Inoue Kosuke, Uchida Kentaro, Tazawa Ryo, Matsumoto Mitsuyoshi, Kenmoku Tomonori, Uekusa Yui, Takaso Masashi
Department of Orthopaedic Surgery, Kitasato University School of Medicine, Sagamihara, JPN.
Cureus. 2025 Jun 18;17(6):e86324. doi: 10.7759/cureus.86324. eCollection 2025 Jun.
Rotator cuff tears are a major cause of shoulder dysfunction and pain, particularly in older adults, with re-tear rates remaining high despite surgical advances. Persistent inflammation and dysregulated extracellular matrix (ECM) remodeling contribute to impaired healing. Tumor necrosis factor-α (TNF-α) is known to drive tendon inflammation, but the downstream mediators linking TNF-α signaling to matrix degradation remain incompletely understood. Netrin-4 (NTN4), a laminin-related protein, has been implicated in inflammatory and ECM-modulating processes. We hypothesized that NTN4 is upregulated following tendon tear in a TNF-α-dependent manner and contributes to sustained ECM degradation.
A rat infraspinatus and supraspinatus full-thickness tendon tear model was established, and tendon tissues were harvested at 0 (intact), 7, 14, 28, and 56 days post-injury (n = 8/time point). Gene expressions of , , , , , and were quantified by qRT-PCR (quantitative real-time reverse transcription polymerase chain reaction). Primary rat tenocytes were stimulated in vitro with recombinant TNF-α (0-10 ng/mL) or NTN4 (0-500 ng/mL), and target gene expression and protein secretion were assessed by qRT-PCR and ELISA (enzyme-linked immunosorbent assay). Statistical comparisons were performed using Kruskal-Wallis with Dunn's post-hoc tests.
In vivo, expression significantly increased from day 14 onward (P < 0.001), peaking at day 28 and remaining elevated at day 56. and were upregulated earlier (days 7-28), and showed sustained induction (days 7-28), while rose later (day 28). In vitro, TNF-α induced in a dose-dependent manner (P < 0.001). Exogenous NTN4 induced MMP-3 expression at both transcript and protein levels, while no significant changes were observed in MMP-1, TNF-α, or IL-6 protein levels under these experimental conditions.
Our findings identify NTN4 as a TNF-α-induced effector that contributes to sustained MMP-3-mediated matrix degradation following rotator cuff tear. By linking inflammatory cytokine signaling to prolonged ECM remodeling, the TNF-α/NTN4/MMP-3 axis may represent a potential therapeutic target for improving tendon healing and reducing re-tear risk.
肩袖撕裂是肩部功能障碍和疼痛的主要原因，尤其是在老年人中，尽管手术技术有所进步，但再撕裂率仍然很高。持续的炎症和细胞外基质（ECM）重塑失调导致愈合受损。肿瘤坏死因子-α（TNF-α）已知会引发肌腱炎症，但将TNF-α信号与基质降解联系起来的下游介质仍未完全了解。Netrin-4（NTN4）是一种层粘连蛋白相关蛋白，与炎症和ECM调节过程有关。我们假设NTN4在肌腱撕裂后以TNF-α依赖的方式上调，并导致ECM持续降解。
建立大鼠冈下肌和冈上肌全层肌腱撕裂模型，并在损伤后0（完整）、7、14、28和56天采集肌腱组织（每个时间点n = 8）。通过qRT-PCR（定量实时逆转录聚合酶链反应）对 、 、 、 、 和 的基因表达进行定量。用重组TNF-α（0 - 10 ng/mL）或NTN4（0 - 500 ng/mL）体外刺激原代大鼠肌腱细胞，并通过qRT-PCR和ELISA（酶联免疫吸附测定）评估靶基因表达和蛋白质分泌。使用Kruskal-Wallis检验和Dunn事后检验进行统计比较。
在体内， 表达从第14天起显著增加（P < 0.001），在第28天达到峰值，并在第56天保持升高。 和 上调较早（第7 - 28天）， 表现出持续诱导（第7 - 28天），而 升高较晚（第28天）。在体外，TNF-α以剂量依赖的方式诱导 （P < 0.001）。外源性NTN4在转录和蛋白质水平上均诱导MMP-3表达，而在这些实验条件下，MMP-1、TNF-α或IL-6蛋白水平未观察到显著变化。
我们的研究结果确定NTN4是一种TNF-α诱导的效应物，它在肩袖撕裂后导致MMP-3介导的基质持续降解。通过将炎性细胞因子信号与延长的ECM重塑联系起来，TNF-α/NTN4/MMP-3轴可能代表改善肌腱愈合和降低再撕裂风险的潜在治疗靶点。
