Blue Light Irradiation Elicits Senescence of Corneal Endothelial Cells In Vitro by Provoking Energy Crisis, Inflammasome Assembly and DNA Damage.

Abstract

Purpose: The blue light from the digital screens endangers the visual system among which the corneas at the outmost of eyes are vulnerable to the irradiation. Therein, the human corneal endothelial (HCE) cells are crucial to maintain corneal transparency and their damage leads to HCE decompensation resulting in blindness ultimately. Thus, understanding the phototoxic effects of the blue light on the HCE cells and the underlying mechanisms is important for taking measures to protect the vision clarity from the blue-light hazard.

Methods: We pulse-irradiated the HCE cell line cells at logarithmic phase for 3 passages using 440 nm blue light and examined the levels of reactive oxygen species (ROS), ATP, nicotinamide adenine dinucleotide (NAD⁺) and autophagy using cytochemistry assay to investigate the alterations of energy metabolism. Moreover, we examined the γH2AX⁺ cells using immunofluorescence and expression of poly(ADP-Ribose)polymerase1 (PARP1) using western blotting to investigate the degrees of DNA damage and repair. We also monitored the levels of inflammasome using western blotting and senescence associated secretory phenotypes (SASPs) of interleukin (IL)-8, IL-1β and IL-6 using qPCR and ELISA to investigate the inflammasome assembly and secretion of SASPs. We detected the senescent features with senescence-associated-β-galactosidase assay, p16 levels by western blotting, Lamin B1 localization by immunofluorescence observation, cell growth by EdU incorporation assay and confluence forming time and alterations of the cell morphology and relative areas by microscopy observation.

Results: The HCE cells exhibited senescent features after blue-light-pulse-irradiation. The blue light provokes overproduction of ROS to decrease the levels of ATP, NAD⁺ and autophagy leading to energy crisis. Moreover, the excess ROS injure DNA and downregulate PARP1 resulting in stable cell-cycle arrest. The excess ROS also facilitate inflammasome assembly leading to hypersecretion of SASPs.

Conclusion: The blue light elicits HCE cell senescence via inducing energy crisis, stable cell-cycle arrest and SASP hypersecretion.