Department of Orthopaedic Oncology, Spinal Tumor Center, Changzheng Hospital, Second Military Medical University, Shanghai, 200003, China.
Shanghai University of Medicine and Health Sciences Affiliated Zhoupu Hospital, Shanghai, 201318, China.
Spine J. 2018 Sep;18(9):1669-1677. doi: 10.1016/j.spinee.2018.05.012. Epub 2018 Jun 7.
Endothelin-1 (ET-1) is an inflammatory mediator associated with cartilage end plate (CEP) degeneration in the intervertebral disc (IVD). SOX9 is downregulated during CEP degeneration, along with its targets, collagen II and aggrecan. Wnt/β-catenin signaling is associated with CEP degeneration and a downstream target of SOX9; however, the precise mechanism of CEP degeneration and the role of ET-1 are largely unknown.
The purpose of the study was to evaluate the influence of the endothelin-A receptor antagonist, BQ-123, on ET-1-induced effects on cartilaginous end plate cells (CECs) associated with CEP degeneration via the Wnt/β-catenin signaling pathway.
STUDY DESIGN/SETTING: The influence of ET-1 on the expression levels of collagen II, aggrecan, and SOX9 in CECs and the effect of BQ-123 in this context were investigated.
To establish a model for CEP degeneration, three lumbar discs (L3-L4, L4-L5, and L5-L6 levels) in New Zealand white rabbits were punctured close to the vertebral end plate using a 14G needle. Intervertebral disc degeneration was evaluated by magnetic resonance imaging 4 weeks after vertebral end plate injury. CECs were then isolated from the degenerated CEPs to allow evaluation of the role of ET-1 and BQ-123 and to investigate their effects on the Wnt/β-catenin signaling pathway. The expression of ET-1 in CECs from degenerated CEPs was analyzed by immunofluorescent staining. Changes in the levels of collagen II, aggrecan, and SOX9 were evaluated in CECs by real-time polymerase chain reaction and by Western blotting. The Wnt/β-catenin signaling pathway was also investigated by Western blotting.
After 4 weeks, IVDs with vertebral end plate injury exhibited clear signs of disc degeneration. Immunofluorescent staining showed that ET-1 was expressed in the cytoplasm of CECs. Endothelin-1 stimulation significantly inhibited the expression of collagen II, aggrecan, and SOX9 in CECs, whereas BQ-123 increased the levels of these three molecules. In addition, ET-1 stimulation increased the expression of β-catenin, cyclin D1, and Dvl1 in the Wnt/β-catenin signaling pathway of CECs from degenerated discs and reduced the expression of GSK-3β, whereas BQ-123 had the opposite effect.
Endothelin-1 can reduce levels of collagen II, aggrecan, and SOX9 in CECs through activation of the Wnt/β-catenin signaling pathway, whereas BQ-123 attenuates these negative effects, highlighting a new molecular mechanism with potential for exploitation for treatment of CEP degeneration.
内皮素-1（ET-1）是一种与椎间盘（IVD）软骨终板（CEP）退化相关的炎症介质。SOX9 在 CEP 退化过程中及其靶标胶原 II 和聚集蛋白聚糖的表达下调。Wnt/β-catenin 信号通路与 CEP 退化有关，是 SOX9 的下游靶点；然而，CEP 退化的确切机制和 ET-1 的作用在很大程度上尚不清楚。
本研究旨在评估内皮素-A 受体拮抗剂 BQ-123 对 ET-1 诱导的软骨终板细胞（CEC）作用的影响，这些作用与 CEP 退化相关，涉及 Wnt/β-catenin 信号通路。
研究设计/设置：研究了 ET-1 对 CEC 中胶原 II、聚集蛋白聚糖和 SOX9 表达水平的影响，以及 BQ-123 在这种情况下的作用。
通过使用 14G 针在接近椎骨终板的位置穿刺三个新西兰白兔的腰椎（L3-L4、L4-L5 和 L5-L6 水平），建立 CEP 退化模型。在椎骨终板损伤后 4 周通过磁共振成像评估椎间盘退变。然后从退化的 CEPs 中分离 CECs，以评估 ET-1 和 BQ-123 的作用及其对 Wnt/β-catenin 信号通路的影响。通过免疫荧光染色分析 CECs 中 ET-1 的表达。通过实时聚合酶链反应和 Western blot 评估 CECs 中胶原 II、聚集蛋白聚糖和 SOX9 的水平变化。还通过 Western blot 研究了 Wnt/β-catenin 信号通路。
4 周后，有椎骨终板损伤的 IVD 显示出明显的椎间盘退变迹象。免疫荧光染色显示 ET-1 在 CEC 的细胞质中表达。ET-1 刺激显著抑制 CEC 中胶原 II、聚集蛋白聚糖和 SOX9 的表达，而 BQ-123 增加了这三种分子的水平。此外，ET-1 刺激增加了退化椎间盘 CECs 中 Wnt/β-catenin 信号通路中β-catenin、细胞周期蛋白 D1 和 Dvl1 的表达，降低了 GSK-3β 的表达，而 BQ-123 则产生相反的效果。
内皮素-1 可以通过激活 Wnt/β-catenin 信号通路降低 CEC 中胶原 II、聚集蛋白聚糖和 SOX9 的水平，而 BQ-123 则减轻了这些负面影响，突出了一种具有治疗 CEP 退化潜力的新分子机制。
