Zhou Xuewu, Shou Kangquan, Yao Hao
Department of Orthopedic, The First Clinical Medical College of China Three Gorges University, Yichang Central People's Hospital, Yichang, HuBei, China.
Clin Exp Pharmacol Physiol. 2025 Sep;52(9):e70062. doi: 10.1111/1440-1681.70062.
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.
骨关节炎（OA）是一种与衰老密切相关的退行性关节疾病，其中软骨细胞衰老在软骨降解中起关键作用。垂体腺苷酸环化酶激活多肽（PACAP）是一种以其抗炎和细胞保护作用而闻名的神经肽；然而，其在软骨细胞衰老中的作用仍知之甚少。本研究探讨了PACAP38对肿瘤坏死因子-α（TNF-α）诱导的软骨细胞衰老的保护作用及其潜在机制。使用2',7'-二氯二氢荧光素二乙酸酯（DCFH-DA）染色测量细胞内活性氧（ROS）水平，通过8-羟基-2'-脱氧鸟苷（8-OHdG）酶联免疫吸附测定（ELISA）评估氧化DNA损伤，并通过衰老相关β-半乳糖苷酶（SA-β-Gal）染色、端粒酶活性测定和端粒长度分析评估衰老标志物。通过实时聚合酶链反应（PCR）和蛋白质印迹法检测基因和蛋白质表达。我们发现，PACAP的I型受体（PAC1R）在小鼠和人类软骨细胞中均有表达，并被TNF-α刺激下调。PACAP38处理显著降低了ROS和8-OHdG水平，恢复了端粒酶活性和端粒长度，并降低了SA-β-Gal活性。机制上，PACAP38上调了沉默调节蛋白3（SIRT3），一种调节氧化应激和代谢的线粒体去乙酰化酶，同时抑制乙酰化p53（ac-p53）和纤溶酶原激活物抑制剂-１（PAI-1）的表达。此外，PACAP38逆转了TNF-α诱导的AMP激活蛋白激酶α（AMPK-α）及其下游靶点乙酰辅酶A羧化酶（ACC）的去磷酸化。重要的是，化合物C对AMPK的抑制消除了这些保护作用，证实了AMPK-SIRT3信号通路的重要作用。总之,我们的研究结果表明，PACAP38通过激活AMPK-SIRT3信号轴减轻TNF-α诱导的软骨细胞衰老，提示OA的一种潜在治疗策略。
