The Impairment of Cell Metabolism by Cardiovascular Toxicity of Doxorubicin Is Reversed by Bergamot Polyphenolic Fraction Treatment in Endothelial Cells

Abstract

The beneficial effects of bergamot polyphenolic fraction (BPF) on the mitochondrial bioenergetics of porcine aortic endothelial cells (pAECs) were verified under the cardiotoxic action of doxorubicin (DOX). The cell viability of pAECs treated for 24 h with different concentrations of DOX was reduced by 50%, but the negative effect of DOX was reversed in the presence of increasing doses of BPF (100 µg/mL and 200 µg/mL BPF). An analysis of the protective effect of BPF on the toxic action of DOX was also carried out on cell respiration. We observed the inhibition of the mitochondrial activity at 10 µM DOX, which was not restored by 200 µg/mL BPF. Conversely, the decrease in basal respiration and ATP production caused by 0.5 or 1.0 µM DOX were improved in the presence of 100 or 200 µg/mL BPF, respectively. After 24 h of cell recovery with 100 µg/mL or 200 µg/mL BPF on pAECs treated with 0.5 µM or 1.0 µM DOX, respectively, the mitochondrial parameters of oxidative metabolism impaired by DOX were re-boosted.

Keywords: bergamot polyphenolic fraction; cell metabolism; doxorubicin; mitochondria; porcine aortica endothelial cells.

Figure 1

Effect of BPF on DOX-induced pAEC cytotoxicity. (A–E) Representative images of pAECs treated with DOX 0.5, 1, 10 µM (B,C,D, respectively) in the absence or presence of 100 µg/mL BPF (E). (F) Cell viability quantification: each bar represents the mean ± SD of three independent experiments. Scale bar (―) 100 µm. Different letters above the bars indicate significant differences (p < 0.05, one-way ANOVA, post hoc Tuckey comparison test) between each DOX treatment with and without BPF and the control (CTR) group.

Figure 2

Effect of 200 µg/mL BPF on the cell metabolism of pAECs treated with 10 µM DOX. The mitochondrial respiration profile was obtained from the oxygen consumption rate (OCR) in control (●, blue), 200 µg/mL BPF (■, orange), 10 µM DOX (▲, gray) and DOX plus BPF (♦, gold) treatment under basal respiration conditions and after the addition of 1.5 μM oligomycin (olig), 1.0 μM FCCP and a mixture of 0.5 μM rotenone plus antimycin A (Rot + AA). Inhibitor injections are shown as dotted lines (A). Mitochondrial parameters (basal respiration, proton leak, maximal respiration, spare respiratory capacity, ATP production) in control (█, blue), 200 µg/mL BPF (█, orange), 10 µM DOX (█, gray) and DOX plus BPF (█, gold) samples (B). Data expressed as points (A) and column charts (B) represent the means ± SD (vertical bars) from three experiments carried out on different cell preparations. Different letters indicate significant differences (p ≤ 0.05) among treatments within the same bioenergetic parameters.

Figure 3

Effect of 200 µg/mL BPF on the cell metabolism of pAECs treated with 1 µM DOX. The action of DOX on mitochondrial respiration without (A,B) and with 24 h of recovery (C,D) in the presence of BPF. The mitochondrial respiration profile was obtained from the oxygen consumption rate (OCR) in control (●, blue), 200 µg/mL BPF (■, orange), 1 µM DOX (▲, gray) and 1 µM DOX plus BPF (♦, gold) treatments under basal respiration conditions and after the addition of 1.5 μM oligomycin (olig), 1.0 μM FCCP and a mixture of 0.5 μM rotenone plus antimycin A (Rot + AA). Inhibitor injections are shown as dotted lines (A,C). Mitochondrial parameters (basal respiration, proton leak, maximal respiration, spare respiratory capacity, ATP production) in control (█, blue), 200 µg/mL BPF (█, orange), 1 µM DOX (█, gray), and 1 µM DOX plus BPF (█, gold) samples (B,D). Data expressed as points (A,C) and column charts (B,D) represent the means ± SD (vertical bars) from three experiments carried out on different cell preparations. Different letters indicate significant differences (p ≤ 0.05) among treatments within the same bioenergetic parameters.

Figure 4

Effect of 100 µg/mL BPF on cell metabolism of pAECs treated with 0.5 µM DOX. The action of DOX on mitochondrial respiration without (A,B) and with 24 h of recovery (C,D) in the presence of BPF. The mitochondrial respiration profile was obtained from the oxygen consumption rate (OCR) in control (●, blue), 100 µg/mL BPF (■, orange), 0.5 µM DOX (▲, gray), and DOX plus BPF (♦, gold) treatments under basal respiration conditions and after the addition of 1.5 μM oligomycin (olig), 1.0 μM FCCP and a mixture of 0.5 μM rotenone plus antimycin A (Rot + AA). Inhibitor injections are shown as dotted lines (A,C). Mitochondrial parameters (basal respiration, proton leak, maximal respiration, spare respiratory capacity, and ATP production) in control (█, blue), 100 µg/mL BPF (█, orange), 0.5 µM DOX (█, gray) and DOX plus BPF (█, gold) samples (B,D). Data expressed as points (A,C) and column charts (B,D) represent the means ± SD (vertical bars) from three experiments carried out on different cell preparations. Different letters indicate significant differences (p ≤ 0.05) among treatments within the same bioenergetic parameters.