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Abstract：
This study aimed to reveal the specific role of early growth response protein 1 (EGR1) and nuclear receptor 4A3 (NR4A3) in nucleus pulposus cells (NPCs) and the related molecular mechanism and to identify a new strategy for treating intervertebral disc degeneration (IVDD). Bioinformatics analysis was used to explore and predict IVDD-related differentially expressed genes, and chromatin immunoprecipitation sequencing (ChIP-seq) revealed NR4A3 as the EGR1 target gene. An in vitro NPC model induced by tributyl hydrogen peroxide (TBHP) and a rat model induced by fibrous ring acupuncture were established. Western blotting, quantitative real-time polymerase chain reaction (qRT-PCR), immunohistochemical staining, immunofluorescence staining, and flow cytometry were used to detect the effects of EGR1 and NR4A3 knockdown and overexpression on NPC apoptosis and the expression of extracellular matrix (ECM) anabolism-related proteins. Interactions between EGR1 and NR4A3 were analyzed via ChIP-qPCR and dual luciferase assays. EGR1 and NR4A3 expression levels were significantly higher in severely degenerated discs (SDD) than in mildly degenerated discs (MDD), indicating that these genes are important risk factors in IVDD progression. ChIP-seq and RNA-seq revealed NR4A3 as a direct downstream target of EGR1, and this finding was verified by ChIP-qPCR and dual luciferase reporter experiments. Remarkably, the rescue experiments showed that EGR1 promotes TBHP-induced NPC apoptosis and impairs ECM anabolism, dependent on elevated NR4A3 expression. In summary, the EGR1-NR4A3 axis mediates the progression of NPC apoptosis and ECM impairment and is a potential therapeutic target in IVDD.
摘要：
本研究旨在揭示早期生长反应蛋白1(EGR1)和核受体4A3(NR4A3)在髓核细胞(NPCs)中的具体作用及相关分子机制,为椎间盘退变(IVDD)治疗提供新策略。生物信息学分析用于探索和预测IVDD相关的差异表达基因，染色质免疫沉淀测序（ChIP-seq）显示NR4A3为EGR1靶基因。建立了三丁基过氧化氢（TBHP）诱导的体外NPC模型和纤维环针刺诱导的大鼠模型。西方印迹,定量实时聚合酶链反应（qRT-PCR），免疫组织化学染色，免疫荧光染色，流式细胞术检测EGR1和NR4A3敲低和过表达对NPC凋亡和细胞外基质（ECM）合成代谢相关蛋白表达的影响。通过ChIP-qPCR和双荧光素酶测定分析EGR1和NR4A3之间的相互作用。EGR1和NR4A3表达水平在严重退化的椎间盘(SDD)中显著高于轻度退化的椎间盘(MDD),表明这些基因是IVDD进展的重要危险因素。ChIP-seq和RNA-seq显示NR4A3是EGR1的直接下游靶标，这一发现已通过ChIP-qPCR和双荧光素酶报告基因实验得到验证。值得注意的是,拯救实验表明，EGR1促进TBHP诱导的NPC细胞凋亡，损害ECM合成代谢，依赖于NR4A3表达升高。总之,EGR1-NR4A3轴介导NPC凋亡和ECM损伤的进展,是IVDD的潜在治疗靶点.
