D-Galactose induces the senescence and senescence-related secretory phenotype switch of nucleus pulposus cells

Senescence of nucleus pulposus cells (NPCs) in degenerative intervertebral discs is related to the development and progression of intervertebral disc degeneration (IVDD). The aim of this study was to establish a reliable and robust cell senescent model using D-galactose (D-Gal) to induce oxidative stress in NPCs. NPCs were isolated from Sprague Dawley rats and incubated with increasing concentrations of D-Gal. Cell viability, cell cycle, senescence-associated markers, and extracellular matrix were detected to evaluate the effect of D-Gal on NPCs. The migration and polarization of macrophage were observed by Transwell assay and flow cytometry. Finally, the oxidative stress mechanism of D-Gal induced NPCs senescence was analyzed by flow cytometry and absorbance analysis. Cell viability and cell cycle analyses revealed that D-Gal induced senescence by blocking DNA synthesis and decreasing NPC proliferation. Furthermore, a dose-dependent increase was observed in senescence-associated makers in D-Gal induced NPCs. RT-qPCR analysis revealed an increase in mRNA expression of key senescence related secretory phenotype (SASP) components. Extracellular matrix was significantly decreased along with cell senescence. Transwell assay and flow cytometry revealed that senescent NPC-conditioned medium triggered macrophage migration and polarization. Increased levels of reactive oxygen species (ROS), advanced glycation end products, and the lipid peroxidation product malondialdehyde, along with decreased superoxide dismutase activity, were associated with the senescence of NPCs induced by D-Gal. The data imply that D-Gal treatment successfully triggered senescence in NPCs and SASP switch, establishing it as a reliable model for studying IVDD.