Abstract
Purpose :
Previously, we found that airborne particulate with a diameter of <10μm (PM10) disrupts the Nrf2 pathway in vivo and confirmed this in primary and transformed human corneal epithelial cells. Therefore the purpose of this study is to determine the effects of PM10 and SKQ1 on immortalized human corneal epithelial cells (HCET).
Methods :
HCET were treated with 50nM SKQ1 for 1h prior to treatment with 100μg/ml PM10 and incubated for 24h. Cell viability was measured by a MTT assay, malondialdehyde (MDA) and reduced glutathione (GSH) levels were analyzed by assay kits, Nrf2, p-PI3K, PI3K, p-NFκB, NFκB and p21 protein levels by western blot and apoptosis was evaluated by flow cytometry. Nrf2 localization in the cell was determined by immunofluorescence and Nrf2 transcriptional activity by an assay kit. mRNA levels of anti-oxidant enzymes and pro-inflammatory cytokines were measured by RT-PCR. Senescence was confirmed by β-galactosidase staining. Nrf2 inhibitor ML385 was also tested on HCET and its effects on Nrf2 protein and its downstream targets, cell viability, MDA and GSH were evaluated.
Results :
In HCET, PM10 decreased cell viability, GSH and Nrf2 protein, Nrf2 transcriptional activity, mRNA levels of anti-oxidant enzymes, increased p-PI3K, p-NFκB and p21 protein, β-galactosidase staining and mRNA levels of pro-inflammatory cytokines; SKQ1 treatment reversed all of these effects. PM10 did not cause apoptosis but induced senescence in HCET. Nrf2 was localized mainly in the nucleus after PM10 exposure, while it was ubiquitously expressed in both control and SKQ1 treated cells. ML385 lowered Nrf2 protein levels and its transcriptional activity in HCET. Additionally, ML385 treatment also abrogated the protective effects of SKQ1 against PM10 toxicity by failing to restore cell viability as well as GSH, and reducing MDA levels.
Conclusions :
PM10 exposure triggers oxidative stress and disrupts the Nrf2 pathway which regulates antioxidant defenses in corneal epithelial cells and SKQ1 treatment reverses these deleterious effects. Inhibition of the Nrf2 pathway leads to the abrogation of the protective effects of SKQ1 against PM10 toxicity.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.