• Wei Yuan, Zhen-Xi Li, Cheng-Long Zhao, Tian-Hui Hou, Si-Wang Hu, Wei-Bo Liu, Feng-Lai Yuan, and Jian-Ru Xiao.
• Department of Orthopaedic Oncology, Spinal Tumor Center, Changzheng Hospital, Second Military Medical University, Shanghai, 200003, China.
• Spine J. 2018 Sep 1; 18 (9): 1669-1677.

Background ContextEndothelin-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.PurposeThe 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/SettingThe 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.MethodsTo 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.ResultsAfter 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.ConclusionsEndothelin-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.Copyright © 2018 Elsevier Inc. All rights reserved.

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