Zhang Weiye, Zhang Ping, Zhan Jiawen, Wei Xu, Du Yuxuan, Zhao Ke, Zhu Liguo, Xie Rong, Xie Hualong, Zhou Shuaiqi, Wang Gewen, Cai Chuhao
China Academy of Chinese Medical Sciences Wangjing Hospital, No. 6 Zhonghuan South Road, Chaoyang District, Beijing, China.
Curr Mol Med. 2025 Jun 23. doi: 10.2174/0115665240370788250617070218.
Studies have shown that abnormal stress is a significant inducer of Intervertebral Disc Degeneration (IVDD). Although traction force is commonly used to delay IVDD, its effects on Nucleus Pulposus Cells (NPCs) and their secreted exosomes remain unclear. In addition, this study systematically revealed the relationship between miR-8485 and IVDD for the first time.
Cellular experiments were performed using a Flexcell cell stretching platform to apply traction force to NPCs. After optimizing loading parameters, NPCderived exosomes (NPCs-exo) were isolated and subjected to miRNA high-throughput sequencing. Differentially expressed miRNAs were identified, and their regulatory effects on the Wnt/β-catenin pathway were investigated. Ex vivo rabbit spinal samples were used to validate the cellular experimental results under traction force loading.
NPCs-exo were found to be internalized by NPCs, and traction force promoted NPCs-exo secretion. High-throughput sequencing and differential expression analysis identified miR-8485 as a differentially expressed miRNA in NPCsexo secreted under Cyclic Mechanical Tension (CMT) conditions. Dual-luciferase reporter assays confirmed the targeted regulatory relationship between miR-8485 and GSK-3β, as well as its involvement in the Wnt/β-catenin pathway-mediated regulation of NPCs degeneration. Ex vivo experiments, including morphological and immunofluorescence analyses, revealed that the traction group exhibited better morphology than the pressure group, with a more organized AF, NP, and higher NPCs content, though some loss persisted. Both groups showed significant differences in ECM markers (Collagen II, Aggrecan, MMP3) compared to the control (p < 0.05). Additionally, the traction group had significantly higher Collagen II and Aggrecan levels than the pressure group (p < 0.05).
CMT can promote the secretion of NPCs-exo, which are internalized by the NPCs. Through the delivery of miR-8485, NPCs-exo target and regulate GSK-3β, thereby enhancing Wnt/β-catenin pathway activity. This mechanism increases NPCs viability and extracellular matrix synthesis while suppressing apoptosis, ultimately delaying IVDD progression. Immunofluorescence staining in animal experiments confirmed that traction force effectively improves extracellular matrix expression in the IVD and mitigates stress-induced morphological alterations of the IVD.
研究表明，异常应力是椎间盘退变（IVDD）的重要诱因。尽管牵引力常用于延缓IVDD，但其对髓核细胞（NPCs）及其分泌的外泌体的影响仍不清楚。此外，本研究首次系统揭示了miR-8485与IVDD之间的关系。
使用Flexcell细胞拉伸平台对NPCs施加牵引力进行细胞实验。优化加载参数后，分离NPCs来源的外泌体（NPCs-exo）并进行miRNA高通量测序。鉴定差异表达的miRNA，并研究其对Wnt/β-连环蛋白通路的调控作用。使用离体兔脊柱样本验证牵引力加载下的细胞实验结果。
发现NPCs-exo被NPCs内化，牵引力促进NPCs-exo分泌。高通量测序和差异表达分析确定miR-8485是在循环机械张力（CMT）条件下分泌的NPCs-exo中差异表达的miRNA。双荧光素酶报告基因实验证实了miR-8485与GSK-3β之间的靶向调控关系，以及其参与Wnt/β-连环蛋白通路介导的NPCs退变调控。包括形态学和免疫荧光分析在内的离体实验表明，牵引组的形态比压力组更好，纤维环、髓核更有组织，NPCs含量更高，尽管仍有一些损失。与对照组相比，两组的细胞外基质标志物（胶原蛋白II、聚集蛋白聚糖、基质金属蛋白酶3）均有显著差异（p < 0.05）。此外，牵引组的胶原蛋白II和聚集蛋白聚糖水平显著高于压力组（p < 0.05）。
CMT可促进NPCs-exo的分泌，后者被NPCs内化。通过递送miR-8485，NPCs-exo靶向并调节GSK-3β，从而增强Wnt/β-连环蛋白通路活性。该机制增加了NPCs的活力和细胞外基质合成，同时抑制细胞凋亡，最终延缓IVDD进展。动物实验中的免疫荧光染色证实，牵引力可有效改善IVD中的细胞外基质表达，并减轻应力诱导的IVD形态改变。
