The Scavenging Activity of Coenzyme Q10 Plus a Nutritional Complex on Human Retinal Pigment Epithelial Cells

Abstract

Age-related macular degeneration (AMD) and diabetic retinopathy (DR) are common retinal diseases responsible for most blindness in working-age and elderly populations. Oxidative stress and mitochondrial dysfunction play roles in these pathogenesis, and new therapies counteracting these contributors could be of great interest. Some molecules, like coenzyme Q₁₀ (CoQ₁₀), are considered beneficial to maintain mitochondrial homeostasis and contribute to the prevention of cellular apoptosis. We investigated the impact of adding CoQ₁₀ (Q) to a nutritional antioxidant complex (Nutrof Total^®; N) on the mitochondrial status and apoptosis in an in vitro hydrogen peroxide (H₂O₂)-induced oxidative stress model in human retinal pigment epithelium (RPE) cells. H₂O₂ significantly increased 8-OHdG levels (p < 0.05), caspase-3 (p < 0.0001) and TUNEL intensity (p < 0.01), and RANTES (p < 0.05), caspase-1 (p < 0.05), superoxide (p < 0.05), and DRP-1 (p < 0.05) levels, and also decreased IL1β, SOD2, and CAT gene expression (p < 0.05) vs. control. Remarkably, Q showed a significant recovery in IL1β gene expression, TUNEL, TNFα, caspase-1, and JC-1 (p < 0.05) vs. H₂O_2, and NQ showed a synergist effect in caspase-3 (p < 0.01), TUNEL (p < 0.0001), mtDNA, and DRP-1 (p < 0.05). Our results showed that CoQ₁₀ supplementation is effective in restoring/preventing apoptosis and mitochondrial stress-related damage, suggesting that it could be a valid strategy in degenerative processes such as AMD or DR.

Keywords: ARPE-19; DRP-1; age-related macular degeneration (AMD); caspase-3; coenzyme Q10; diabetic retinopathy (DR); mitochondrial stress; oxidative stress.

Figure 1

DNA oxidative damage analyzed as 8-OHdG levels in ARPE-19 cells’ supernatants by ELISA in basal conditions (A) and after the addition of H₂O₂ (600 µM, 1 h) and antioxidant treatments in concomitance for 30 min (B) (* p < 0.05 vs. control) (n = 3). The application of NQ showed a tendency to significantly reduce 8-OHdG levels vs. H₂O₂ control group (p = 0.0550).

Figure 2

Percentage of the fluorescence intensity of caspase-3 (green) immunolabeling in basal conditions after Q, N, and NQ showed statistical differences between control and Q (p < 0.05) (A). Oxidative environment induced by H₂O₂ increased caspase-3 immunofluorescence vs. control group (p < 0.001) (B) (n = 3). After N and NQ with oxidative stress, significant differences were observed vs. H₂O₂ group (* p < 0.05, ** p < 0.01, *** p < 0.001). Nuclei were labeled with 4′,6-diamidino-2-phenylindole (DAPI, blue). Scale bar: 20 µm.

Figure 3

Percentage of TUNEL fluorescence intensity (red) in basal conditions after Q, N, and NQ showed statistical differences between control and Q (* p < 0.05) (A). H₂O₂ group showed a significant increase vs. control group (*** p < 0.001). After Q, N, and NQ treatments in concomitance with oxidative stress, a significant reduction was observed in Q and NQ vs. H₂O₂ group (* p < 0.05 and *** p < 0.001) (B) (n = 3). Nuclei were labeled with 4′,6-diamidino-2-phenylindole (DAPI, blue). Scale bar: 20 µm.

Figure 4

Quantification of cytokine levels in which changes have been observed in standard conditions and under oxidative stress with treatments Q, N, and NQ (n = 4). Levels of IL17A, IL6, and RANTES in ARPE-19 cells supernatant (A–C) in standard conditions. Levels of caspase-1, IL12-p70, and RANTES in ARPE-19 cells supernatant after oxidative stress conditions (D–F) and TNFα levels in lysates after oxidative stress conditions (G). Lysates’ data are presented as pg/µg protein and supernatants’ data are presented as pg/mL. RANTES data are presented as RFU. For all data mean ± SEM are presented. * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. H₂O₂. Q—coenzyme Q₁₀, N—Nutrof total, NQ—Nutrof total + CoQ₁₀.

