Abstract：
Metabolic dysfunction of the extracellular matrix (ECM) is one of the primary causes of intervertebral disc degeneration (IVDD). Previous studies have demonstrated that the transcription factor Brachyury (Bry) has the potential to promote the synthesis of collagen II and aggrecan, while the specific mechanism is still unknown. In this study, we used a lipopolysaccharide (LPS)-induced model of nucleus pulposus cell (NPC) degeneration and a rat acupuncture IVDD model to elucidate the precise mechanism through which Bry affects collagen II and aggrecan synthesis in vitro and in vivo. First, we confirmed Bry expression decreased in degenerated human nucleus pulposus (NP) cells (NPCs). Knockdown of Bry exacerbated the decrease in collagen II and aggrecan expression in the lipopolysaccharide (LPS)-induced NPCs degeneration in vitro model. Bioinformatic analysis indicated that Smad3 may participate in the regulatory pathway of ECM synthesis regulated by Bry. Chromatin immunoprecipitation followed by quantitative polymerase chain reaction (ChIP-qPCR) and luciferase reporter gene assays demonstrated that Bry enhances the transcription of Smad3 by interacting with a specific motif on the promoter region. In addition, Western blot and reverse transcription-qPCR assays demonstrated that Smad3 positively regulates the expression of aggrecan and collagen II in NPCs. The following rescue experiments revealed that Bry-mediated regulation of ECM synthesis is partially dependent on Smad3 phosphorylation. Finally, the findings from the in vivo rat acupuncture-induced IVDD model were consistent with those obtained from in vitro assays. In conclusion, this study reveals that Bry positively regulates the synthesis of collagen II and aggrecan in NP through transcriptional activation of Smad3.NEW & NOTEWORTHY Mechanically, in the nucleus, Bry enhances the transcription of Smad3, leading to increased expression of Smad3 protein levels; in the cytoplasm, elevated substrate levels further lead to an increase in the phosphorylation of Smad3, thereby regulating collagen II and aggrecan expression. Further in vivo experiments provide additional evidence that Bry can alleviate IVDD through this mechanism.
摘要：
细胞外基质（ECM）的代谢功能障碍是椎间盘退变（IVDD）的主要原因之一。先前的研究表明，转录因子Brachyury（Bry）具有促进II型胶原和聚集蛋白聚糖合成的潜力，而具体机制仍然未知。在这项研究中,我们使用脂多糖（LPS）诱导的NPC变性模型和大鼠针刺IVDD模型来阐明Bry在体内外影响II型胶原和聚集蛋白聚糖合成的确切机制。首先,我们证实了在退化的人髓核（NP）细胞（NPCs）中Bry表达降低。在脂多糖（LPS）诱导的NPCs变性的体外模型中，Bry的敲除加剧了II型胶原和聚集蛋白聚糖表达的降低。生物信息学分析表明Smad3可能参与了Bry调控的ECM合成的调控途径。染色质免疫沉淀，然后进行定量聚合酶链反应（ChIP-qPCR）和荧光素酶报告基因测定，表明Bry通过与启动子区域上的特定基序相互作用来增强Smad3的转录。此外,westernblot和逆转录-qPCR分析显示Smad3正调节NPCs中聚集蛋白聚糖和胶原II的表达。以下拯救实验表明，Bry介导的ECM合成调节部分依赖于Smad3磷酸化。最后,大鼠体内针刺诱导IVDD模型的结果与体外实验结果一致.总之,这项研究表明，Bry通过转录激活Smad3正向调节NP中胶原蛋白II和聚集蛋白聚糖的合成。
