ABSTRACT: Corneal endothelial cells (CECs) are essential for maintaining corneal hydration, transparency, and visual function. However, mature CECs have extremely limited proliferative capacity in vivo, and their progressive senescence with age contributes to oxidative stress, aberrant extracellular matrix (ECM) remodeling, loss of cell density, and corneal endothelial dysfunction. Therefore, promoting CEC proliferation while delaying cellular senescence represents an important therapeutic challenge. Here, we identified chondroitin sulfate (CS), a naturally occurring glycosaminoglycan, as a potential regulator of CEC proliferation and senescence. Using a transforming growth factor-β (TGF-β)-induced senescence model in B4G12 cells, together with human primary and rabbit CEC models, we demonstrated that CS markedly promoted CEC proliferation, reduced SA-β-gal-positive cells, and suppressed the senescence-associated markers p16, p21, and p53. Transcriptomic analysis indicated that the protective effects of CS were primarily associated with ECM remodeling, oxidative stress, and senescence-related signaling pathways. Mechanistically, CS inhibited aberrant TGF-β/Smad activation and reduced ECM-associated protein deposition, including collagen II. Meanwhile, CS enhanced antioxidant defenses by upregulating SOD, PRDX2, GPX4, and CAT, thereby reducing intracellular reactive oxygen species (ROS) accumulation and oxidative stress-induced DNA damage. These effects were accompanied by reduced NF-κB/p38-MAPK activation and alleviation of p53–p21/p16-mediated cell-cycle arrest. Collectively, our findings demonstrate that CS promotes CEC proliferation and delays cellular senescence through coordinated regulation of the TGF-β/Smad, ECM, oxidative stress, and inflammatory senescence-associated pathways. This study identifies CS as a potential therapeutic candidate for restoring CEC regenerative capacity and treating age-associated corneal endothelial dysfunction.
