Oxidative Stress Induces Apoptosis and Inflammation in in vitro Dry Eye Models Which Can Be Reversed by Antioxidant SkQ1

Abstract

Purpose : Oxidative stress and inflammation are common phenomena in Dry Eye Disease (DED). However, their roles in the pathogenesis of DED need to be clarified. In our study, we illustrated the relationship between oxidative stress and DED in vitro, with a mitochondria-targeted antioxidant, SkQ1.

Methods : Tert-butyl hydroperoxide (TBHP) was added in cultured human corneal epithelial cell (HCEC) for oxidant damage model. 4 hydroxynonenal (4-HNE) and Malondialdehyde (MDA) were determined as indicated biomarker of oxidant damage. The antioxidant effect of SkQ1 was evaluated. Two dry eye in vitro models were set up by culturing HCEC in hypertonic medium or media with 0.0002% benzalkonium chloride (BAK). Cell statuses were monitored by viability assay (MTT), Reactive Oxygen Species (ROS) assay (FACS and fluorescent staining) and apoptosis assay (FACS), cytokines and matrix metalloproteases were quantified with ELISA.

Results : ROS, 4-HNE and MDA level were increased in TBHP model. SkQ1 effectively eliminated ROS, associated with reduced apoptosis and IL-6, MMP3 production. The relationship between oxidant damage and DED was explored using hyperosmolarity and BAK-induced in vitro models. Excessive mitochondrial ROS was found in both models and happened earlier than cell apoptosis and viability loss. SkQ1 significantly reduced level of mitochondrial ROS, followed by decreased cell apoptosis and reversed cell viability. Upregulation of inflammatory factors production was determined in each model (IL-6, MMP3, MMP9 in hyperosmotic model; IL-1α, IL-6, MMP9 in BAK model), while SkQ1 effectively down-regulates the protein level of these DED-associated cytokines and matrixes.

Conclusions : Our finding indicates that increased mitochondrial ROS level widely occurs in DED models which orchestrates cell apoptotic and inflammatory responses. Antioxidant SkQ1 reverts these cell damages, offering an universal therapeutic potential. 4-HNE and MDA, as widely accepted biomarkers in DED, were significantly upregulated in dry eye patients' tears, suggesting a perspective clinical application of antioxidant agent for DED.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.

 

SkQ1 modulates cell status and IL-6 & MMP3 level in TBHP model (A-C). Dynamic change of cell status in in vitro dry eye models (D).

 

SkQ1 modulates cell status, downregulate inflammatory factor levels in in vitro dry eye models (A-D). 4-HNE and MDA level in dry eye patients' tears (E).