Abstract

Pressure injuries develop when prolonged compression of the skin and subcutaneous tissue impairs blood circulation, leading to localized tissue ischemia, degeneration, and ultimately necrosis. Ischemia-reperfusion(I/R) injury is one of the key pathological mechanisms underlying pressure injury formation. While cellular senescence has been implicated in I/R-related pathologies, its role in pressure injury development remains unclear. We aimed to elucidate the mechanisms of keratinocyte senescence in cutaneous I/R injury. An in vitro hypoxia/reoxygenation (H/R) model was employed to simulate I/R injury using human immortalized keratinocytes (HaCaT). H/R induction significantly exacerbated the senescence response, as characterized by reduced cell proliferation, increased apoptosis, elevated SA-β-galactosidase (SA-β-gal) activity, and upregulated expression of senescence markers (p16 and p21). Quantitative proteomic analysis identified CDC6 as a prominently upregulated protein under H/R conditions. siRNA-mediated CDC6 knockdown attenuated keratinocyte senescence, restored G1/S phase cyclin-D1 (CCND1) expression, and suppressed p53 levels, demonstrating its regulatory role in senescence via the p53/CCND1 pathway. Furthermore, protein interaction network analysis and experimental validation revealed CDC6's direct binding with origin recognition complex 2 (ORC2), evidenced by nuclear colocalization (immunofluorescence) and physical interaction (co-immunoprecipitation). Collectively, our findings pioneers the mechanistic elucidation of CDC6 in pressure injury pathology, proposing senescence-targeted interventions as a novel therapeutic strategy for ulcer management.