Abstract
Introduction: Excessive senescent cell accumulation impairs wound healing. While in vitro models are crucial, not all cells in hard-to-heal wounds are senescent, and models reflecting delayed healing without complete senescence are lacking. This study aimed to establish a hydrogen peroxide (H2O2)-induced senescence model and evaluate whether increased senescent cell proportions delay healing in a scratch assay. Methods: NIH/3T3 cells were treated with 0-1,200μM H2O2. SA-β-gal activity, proliferation, and senescence-related gene/protein expression were assessed. Untreated (U) and H2O2-treated (H) cells were mixed at ratios U100 (0:100), U50H50 (50:50), and H100 (100:0), and scratch assays were performed. Senescence marker expression was analyzed 48 h after medium replacement. Results: 1,200μM H2O2 increased SA-β-gal activity (p < 0.01), reduced proliferation (p < 0.01), and elevated Cdkn1a gene (p < 0.01) and p53 protein expression (p =0.03). Scratch assays showed larger wound areas (U100 < U50H50 < H100, p < 0.01 at 48 h) with a proportion-dependent increase in Cdkn1a expression (p < 0. 01). Conclusions: An H2O2 -induced senescence model in NIH/3T3 cells was established. Increased senescent cell proportions impaired wound healing, and partial senescence contributed to delayed closure.
