Abstract: Drug synergy is a promising new anti-aging strategy. Construction of the aging model induced by D-galactose in HUVEC cells was aimed to explore the potential synergistic anti-aging effect of acetylsalicylic acid and acetylcysteine, and to investigate the underlying mechanisms of their synergy in anti-aging. Network pharmacology was used to screen a group of small molecules with potential anti-aging activity. D-galactose-induced HUVEC cells was employed to detect the anti-aging activity of small molecules, and Chou-Talalay model was used to screen two small molecule combinations with synergistic anti-aging effects. Further investigation of the mechanisms underlying the anti-aging activity mediated by the selected small molecule combinations was conducted using biochemical and cell biological approaches. Results showed that acetaminophen acid and acetylcysteine were a highly effective pair of small molecules with anti-aging activity, which could produce synergistic effects in mediating anti-aging. The synergistic combination significantly reduced intracellular reactive oxygen species levels, effectively relieved cell cycle arrest induced by D-galactose, and significantly reduced the transcription levels of early inflammatory factors (IL-1β, COX-2). In addition, the synergistic combination significantly reduced the protein levels of cell senescence markers (p53, p16), as well as p-p38 and nuclear p65 protein levels, indicating that the acetylsalicylic acid and N-acetylcysteine synergistic combination may inhibit cell aging via the MAPK/NF-κB pathway. These results provide a theoretical basis for the further development and utilization of acetylsalicylic acid and N-acetylcysteine as a synergistic anti-aging combination.

Key words: cell senescence, acetaminophen, acetylcysteine, synergies, MAPK/NF-κB