PACAP Inhibits TNF‐α‐ Induced Senescence in Primary Chondrocytes by SIRT3: 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.