Identification and experimental validation of biomarkers associated with exercise in intervertebral disc degeneration through bulk RNA and single-cell RNA sequencing analysis

Long-term running positively affects the intervertebral disc. This study aimed to identify and validate biomarkers associated with exercise-related genes (ERGs) in intervertebral disc degeneration (IDD), potentially guiding the development of targeted therapies. IDD-related datasets and ERGs were extracted from public databases. Biomarkers were identified through differential expression analysis, weighted gene co-expression network analysis (WGCNA), protein-protein interaction (PPI) networks, machine learning techniques, and gene expression analyses. A nomogram based on these biomarkers was developed and evaluated. Subsequently, functional enrichment, RNA methylation, drug prediction, molecular docking, and single-cell RNA sequencing (scRNA-seq) analyses were performed. Finally, reverse transcription quantitative PCR (RT-qPCR) was conducted to validate the expression of biomarkers. TNFAIP6 and CHI3L1 were identified as biomarkers for IDD, both exhibiting significantly higher expression in IDD samples. These findings were corroborated by RT-qPCR. A nomogram was developed and validated, with the calibration curve indicating its effectiveness in predicting IDD risk (P = 0.798 in the Hosmer-Lemeshow test). All biomarkers were significantly co-enriched in the "spliceosome" pathway. Additionally, compound 16 was found to target CHI3L1, while acetylsalicylic acid was predicted to target TNFAIP6. Molecular docking analysis revealed favorable binding energies between the compounds and biomarkers, including a binding energy of -9.1 kcal/mol between compound 16 and CHI3L1. Finally, scRNA-seq analysis identified homeostatic fibroblasts, progenitor cells, fibrochondrocytes, and regulatory chondrocytes as key cell types in IDD, with dynamic expression patterns of CHI3L1 observed during the differentiation of these key cells. Biomarkers associated with exercise, specifically TNFAIP6 and CHI3L1, were identified. These findings provide valuable insights for the development of targeted therapies for patients with IDD.