Fibulin-3 Regulates Tissue Inhibitor of Metalloproteinases 3 to Inhibit Senescence in Intervertebral Disc Nucleus Pulposus Cells

Abstract

To investigate the effect of fibulin-3 on the senescence of intervertebral disc nucleus pulposus cells (NPCs) through the regulation of tissue inhibitor of metalloproteinases 3 (TIMP-3) expression and to elucidate the molecular mechanisms involved.

Methods 

 1). The nucleus pulposus tissues and imaging data of 37 patients who had undergone intervertebral disc surgery were collected. The degree of degeneration of the intervertebral discs were classified according to the Pfirrmann grading system. The senescence degree of NPCs was determined using senescence-associated β-galactosidase (SA-β-gal) staining. Fibulin-3 expression levels were determined using Western blot and ELISA. The relationship between fibulin-3 and disc degeneration and NPCs senescence was investigated. 2). Human intervertebral disc NPCs were cultured in vitro. The proliferation and senescence of NPC across continuous passage were observed via CCK-8 assay and SA-β-gal staining, respectively. Fibulin-3 expression levels and the expression of inflammatory cytokines and matrix metalloproteinases were assessed. Exogenous fibulin-3 was added to verify its effect on the proliferation and senescence of NPCs. 3). The effect of fibulin-3 on the apoptosis and proliferation of NPCs was verified through gene overexpression, which was used in combination with an apoptosis inhibitor for bidirectional verification. 4). Bioinformatics analysis was performed to explore the relationship between fibulin-3 and the TIMP family. Experiments overexpressing fibulin-3 and silencing the TIMP-3 gene were performed to verify their role in NPCs senescence.

Results 

 1). The intervertebral disc degeneration samples from 37 patients were classified according to the Pfirrmann grading system. The higher the degeneration grade, the lower fibulin-3 expression. Spearman correlation analysis showed that the disc grade was negatively correlated with the NPC senescence grade (r=－0.87, P<0.001) and fibulin-3 expression (r=－0.79, P<0.001). 2). As the passage number of NPCs increased, fibulin-3 expression gradually decreased, cell proliferation ability weakened, and the expression of inflammatory cytokines and matrix metalloproteinases increased. After exogenous fibulin-3 was added, cell morphology and growth status were maintained, cell senescence was significantly inhibited, and the expression of inflammatory cytokines and matrix metalloproteinases was markedly reduced. 3). Gene overexpression experiments showed that fibulin-3 reduced NPC apoptosis and promoted cell proliferation, thereby inhibiting NPC senescence. 4). Bioinformatics analysis revealed a significant association between fibulin-3 and TIMP-3 of the TIMP family. Further experiments confirmed that overexpressing fibulin-3 enhanced TIMP-3 expression, while silencing the TIMP-3 gene significantly weakened the inhibitory effect of fibulin-3 on NPCs senescence. This indicates that, through regulating TIMP-3, fibulin-3 inhibits the activity of matrix metalloproteinases, affects the synthesis and degradation of the extracellular matrix, and ultimately inhibits NPCs senescence.

Conclusion 

 This study demonstrates that fibulin-3 plays a crucial role in inhibiting the senescence of intervertebral disc NPCs by regulating TIMP-3. The specific mechanisms involved are as follows, fibulin-3 upregulates TIMP-3 expression, inhibits matrix metalloproteinase activity, and reduces extracellular matrix degradation, thereby promoting extracellular matrix synthesis. Additionally, fibulin-3 inhibits NPCs senescence by reducing apoptosis and promoting cell proliferation. Therefore, fibulin-3 and TIMP-3 have potential therapeutic significance in maintaining intervertebral disc health and delaying degeneration.

Keywords: Fibulin-3, Nucleus pulposus cells, Intervertebral disc degeneration, Tissue inhibitor of metalloproteinases 3