Addition of CoQ₁₀ to a nutritional complex improves mitochondrial status in oxidative stress-induced retinal pigment epithelial cells

Abstract

Purpose : Mitochondria are abundant in retinal pigment epithelium (RPE) and constitute the main source of energy. They are highly dynamic and an imbalance in their dynamics (fusion, fission, biogenesis, mitophagy) along with alterations in mitochondrial DNA (mtDNA) is associated with early changes in some retinal pathologies. Some molecules, like coenzyme Q₁₀ (CoQ₁₀), are considered beneficial to keep mitochondrial homeostasis. Our objective was to study the effect of CoQ₁₀ addition to a nutritional complex (Nutrof Total) on mitochondrial status in an in vitro hydrogen peroxide (H₂O₂)-induced oxidative stress model.

Methods : ARPE19 cells (cultured for 2.5 months) were subjected to 600 µM H₂O₂ for 1-3 h (H) in concomitance with CoQ₁₀ (Q, 0.1 µM CoQ₁₀), Nutrof Total (N, 0.01 mg/ml) or CoQ₁₀+Nutrof Total (QN) treatments (30 min-1 h; n=3). Phenotype was confirmed by immunofluorescence (IF) and flow cytometry for RPE-65, cytokeratin-18 and zonnule occludens-1. Early (Caspase-3) and late (TUNEL) apoptosis were evaluated and DNA damage (8OHdG) was detected. Specific mitochondrial markers were: PCR from 12S for mtDNA evaluation, dynamin related protein (DRP1) was detected by IF and superoxide release was analyzed using MitoSOX in live cells. ANOVA test followed by post-hoc were applied and p<0.05 (2-tailed) was considered statistically significant (GraphPad v. 8.0.1).

Results : H₂O₂ significantly enhanced stress markers in RPE cells by increasing 8OHdG levels (p<0.05), caspase-3 (p<0.0001) and TUNEL intensity (p<0.01) vs. saline. Mitochondrial markers were also significantly increased by oxidative stress: DRP1 (p<0.01), superoxide (p<0.01) and mtDNA (p<0.05) vs. saline. Remarkably, QN group showed a statistically significant TUNEL (p<0.01), caspase-3 (p<0.05), DRP1 (p<0.05), superoxide (p<0.05) and mtDNA (p<0.05) reduction, and 8OHdG levels were almost significantly reduced (p=0.055) vs. H. N treatment reduced caspase-3 (p=0.07), and Q showed significant reduction in TUNEL (p<0.05) and mtDNA (p<0.05) vs. H.

Conclusions : Our results showed that adding CoQ₁₀ to Nutrof Total formula is more effective than individual supplementation in restoring/preventing mitochondrial stress related damage, suggesting that it could be a valid strategy for limiting early mitochondrial changes in RPE cells in degenerative processes such as age-related macular degeneration or diabetic retinopathy.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.