补肾活血方调控Wnt/β-catenin-VEGF信号通路改善终板内血管芽损伤延缓椎间盘退变的机制研究

The nutritional metabolic disorder caused by vascular buds injury in the vertebral endplate is the initial factor of intervertebral disc degeneration (IDD) , according to the recent studies, the vascular endothelial growth factor (VEGF) is closely related to vascular buds in the vertebral endplate, and the VEGF can be regulated by Wnt/β-catenin signaling pathway. Our previous clinical study confirmed that the BSHX recipe is effective in treatment of degenerative diseases of intervertebral disc. Meanwhile, the previous basic trails verify the recipe be capable of delaying the degeneration process of IDD models. According to the literature analysis, the mechanism of the BSHX recipe delaying IDD would likely be through regulating the Wnt/β-catenin-VEGF signaling pathway of endplate chondrocytes, then improving the injury of vascular buds in the vertebral endplate. Based on this hypothesis, from the aspects of intervertebral disc nutrition supplying, angiogenesis and molecular regulation of endplate chondrocytes, the research focuses on the molecular mechanism of vascular buds injury and the mechanism of BSHX recipe improving the injury. In the research, the in vivo rabbit IDD model and ex vivo organ culturing rabbit IDD model were developed. By using multiple advanced medical technologies of tissue culture, molecular biology and electron microscopy, we will evaluate the changes of vascular buds and related proteins expression to find out the mechanism. This research deepens the understanding of the IDD mechanism from the view of the nutrition supplying, and provides new thoughts for TCM in the prevention and treatment of IDD.

终板内血管芽损伤引起的营养代谢障碍是椎间盘退变（IDD）的初始诱发因素，已有研究表明血管内皮生长因子（VEGF）与终板内血管芽关系密切，而Wnt/β-catenin信号通路对VEGF的表达具有调控作用。课题组前期研究证实补肾活血方治疗椎间盘退行性疾病具有较好疗效，能够延缓退变模型IDD的进程。根据文献分析，本方延缓IDD的机制可能是通过调控终板软骨细胞Wnt/β-catenin-VEGF信号通路，从而改善了终板内血管芽损伤。围绕此假说，本项目从椎间盘营养供应、血管生成、终板软骨细胞的分子调控等方面展开研究，重点阐述终板内血管芽损伤的分子机制及补肾活血方改善损伤的作用机制。项目拟采用在体与离体IDD模型，通过组织培养、分子生物学、电镜等技术，检测干预前后终板内血管芽变化及通路关键蛋白和基因的表达情况。本项目从营养供应角度深化对IDD发生机制的理解，为中医药防治IDD提供新思路。

目的：本课题旨在明确终板软骨细胞中Wnt/β-catenin信号通路调控VEGF表达在终板内血管芽损伤中的作用，以及补肾活血方对终板细胞“Wnt/β-catenin-VEGF”信号通路的调控作用，以希从营养供应角度探讨补肾活血方改善终板内血管芽损伤，延缓IDD的作用机制。本课题延续前期研究成果，进行了以下研究工作：1、建立在体椎间盘压力退变模型，观察在体椎间盘应力退变模型椎间盘的变化情况。进一步比较椎间盘退变过程中及补肾活血方干预前后终板内血管芽相对数量及面积、通过造影结合mirco-CT扫描观察终板内血管芽超微结构的变化情况，同时检测干预前后终板组织中Wnt3α、β-catenin、VEGF、VEGFR蛋白和基因的表达情况。2、建立离体脊柱运动节段压力退变模型，应用补肾活血方含药血清干预模型，并在干预基础上加入通路激活剂，验证补肾活血方对通路的调控作用。检测Wnt/β -catenin-VEGF信号途径关键因子Wnt3α、GSK-3β、β -catenin及VEGF、VEGFR蛋白及基因的表达变化情况，并检测不同干预前后血管芽的变化情况。结果表明：随着在体加压时间的延长，终板退变情况逐渐加重，伴随终板内血管芽数量下降、VEGFA表达相应降低，而补肾活血方各剂量组短期内均对终板退变有延缓作用，以中剂量组最为显著，可维持终板内血管芽数量，在此过程中Wnt/β-catenin通路与VEGFA表达呈负相关。在此基础上，进一步通过离体模型验证，加入GSK-3β抑制剂激活Wnt/β-catenin通路后逆转了补肾活血方延缓椎间盘终板退变的改善作用，并降低了终板内VEGFA的表达，表明补肾活血方通过调控抑制Wnt/β-catenin通路，提高VEGFA表达，改善终板内血管芽损伤，延缓椎间盘退变。结论：椎间盘应力退变过程中Wnt/β-catenin信号通路异常激活，抑制了终板软骨细胞内VEGF表达，进而引起终板内血管芽损伤，造成椎间盘退变。补肾活血方很通过抑制Wnt/β-catenin信号通路，调控了终板软骨细胞VEGF的表达，维持了终板内血管芽数量，从而实现延缓椎间盘退变的作用。