Comprehensive Transcriptomic Analysis and Experimental Validation of Notochordal Cells and Nucleus Pulposus Cells: Uncovering Novel Therapeutic Targets for Intervertebral Disc Degeneration

Current therapeutic strategies for intervertebral disc degeneration (IDD)-related low back pain are limited to symptomatic alleviation. Notochordal cells (NCs), as progenitor cells of the nucleus pulposus (NP), lead us to develop innovative NC-based new therapies for IDD. A total of 40 NP specimens, obtained according to IDD criteria with defined Pfirrmann grades and histological degeneration score, were categorized as either normal (Grade II) or degenerated (Grade IV). An IDD model was established in SD rats by needle puncture of the annulus fibrosus. Degenerated NP tissue was identified using MRI, H&E, Safranin O, and Masson staining. NCs and NP cells (NPCs) were isolated and identified based on specific cellular markers. Furthermore, mRNA-seq was performed to profile gene expression in these cells. GO annotation and KEGG pathway analysis were employed to perform functional enrichment analysis of the differentially expressed genes (DEGs). Cell viability was assessed using the CCK-8 assay. An in vitro cell degeneration model was established by treating NPCs with TBHP. Analysis of specific marker expression was performed using Western blotting, immunohistochemistry, and immunofluorescence. We found that the number of NCs in degenerated NP tissues was significantly reduced compared to those in normal NP tissues, but a small amount of notochordal cell markers could still be detected. Analysis of sequencing data identified 2391 upregulated and 3813 downregulated DEGs. GO enrichment analysis indicated that these DEGs were significantly associated with regulatory signals including cellular senescence and oxidative stress. KEGG pathway analysis further revealed that the DEGs were primarily enriched in the TNF and HIF-1 signaling pathways. Subsequent screening identified the top 10 key genes potentially related to IDD: <i>Sod2</i>, <i>Cxcl12</i>, <i>Spp1</i>, <i>Fn1</i>, <i>Cat</i>, <i>Il6</i>, <i>Ccl2</i>, <i>Igf1</i>, <i>Fgf2</i>, and <i>Acta2</i>. Collectively, our findings establish a clear link between SOD2/CAT and the pathogenesis of IDD. SOD2 and CAT may serve as promising new potential therapeutic targets for IDD by inhibiting oxidative stress and cellular senescence in NPCs.