补肾中药调控Hedgehog信号防治激素性股骨头坏死的机制研究

Gluco-corticoid(GC) induced Osteonecrosis of femoral head (SONFH) is a puzzle in the clinic of orthopedics. It has been confirmed that GC treatment could promote the adipogenic differnetiation of bone marrow mesenchymal stem cells (BMSC) instead of osteogenic differentiation, which would induce or aggravate the Osteonecrosis. Hedgehog (Hh) signaling played an important role in the skeletal development and it could regulate the osteogenic differentiation of BMSC as well as the pathogenesis of bone and joint diseases. The previous results of our team showed that SONFH could be prevented when treated with the decoction of kidney tonifying Chinese medicine, You-Gui-Yin, whereas the mechanism beneath it are still unravelled. Our unpublished data indicated that the proliferation and osteogenic differentiaion of BMSC were promoted by You-Gui-Yin treatment, accompanied with activation of Hh signaling. Blockade of Hh signaling by Hh signaling inhibitor could inhibit You-Gui-Yin induced osteogenic differentiation. Therefore, we hypothesized that You-Gui-Yin could regulates the proliferation and osteogenic differentiation of BMSC to prevent SONFH throught activating Hh signaling. In this project, a comparative analysis of the effect of You-Gui-Yin on Hh signal was conducted on BMSC and mouse SONFH models, and the effect and mechanism of Hh signaling changes on You-Gui-Yin induced BMSC differentiation were investigated and verified its function in transgenic animals. Taken together, explore the key targets and events of the prevention of You-Gui-Yin on SONFH will provide powerful evidence for the pathogenesis of SONFH and clinical application of You-Gui-Yin.

激素诱发股骨头坏死（SONFH）是骨科难题之一，研究认为激素能导致骨髓间充质干细胞（BMSC）成骨、成脂分化失衡，从而诱发或加重骨坏死。Hedgehog（Hh）信号是调控骨发育的一个重要通路，其能调控BMSC成骨分化，并参与多种骨关节疾病的发生发展。申请人前期结果表明，补肾中药右归饮均能有效改善SONFH症状，降低股骨头塌陷发生率，同时还能促进BMSC增殖与成骨分化，但其作用机制尚不清楚。预实验表明右归饮能上调BMSC的Hh信号，而阻断Hh信号可抑制右归饮促成骨作用。因此我们假设右归饮通过Hh信号调控BMSC增殖、成骨分化防治SONFH。本项目拟在动物模型和原代BMSC中研究右归饮对Hh信号的影响，继而探究Hh信号改变对右归饮诱导BMSC增殖分化的影响，并在转基因动物体内验证。通过上述研究揭示右归饮防治SONFH的关键靶点和效应环节，为SONFH的发生机制及临床应用右归饮治疗提供有力依据。

为探究Hedgehog信号在右归饮治疗激素性股骨头坏死中的作用及具体作用机制。本项目首先观察分析右归饮干预对激素性股骨头坏死（SONFH）模型成骨细胞和原代分离BMSC中Hh信号的影响，分析其变化与SONFH模型股骨头骨代谢情况和BMSC成骨分化情况的相关性，结果表明右归饮干预可以显著提高股骨头组织中Hh信号，并增加间充质干细胞的增殖能力。在此基础上，给予间充质干细胞以Hh信号抑制剂Cyclopamin或特异性敲降Hh信号关键转录因子Gli2来阻断Hh信号，观察Hh信号在右归饮诱导BMSC成骨分化能力的影响。结果表明，Hh信号抑制剂Cyclopamin或特异性敲降Hh信号关键转录因子Gli2均可以显著抑制间充质干细胞的增殖能力和成骨分化能力。上述结果表明，Hh信号缺失显著抑制右归饮对间充质干细胞的促增殖作用。随后，我们构建间充质干细胞特异性Hh缺失小鼠（条件性缺失Hh信号关键蛋白Smo小鼠SmoGliCreER），并制备SONFH模型来在体动物验证Hh信号在右归饮治疗股骨头坏死的分子机制。结果表明，右归饮可以显著降低SONFH模型小鼠骨小梁间隙，提高骨小梁数量、厚度及骨体积分数；而Smo缺失显著损伤上述右归饮对SONFH模型小鼠的保护作用。上述体内外研究结果表明，Hh信号介导右归饮对SONFH的治疗作用。