From antioxidant to senotherapeutic: Repurposing n-acetylcysteine to counteract senescence via the HuR pathway in human endothelial cells

Cellular senescence plays a dual role in physiology and pathology. While it contributes to tumour suppression and tissue repair, its persistence promotes chronic inflammation, tissue dysfunction, and age-related diseases. Oxidative stress is a major driver of this process, and the RNA-binding protein HuR serves as a regulator by stabilizing transcripts encoding antioxidant and stress-response proteins. In this study, we established an in vitro model of replicative senescence using human umbilical vein endothelial cells (HUVECs) and investigated the potential anti-aging effects of N-acetylcysteine (NAC), an antioxidant and glutathione precursor. Late-passage HUVECs exhibited a senescent phenotype, including enlarged morphology, increased senescence-associated (SA)-β-galactosidase activity, elevated reactive oxygen species (ROS) levels, and upregulation of p16 and p21 proteins. We observed a progressive decline in HuR expression from early to late passages. NAC treatment counteracted these hallmarks, significantly decreasing ROS and p21 levels. Notably, NAC restored HuR expression and its protective targets, MnSOD and HSP70, and reduced inflammatory markers (IL-6 and TNF-α) levels. These findings suggest that the decline of the HuR-protective axis is a critical driver of endothelial senescence and demonstrate that NAC can mitigate aging hallmarks, representing a promising therapeutic strategy to target aging-related dysfunctions.