Decoding Glycolysis Mechanisms and Cellular Heterogeneity in Intervertebral Disc Degeneration via scRNA-seq and Bulk RNA-seq

<title>Abstract</title> <p>Intervertebral disc degeneration (IDD) is a major contributor to chronic low back pain; however, the metabolic alterations and cellular mechanisms driving its progression remain incompletely understood. Increasing evidence indicates that glycolytic reprogramming is closely associated with inflammatory responses, oxidative stress, and extracellular matrix (ECM) degradation during degeneration. Here, we integrated bulk RNA sequencing and single-cell RNA sequencing analyses to systematically characterize glycolysis-related molecular changes and cellular heterogeneity in IDD. Through differential expression analysis, protein–protein interaction network analysis, and machine learning approaches, 39 glycolysis-associated candidate genes were identified, and FOS and CEBPB were subsequently recognized as key regulatory genes. Both genes were significantly upregulated in IDD tissues and demonstrated high diagnostic performance in independent datasets (AUC ≥ 0.90). Single-cell analysis revealed that FOS and CEBPB exhibited distinct expression patterns across nucleus pulposus cell populations, particularly in pre-chondrocytes, stem cells, and fibroblast progenitor cells. Immune infiltration and regulatory network analyses suggested that these genes may participate in metabolic-inflammatory regulation during IDD progression. Importantly, immunohistochemical analysis of human degenerative disc specimens and immunofluorescence validation in a rat puncture-induced IDD model consistently confirmed the upregulation of FOS and CEBPB, accompanied by decreased expression of anabolic ECM markers (Aggrecan and Collagen II) and increased expression of catabolic markers (MMP3 and MMP9). Collectively, this study reveals that glycolytic dysregulation contributes to IDD progression through metabolic reprogramming and cellular heterogeneity, and identifies FOS and CEBPB as potential biomarkers and therapeutic targets for metabolic intervention in IDD.</p>