Figure 5

Quantification of SOD2 gene expression of cultured ARPE-19 cells in standard conditions and under oxidative stress with Q, N, and NQ treatments (n = 4). SOD2 expression in standard conditions showed a significant reduction with all antioxidant treatments (A). H₂O₂ group showed a significant decrease vs. control group (* p < 0.05). SOD2 expression in ARPE-19 cells with 2 h of H₂O₂ in concomitance showed no significant reduction with treatments (B). For all data, mean ± SEM are presented. * p < 0.05 vs H₂O₂ group. Q—coenzyme Q₁₀, N—Nutrof total, NQ—Nutrof total + CoQ₁₀.

Figure 6

Quantification of ILβ1 expression of cultured ARPE-19 cells in standard conditions and under oxidative stress with treatments Q, N and NQ (n = 4). ILβ1 expression significantly decreased with N antioxidant treatment * p < 0.05 vs. control (A). ILβ1 expression in ARPE-19 cells with 1 h of H₂O₂ in concomitance decreased after Q and N treatment (B) (* p < 0.05) vs. H₂O₂ group. Q—coenzyme Q₁₀, N—Nutrof total, NQ—Nutrof total + CoQ₁₀.

Figure 7

Quantification of CAT expression of cultured ARPE-19 cells in standard conditions and under oxidative stress with treatments Q, N, and NQ (n = 4). No changes were observed in CAT expression in basal conditions with antioxidant treatments (A). CAT expression in ARPE-19 cells with 1 h of H₂O₂ concomitance showed a decrease only in H₂O₂ group vs. control (* p < 0.05) (B). Q—coenzyme Q₁₀, N—Nutrof total, NQ—Nutrof total + CoQ₁₀.

Figure 8

Percentage of mitochondrial superoxide indicator in live ARPE-19 cells measured by MitoSOX (red) in standard conditions and under oxidative stress with treatments Q, N, and NQ (n = 3). No changes in basal conditions were observed (A). H₂O₂ group showed a significant increase vs. control group (* p < 0.05), (B) and after H₂O₂ in concomitance, only the NQ treatment decreased MitoSOX (p = 0.0533) (B). Q—coenzyme Q₁₀, N—Nutrof total, NQ—Nutrof total + CoQ₁₀. * p < 0.05. Nuclei were labeled with 4′,6-diamidino-2-phenylindole (DAPI) (blue). Scale bar: 20 µm.

Figure 9

Mitochondrial DNA amount of cultured ARPE-19 cells measured by 12S RT-PCR under standard conditions and under oxidative stress with treatments Q, N, and NQ (n = 4). No changes were observed in the mitochondrial DNA amount in cells treated with different treatments under basal conditions (A). H₂O₂ group showed an almost significant increase vs. the control group (p = 0.0690) (B) and the NQ group in concomitance with H₂O₂ significantly decreased mtDNA vs. the H₂O₂ group * p < 0.05 (B). Q—coenzyme Q₁₀, N—Nutrof total, NQ—Nutrof total + CoQ₁₀.

Figure 10

Mitochondrial membrane potential (mtΔψ) determined by live JC-1 measurement in ARPE-19 cells under basal conditions (A,B) and in concomitance with oxidative stress conditions with antioxidants treatments (C,D) (n = 3). J-monomers, green; J-aggregates, red. No changes were observed in JC-1 under basal conditions (A); however, in concomitance with H₂O₂ only, the Q treatment significantly decreased the mtΔψ vs. H₂O₂ group (B) (* p < 0.05). Q—coenzyme Q₁₀, N—Nutrof total, NQ—Nutrof total + CoQ₁₀. Nuclei were labeled with 4′,6-diamidino-2-phenylindole (DAPI) (blue). Scale bar: 20 µm.

Figure 11

Percentage of mitochondrial DRP-1 (red) measurement in ARPE-19 cells under basal conditions (A,B) and under oxidative stress conditions with treatments Q, N, and NQ (C,D) (n = 4). Q treatment significant increased DRP-1 under basal conditions (B) (** p < 0.01). H₂O₂ group showed a significant increase vs. control group (* p< 0.05) (B). After concomitance with H₂O_2, only NQ treatment showed a significant decrease vs. H₂O₂ group (* p < 0.05). Q—coenzyme Q₁₀, N—Nutrof total, NQ—Nutrof total + CoQ₁₀. Nuclei were labeled with 4′,6-diamidino-2-phenylindole (DAPI) (blue). Scale bar: 20 µm.