Integrative analysis of single-cell RNA sequencing, bulk RNA sequencing, and proteomic data identified NOTCH3 as a hub gene contributing to human intervertebral disc fibrosis.

Intervertebral disc degeneration (IDD) is marked by nucleus pulposus (NP) fibrosis, but its molecular drivers remain unclear. This study investigated NP cellular heterogeneity and key regulators using integrative multi-omics. NP specimens from IDD patients and controls were analyzed by scRNA-seq, bulk RNA-seq, proteomics, and fibrosis histological staining. Bioinformatics identified NP subtypes, pseudotime trajectories, and intercellular communication. Validation included immunostaining, qRT-PCR, Western blotting, and functional assays following NOTCH3 knockdown or over-expression. Fibrosis staining confirmed increased collagen deposition in degenerated NP tissues. scRNA-seq revealed eight NP subpopulations, with fibroblast-like NP cells enriched in degeneration. Degenerated samples showed enhanced NOTCH, IGF, and TGF-β signaling. Multi-omics consistently identified NOTCH3 as a hub gene. Functional experiments showed NOTCH3 promoted apoptosis, senescence, fibroblast differentiation, and ER stress–associated fibrosis, while silencing reversed these effects. Immunohistochemistry confirmed elevated NOTCH3 in degenerated tissues. NOTCH3 is a central regulator of NP fibrosis and degeneration, acting via ER stress and fibrotic pathways. Targeting NOTCH3 may represent a novel therapeutic strategy for restoring disc homeostasis in IDD.