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

Background Long-term running positively impacts the intervertebral disc. This study aimed to ascertain and validate biomarkers linked to exercise-related genes (ERGs) in intervertebral disc degeneration (IDD), potentially guiding the development of targeted therapies. Methods In this study, IDD related datasets and ERGs were mined from public databases. Biomarkers were ascertained through differential expression analysis, weighted gene co-expression network analysis (WGCNA), protein-protein interaction (PPI), machine learning, and gene expression analyses. A nomogram was developed and evaluated based on these biomarkers. Subsequently, functional enrichment, RNA methylation, drug prediction, molecular docking, and single-cell RNA sequencing (scRNA-seq) analyses were conducted. Finally, to validate the expression of biomarkers, reverse transcription quantitative PCR (RT-qPCR) analysis was executed. Results In our study, TNFAIP6 and CHI3L1 were identified as biomarkers for IDD, both of which exhibited significantly higher expression in IDD samples. These findings were further supported by RT-qPCR. A nomogram was developed and validated, with the calibration curve demonstrating its effective ability to predict IDD risk (P = 0.798 in Hosmer-Lemeshow test). All biomarkers were significantly co-enriched in "spliceosome" pathway. Additionally, compound 16 was found to target CHI3L1, while acetylsalicylic acid was predicted as a compound targeting TNFAIP6. Molecular docking analysis showed favorable binding energies between compound and biomarker, such as 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 key cells differentiation. Conclusion Biomarkers (TNFAIP6 and CHI3L1) associated with exercise were identified. These findings offered valuable insights for developing targeted therapies for IDD patients.