Abstract
Purpose :
Recent studies have demonstrated that oxidative stress is involved in pathogenesis of dry eye disease. Higher levels of reactive oxygen species (ROS), lipid oxidative stress markers and inflammatory cells were found in the conjunctiva and tear film of Sjögren syndrome patients. Polyols erythritol and glycerin have demonstrated osmoprotective properties in vitro. Evidence indicates these polyols also have antioxidant properties. The purpose of this study was to develop a cellular assay for evaluating the antioxidant properties of these polyols in Riken human corneal epithelial cells (R-HCEC).
Methods :
R-HCEC were seeded in a 96 well plate, incubated for 72 hours, the cell culture media was replaced with fresh medium, and the cells were incubated for an additional 24 hours. Cells were pre-exposed to CellROX Green (CRG) for 30 minutes and then glucose oxidase (GOx) from 0.1-50 milliunits (mU) to determine an effective concentration needed to initiate ROS production. For antioxidant testing R-HCEC were preincubated in medium containing a final concentration of 0.8% erythritol and/or glycerin for 30 minutes and CRG was added with the polyols for an additional 30 minutes. Cells were then exposed to GOx for 3.5 hours and fluorescence from oxidized CRG was measured. Cells were assessed for metabolic activity by alamarBlue™ after completion of the assays.
Results :
Metabolic activity and ROS produced fluorescence indicated that 0.5 mU of GOx was an appropriate concentration to evaluate the antioxidant activity. GOx unstimulated cell fluorescence remained at baseline, <1000 relative fluorescent units (RFUs) throughout the time course, while GOx stimulated cells increased from 2800 RFUs to 42,000 RFUs at time 3.5 hours. Cells receiving GOx and erythritol and/or glycerin had significantly reduced RFUs, ranging from 32,000 to 34,000 RFUs (p≤0.05), when compared to cells receiving GOx only.
Conclusions :
This in vitro cell model can be used to initiate oxidative stress in R-HCEC and evaluate the antioxidant potential of ingredients in eye care formulations. Both 0.8% erythritol and 0.8% glycerin individually or in combination (at an equivalent percentage) exhibit antioxidant properties in commercially available eye drop formulations.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.