Table1_Role of ferroptosis and immune infiltration in intervertebral disc degeneration: novel insights from bioinformatics analyses.DOCX

Background: Intervertebral disc degeneration (IVDD), which contributes to stenosis of the spinal segment, commonly causes lower back pain. The process of IVDD degradation entails gradual structural adjustments accompanied by extreme transformations in metabolic homeostasis. However, the molecular and cellular mechanisms associated with IVDD are poorly understood.Methods: The RNA-sequencing datasets GSE34095 and GSE56081 were obtained from the Gene Expression Omnibus (GEO) database. Ferroptosis-related differentially expressed genes (DEGs) were identified from these gene sets. The protein–protein interaction (PPI) network was established and visualized using the STRING database and Cytoscape software, and the key functional modules of ferroptosis-related genes were identified. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed on the DEGs. Weighted gene co-expression network analysis (WGCNA), immune infiltration analysis in the GEO database, and other GSE series were used as validation datasets. The xCELL algorithm was performed to investigate the immune cell infiltration differences between the degenerated IVDD and control groups.Results: The major genes involved in nucleus pulposus tissue immune infiltration and ferroptosis-related genes were mined by bioinformatics analysis. A total of 3,056 DEGs were obtained between the IVDD tissue and control groups. The DEGs were enriched in the cell cycle; apoptosis; necroptosis; and the PI3K-Akt, Hippo, and HIF-1 signaling pathways. PCR and Western blot techniques were utilized to confirm the differential ferroptosis-related genes. The results indicated that the protein expression levels of NCOA4 and PCBP1 were elevated, while the protein expression level of GPX4 was reduced in NPCs following IL-1β treatment. Our study has found that severe disc tissue degeneration leads to a noteworthy increase in the expression of CD8A in naive T cells, CCR7 in memory CD4+ cells, GZMB in natural killer (NK) cells, and CD163 and CD45 in macrophages.Conclusion: Our data demonstrate that ferroptosis occurs in IVDD, suggesting that ferroptosis may also increase IVDD improvement by triggering immune infiltration. This work was conducted to further understand IVDD pathogenesis and identify new treatment strategies.

背景：椎间盘退变（IVDD）是导致椎间段狭窄的重要原因，常引起腰背疼痛。IVDD的退变过程涉及结构的逐步调整，并伴随代谢稳态的剧烈变化。然而，与IVDD相关的分子和细胞机制尚未得到充分理解。方法：从基因表达综合数据库（GEO）中获取了RNA测序数据集GSE34095和GSE56081。从这些基因集中识别了铁死亡相关差异表达基因（DEGs）。利用STRING数据库和Cytoscape软件建立了蛋白质-蛋白质相互作用（PPI）网络并进行可视化，确定了铁死亡相关基因的关键功能模块。对DEGs进行了基因本体（GO）和京都基因与基因组百科全书（KEGG）通路富集分析。采用加权基因共表达网络分析（WGCNA）、GEO数据库中的免疫浸润分析以及GSE系列的其他数据集作为验证数据集。通过xCELL算法研究椎间盘退变与对照组之间的免疫细胞浸润差异。结果：通过生物信息学分析挖掘出与椎间盘组织免疫浸润和铁死亡相关基因的主要基因。在IVDD组织与对照组之间共获得3,056个DEGs。这些DEGs富集于细胞周期、凋亡、坏死性凋亡以及PI3K-Akt、Hippo和HIF-1信号通路。利用PCR和Western blot技术确认了差异铁死亡相关基因。结果表明，在IL-1β处理后，NCOA4和PCBP1的蛋白质表达水平升高，而GPX4的蛋白质表达水平降低。本研究发现，严重的椎间盘组织退变导致初始T细胞中CD8A、记忆CD4+细胞中CCR7、自然杀伤（NK）细胞中GZMB以及巨噬细胞中CD163和CD45的表达显著增加。结论：我们的数据显示，铁死亡在IVDD中发生，这表明铁死亡可能通过触发免疫浸润来提高IVDD的改善。本研究旨在进一步理解IVDD的发病机制并识别新的治疗策略。