Single-Cell Analysis Reveals Aspirin Restores Intervertebral Disc Integrity via Ferroptosis Regulation

Haiyun Niu,1 Hao Qi,2 Peng Zhang,2 Hongyu Meng,3 Ning Liu,3 Di Zhang2 1Orthopedics Department of Joint Surgery, Hebei Medical University Third Hospital, Shijiazhuang City, Hebei Province, People’s Republic of China; 2Orthopedics Department of Spine Surgery, Hebei Medical University Third Hospital, Shijiazhuang City, Hebei Province, People’s Republic of China; 3Orthopedics Department, Hebei Medical University Third Hospital, Shijiazhuang City, Hebei Province, People’s Republic of ChinaCorrespondence: Di Zhang, Email [email protected]: Low back pain represents a major global health issue, with intervertebral disc degeneration (IVDD) being one of its primary causes. Disc degeneration involves complex processes such as inflammation, matrix degradation, and cell death, yet the underlying mechanisms remain poorly understood. Single-cell RNA sequencing offers a powerful approach to elucidate cellular heterogeneity and dynamic changes in IVDD, providing valuable insights for early diagnosis and targeted therapeutic strategies.Methods: The Harmony algorithm was used to integrate four independent single-cell sequencing datasets. Subtype identification, differential expression analysis, enrichment analysis, and cell proportion analysis were conducted to explore functional alterations in various nucleus pulposus cell (NPC) subpopulations. Molecular docking was employed to evaluate the stability of aspirin targeting GPX4. In vitro and in vivo experiments were performed to assess the therapeutic effects of aspirin on IVDD.Results: Eight distinct NPC subtypes were identified based on cellular heterogeneity and their associated marker genes. The CDKN1A⁺aNPC subtype increased progressively with disease severity, while the matrix-supporting ABI3BP⁺mNPC and SOD3⁺mNPC subtypes significantly decreased in advanced degeneration. Concurrently, there was an increase in ECM remodeling-related LTBP1⁺mNPCs. Within the CDKN1A⁺aNPC, GPX4 was notably downregulated, suggesting the activation of ferroptosis. Molecular docking results revealed a high affinity of aspirin for GPX4. Additionally, aspirin inhibited ferroptosis and ameliorated disc structural damage.Conclusion: The increased proportion of CDKN1A⁺aNPC cells serves as an early warning feature for the progression of IVDD. Aspirin stabilizes the targeting of GPX4, thereby inhibiting ferroptosis and exerting therapeutic effects on IVDD.Keywords: low back pain, intervertebral disc degeneration, single-cell analysis, cell death, glutathione peroxidase 4