Ultraviolet B irradiation induces senescence of human corneal endothelial cells in vitro by DNA damage response and oxidative stress

The human corneal endothelial cells (HCEnCs) play a vital role in the maintenance of corneal transparency and visual acuity. In our daily life, HCEnCs are inevitably exposed to ultraviolet B (UVB) radiation leading to de-creases of visual acuity and corneal transparency resulting in visual loss eventually. Therefore, understanding the UVB-induced cytotoxicity in HCEnCs is of importance for making efficient strategies to protect our vision from UVB-damage. However, in-depth knowledge about UVB-induced cytotoxicity in HCEnCs is missing. Herein, we pulse-irradiated the HCEnCs in vitro with 150 mJ/cm2 UVB (the environmental dose) at each subculture for 4 passages to explore the insights into UVB-induced phototoxicity. The results showed that the UVB-treated HCEnCs exhibit typical senescent characteristics, including significantly enlarged relative cell area, increased senescence-associated beta-galactosidase positive staining, and upregulated p16INK4A and senescence associated secretory phenotypes (SASPs) such as CCL-27, IL-1 alpha/6/8/10, TGF-beta 1 and TNF-alpha, as well as decreased cell pro-liferation and Lamin B1 expression, and translocation of Lamin B1. Furthermore, we explored the causative mechanisms of senescence and found that 150 mJ/cm2 UVB pulse-irradiation impairs DNA to activate DNA damage response (DDR) pathway of ATM-p53-p21WAF1/CIP1 with downregulated DNA repair enzyme PARP1, leading to cell cycle arrest resulting in DDR-mediated senescence. Meanwhile, UVB pulse-irradiation also elicits a consistent increase of ROS production to aggravate DNA damage and impose oxidative stress on energy meta-bolism leading to metabolic disturbance resulting in metabolic disturbance-mediated senescence. Altogether, the repeated pulse-irradiation of 150 mJ/cm2 UVB induces HCEnC senescence via both DDR pathway and energy metabolism disturbance.