<scp>PACAP</scp> Inhibits <scp>TNF</scp>‐α‐ Induced Senescence in Primary Chondrocytes by <scp>SIRT3</scp>: An Implication in Osteoarthritis

Osteoarthritis (OA) is a degenerative joint disease closely associated with aging, in which chondrocyte senescence plays a critical role in cartilage degradation. Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide known for its anti-inflammatory and cytoprotective effects; however, its role in chondrocyte senescence remains poorly understood. This study investigated the protective effects of PACAP38 on tumour necrosis factor-α (TNF-α)- induced chondrocyte senescence and the underlying mechanisms. Intracellular reactive oxygen species (ROS) levels were measured using 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) staining, oxidative DNA damage was assessed by 8-hydroxy-2'-deoxyguanosine (8-OHdG) enzyme-linked immunosorbent assay (ELISA), and senescence markers were evaluated by senescence-associated β-galactosidase (SA-β-Gal) staining, telomerase activity assay, and telomere length analysis. Gene and protein expression were examined via real-time polymerase chain reaction (PCR) and Western blotting. We found that PACAP type I receptor (PAC1R), the receptor for PACAP, was expressed in both murine and human chondrocytes and was downregulated by TNF-α stimulation. PACAP38 treatment significantly reduced ROS and 8-OHdG levels, restored telomerase activity and telomere length, and decreased SA-β-Gal activity. Mechanistically, PACAP38 upregulated sirtuin 3 (SIRT3), a mitochondrial deacetylase that regulates oxidative stress and metabolism, while suppressing acetylated p53 (ac-p53) and plasminogen activator inhibitor-1 (PAI-1) expression. Furthermore, PACAP38 reversed TNF-α-induced dephosphorylation of AMP-activated protein kinase alpha (AMPK-α) and its downstream target acetyl-CoA carboxylase (ACC). Importantly, inhibition of AMPK by compound C abolished these protective effects, confirming the essential role of the AMPK-SIRT3 signalling pathway. In conclusion, our findings demonstrate that PACAP38 mitigates TNF-α-induced chondrocyte senescence via activation of the AMPK-SIRT3 signalling axis, suggesting a potential therapeutic strategy for OA.