Inhibition of the P53/P21 Pathway Attenuates the Effects of Senescent Nucleus Pulposus Cell‐Derived Exosomes on the Senescence of Nucleus Pulposus Cells

Abstract

The purpose of this paper is to investigate the effects of senescent nucleus pulposus cell (NPC)-derived exosomes (SNPC-Exo) and the roles of the P53/P21 pathway on the senescence of NPC. Methods: The senescent phenotypes of NPC were induced by interleukin-1β treatment. SNPC-Exo was extracted from the culture medium of senescent NPC and purified by differential centrifugation. The structure of SNPC-Exo was identified by transmission electron microscopy and western blot analysis was used to determine the exosomal marker proteins CD63 and Tsg101. Western blot analysis was performed to determine the relative expression levels of P16, P21, and P53 in NPC. Senescence-associated β-galactosidase (SA-β-gal) staining was used to stain the senescent NPC and a phase contrast microscope was used to observe and count the SA-β-gal staining of NPC. The proliferation of SNPC-Exo-treated NPC was assessed using growth curve analysis and the colony formation assay. The cell cycle of SNPC-Exo-treated NPC was determined by flow cytometry. NPC were transfected with siRNA to knock down P53 and P21 expression. Results: Interleukin-1β-treated NPC had a higher percentage of SA-β-gal positive cells (45%) than the control group (20%) and showed an increase in the relative expression of P16, P21, and P53 (P < 0.05). SNPC-Exo were positive for exosomal marker protein CD63 and Tsg 101 and negative for calnexin, and successfully internalized as previously described. SNPC-Exo-treated NPC showed an increase in the relative expression of P21 and P53 (P < 0.05). Compared with the control group, the SNPC-Exo-treated NPC showed a lower growth rate (3 times lower on the 5th day and 2 times lower on the 7th day), fewer colony-forming units (12.0%), and a higher percentage of SA-β-gal-positive NPC (50.0%). The SNPC-Exo-treated NPC contained more G1 phase cells (68.0%) and fewer S phase (15.5%) cells than the control group (53.0% in G1 phase, 33.5% in S phase). The expression of P21 and P53 significantly decreased in SNPC-exo-treated NPC after siRNA transfection (P < 0.05), followed by a higher growth rate (2 times higher on the 5th day and 1.5 times higher on the 7th day) and lower percentage of SA-β-gal-positive NPC (22.5%). Moreover, the inhibition of the P53/P21 pathway promoted the SNPC-Exo-treated NPC to enter the S phase (from 15.5% to 25.3%). Conclusion: The inhibition of the P53/P21 pathway attenuated the senescence of NPC induced by SNPC-Exo.

Exhausted local lactate accumulation via injectable nanozyme-functionalized hydrogel microsphere for inflammation relief and tissue regeneration

“…The P53 protein is a transcription factor that controls a broad range of cellular processes including cell cycle arrest, DNA repair, and cellular metabolism. The activation of P53 is closely related to the onset of NPC senescence [ 41 ]. Recent study showed activation of Sirt1 might reduce P53 acetylation and potentially protect NP cells against apoptosis and senescence [ 42 ].…”

Cellular Senescence in Intervertebral Disc Aging and Degeneration: Molecular Mechanisms and Potential Therapeutic Opportunities

“…Although the role of inflammatory stress in contributing to disc degeneration is well documented, the role of inflammatory stress in disc CS is unclear. Stimulating NP cells with inflammatory cytokines such as IL-1β or TNF-α can induce premature or acute senescence as marked by increased level of SA-β-gal and other senescence markers p16 INK4a , p21 and p53, and loss of proliferation [ 51 , 52 , 53 , 54 ]. In addition, senescent cells induced by TNF-α treatment activate the senescence of healthy cells through phosphorylation of signal transducer and activator of transcription 3 (STAT3) and the paracrine effect via IL-6 secretion [ 55 ].…”

“…The level of p16 INK4a expression has also been shown to increase with aging in human disc and animal models of aging [ 8 , 13 , 86 ]. Inflammation induced by TNF-α or IL-1β increases the p16 INK4a , p21 CIP1 , and p53 expression in NP cells [ 51 , 52 , 53 , 54 , 55 ]. In cultured NP cells, a combination of low glucose, hypoxia, high osmolality, and absence of serum induces the expression of p16 INK4a , p21 CIP1 , and intercellular adhesion molecule-1 (ICAM-1) [ 35 ].…”

Exosomes and exosomal miRNAs: A new therapy for intervertebral disc degeneration

“…Besides, their enriched exosomal miR-16 could directly inhibit the anti-apoptotic IGF-1/IGF-1R pathway to facilitate the apoptosis of normal NPCs ( Zhang et al, 2021a ; Feng et al, 2022 ). Furthermore, exosomes derived from NPCs with a senescent phenotype induced by IL-1β can accelerate the senescence of normal NPCs by activating the P53/P21 pathway ( Chen et al, 2021 ).…”