Mechanisms of PM Disruption of the Nrf2 Pathway in Cornea.

We have previously shown that PM exposure causes oxidative stress and reduces Nrf2 protein levels, and SKQ1 pre-treatment protects against this damage in human corneal epithelial cells (HCE-2). The current study focuses on uncovering the mechanisms underlying acute PM toxicity and SKQ1-mediated protection. HCE-2 were pre-treated with SKQ1 and then exposed to 100 μg/mL PM. Cell viability, oxidative stress markers, programmed cell death, DNA damage, senescence markers, and pro-inflammatory cytokines were analyzed. Nrf2 cellular location and its transcriptional activity were determined. Effects of the Nrf2 inhibitor ML385 were similarly evaluated. Data showed that PM decreased cell viability, Nrf2 transcriptional activity, and mRNA levels of antioxidant enzymes, but increased p-PI3K, p-NFκB, COX-2, and iNOS proteins levels. Additionally, PM exposure significantly increased DNA damage, phosphor-p53, p16 and p21 protein levels, and -galactosidase (-gal) staining, which confirmed the senescence. SKQ1 pre-treatment reversed these effects. ML385 lowered the Nrf2 protein levels and mRNA levels of its downstream targets. ML385 also abrogated the protective effects of SKQ1 against PM toxicity by preventing the restoration of cell viability and reduced oxidative stress. In conclusion, PM induces inflammation, reduces Nrf2 transcriptional activity, and causes DNA damage, leading to a senescence-like phenotype, which is prevented by SKQ1.