠骨丢失模型及原代培养的破骨细胞，利用Real-time PCR、Western blot、免疫组化、电镜、免疫共沉淀等技术，探讨GC对破骨细胞自噬的影响及其细胞结构和功能变化，以及PI3K/Akt/mTOR信号通路在该过程中的调节机制。本课题从分子、细胞和整体水平上开展整合性研究，以深化对GC诱导的骨丢失的病理生理机制的理解，为其治疗开辟新思路和新途径。

糖皮质激素（GC）诱导的骨量丢失与破骨细胞介导的骨吸收增强密切相关，自噬相关蛋白在破骨细胞皱褶缘形成所介导的骨吸收过程中发挥关键作用，但GC对破骨细胞自噬的影响及其细胞结构和功能变化以及PI3K/Akt/mTOR信号传导通路的分子调节机制却知之甚少。我们发现，采用糖皮质激素10-7M地塞米松能够体外诱导原代培养的小鼠破骨细胞发生自噬，采用荧光标记的方法，在激光共聚焦显微镜下可以观察到自噬小体的形成，同时自噬相关基因Beclin 1和LC 3的mRNA水平及蛋白水平的表达明显增强，自噬复合体启动等相关分子Agt1（ULK1）、Agt13表达增强，体内使用糖皮质激素泼尼松龙后也证实破骨细胞发生自噬，相关自噬分子的表达和时序效应进一步验证了体外试验的结果。PI3K/Akt/mTOR信号通路的关键分子包括PI3K 110γ亚基、p-Akt、Akt、m-TOR的蛋白表达在破骨细胞发生自噬时活性下降，抑制该信号通路会增强破骨细胞的自噬，调节体内骨量。BPs可以改善股骨骨痂的骨密度状况，提供更好的生物力学效能，有效延缓骨重建过程，其机制是破骨细胞的功能抑制。上述结果证实破骨细胞自噬在糖皮质激素诱导的骨量丢失过程中的新机制，为临床治疗糖皮质激素性骨质疏松症提供新的干预靶点和理论基础。全面完成了预期目标，已发表SCI论文2篇。