Department of Orthopaedics, Lanzhou University Second Hospital, Lanzhou, Gansu, 730030, China.
The Key Laboratory of Tumor Molecular Diagnosis and Individualized Medicine of Zhejiang Province, Zhejiang Provincial People's Hospital, Affiliated People's Hospital, Hangzhou Medical College, Hangzhou, Zhejiang, 311300, China.
Biol Direct. 2024 Sep 12;19(1):81. doi: 10.1186/s13062-024-00528-4.
Limited supply of certain nutrients and deregulation of nucleus pulposus (NP) plays a key role in the pathogenesis of intervertebral disc degeneration (IVDD). However, whether nutrient deprivation-induced cell death, particularly disulfidptosis, contributes to the depletion of NP cells and the development of IVDD, is unknown.
RNA-seq, single-cell RNA-seq, and Genome-wide DNA methylation datasets of nucleus pulposus tissue were collected for bioinformatic analysis. Predictive models of disulfidptosis related genes in IVDD were constructed by machine learning and their differential expression was analyzed. In addition, we performed cell subsets identification analysis, cell-cell communications analysis, and functional enrichment analysis of key genes in the core subset based on single-cell RNA-seq data of NP tissues isolated from one normal sample and one IVDD sample. Finally, glucose deprivation-induced disulfidptosis in human NP cells (HNPCs) was verified by various cell death inhibitors and disulfidptosis-related molecular markers.
We found the disulfidptosis signal was significantly activated in the IVDD group. Using single-cell RNA-seq analysis, we focused on the chondrocytes and found that disulfidptosis-related genes significantly highly expressed in the IVDD C4 chondrocyte subset, which was identified as a new disulfidptosis-associated cell subset. Correlation analysis revealed the negative correlation between SLC7A11 (driving gene of disulfidptosis) and the glucose transporter GLUTs (SLC2A1-4) family genes (suppressing genes of disulfidptosis) in the IVDD group. We also found obvious cell death in HNPC upon glucose starvation, while employment of various cell death inhibitors could not inhibit glucose starvation-induced death in HNPCs. Moreover, the accumulation of disulfide bonds in cytoskeletal proteins was indicated by slowed migration in non-reducible protein blotting experiments. 2-DG, a key disulfidptosis inhibitor, significantly rescued cell death caused by glucose starvation through lowering the NADP/NADPH ratio.
We validated the occurrence of disulfidptosis in HPNCs and identified a novel disulfidptosis-associated cell subset, followed by experimental verification of disulfidptosis in a glucose-limited context to mimic a fall in nutrient supply during the development disc degeneration. These findings provided new insights into the pathological mechanisms of IVDD and encourage us to explore potential therapeutic targets involved in the regulation of disulfidptosis for the prevention of intervertebral disc degeneration.
特定营养物质的供应有限和髓核（NP）失调在椎间盘退变（IVDD）的发病机制中起着关键作用。然而，营养剥夺诱导的细胞死亡，特别是二硫键细胞死亡，是否导致 NP 细胞耗竭和 IVDD 的发展尚不清楚。
收集 NP 组织的 RNA-seq、单细胞 RNA-seq 和全基因组 DNA 甲基化数据集进行生物信息学分析。通过机器学习构建 IVDD 相关二硫键细胞死亡基因的预测模型，并分析其差异表达。此外，我们还基于从一个正常样本和一个 IVDD 样本中分离的 NP 组织的单细胞 RNA-seq 数据，进行细胞亚群鉴定分析、细胞间通讯分析和核心亚群关键基因的功能富集分析。最后，通过各种细胞死亡抑制剂和二硫键细胞死亡相关分子标志物验证葡萄糖剥夺诱导的人 NP 细胞（HNPC）中二硫键细胞死亡。
我们发现 IVDD 组中二硫键细胞死亡信号明显激活。通过单细胞 RNA-seq 分析，我们关注软骨细胞，发现二硫键细胞死亡相关基因在 IVDD C4 软骨细胞亚群中显著高表达，该亚群被鉴定为一种新的二硫键细胞死亡相关细胞亚群。相关性分析显示，在 IVDD 组中，SLC7A11（二硫键细胞死亡的驱动基因）与葡萄糖转运体 GLUTs（SLC2A1-4）家族基因（二硫键细胞死亡的抑制基因）呈负相关。我们还发现 HNPC 在葡萄糖饥饿时明显发生细胞死亡，而使用各种细胞死亡抑制剂不能抑制 HNPC 中葡萄糖饥饿诱导的死亡。此外，非还原蛋白印迹实验表明细胞骨架蛋白中二硫键的积累导致迁移速度减慢。2-DG，一种关键的二硫键细胞死亡抑制剂，通过降低 NADP/NADPH 比值，显著挽救了葡萄糖饥饿引起的细胞死亡。
我们验证了 HNPC 中二硫键细胞死亡的发生，并鉴定了一个新的二硫键细胞死亡相关细胞亚群，随后通过葡萄糖限制条件下的实验验证了二硫键细胞死亡，以模拟椎间盘退变过程中营养物质供应的下降。这些发现为 IVDD 的病理机制提供了新的见解，并促使我们探索调节二硫键细胞死亡的潜在治疗靶点，以预防椎间盘退变。
