Abstract
Purpose :
To determine the effect of hydroquinone (HQ), an oxidative component of cigarette smoke, on inflammatory markers in porcine corneal endothelial cells, and to find an optimum treatment time and concentration for hydroquinone as a model of smoking-induced oxidative stress in corneal cells.
Methods :
Four porcine eyes were harvested. The corneoscleral rim was separated from the rest of the eye and treated with 0.5% trypsin for 15 minutes. Corneal endothelial cells were then scraped from Descemet membrane and plated into one well of a 12 well plate. Cells were proliferated to confluency in a low growth factor medium, then passaged three times onto one well of a 6 well plate, two wells of a 6 well plates, then twelve wells of a 12 well plate for treatment. The treatment groups were as follows: HQ for 1, 2, and 4 hours at 200, 250, and 300 µM (9 wells total), and an untreated control group (3 wells). Total RNA was extracted using the RNEasy Mini Kit and then converted to cDNA using the iScript Reverse Transcription Supermix. Expression of inflammatory markers IL-6, TNF-alpha, and p38, and the reference gene GAPDH, were quantified using real-time quantitative PCR (RT-qPCR). The experiment was repeated to obtain 3 biological replicates per treatment group. The delta-delta Ct method was used to calculate relative gene expression, and statistical tests were calculated using delta Ct values.
Results :
Single-factor analysis of variance (ANOVA) revealed a statistically significant effect of HQ on TNF-alpha expression (p<0.005) and a marginally significant effect on IL-6 expression (p<0.10). No significant effect was observed on p38 expression (p=0.51). Pairwise t-tests were then conducted on TNF-alpha and IL-6 expression for each treatment group versus the untreated controls, revealing that for both TNF-alpha and IL-6, increase in expression became significant at treatment time of 2 hours at 250 and 300 µM (p<0.05), and at treatment time of 4 hours for 200, 250, and 300 µM (p<0.05).
Conclusions :
HQ treatment beginning at 2 hours and 250 µM significantly increases expression of the inflammatory markers IL-6 and TNF-alpha, but not p38. Higher treatment times and concentrations should be chosen with caution due to increasing HQ cytotoxicity and low RNA yield. HQ may be useful as a novel smoking-induced oxidative stress model in corneal endothelial cells.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.