Zeaxanthin induces Nrf2-mediated phase II enzymes in protection of cell death

Abstract

Zeaxanthin (Zea) is a major carotenoid pigment contained in human retina, and its daily supplementation associated with lower risk of age-related macular degeneration. Despite known property of Zea as an antioxidant, its underlying molecular mechanisms of action remain poorly understood. In this study, we aim to study the regulation mechanism of Zea on phase II detoxification enzymes. In normal human retinal pigment epithelium cells, Zea promoted the nuclear translocation of NF-E2-related factor 2 (Nrf2) and induced mRNA and protein expression of phase II enzymes, the induction was suppressed by specific knockdown of Nrf2. Zea also effectively protected against tert-butyl hydroperoxide-induced mitochondrial dysfunction and apoptosis. Glutathione (GSH) as the most important antioxidant was also induced by Zea through Nrf2 activation in a time- and dose-dependent manner, whereas the protective effects of Zea were decimated by inhibition of GSH synthesis. Finally, Zea activated the PI3K/Akt and MAPK/ERK pathway, whereas only PI3K/Akt activation correlated with phase II enzymes induction and Zea protection. In further in vivo analyses, Zea showed effects of inducing phase II enzymes and increased GSH content, which contributed to the reduced lipid and protein peroxidation in the retina as well as the liver, heart, and serum of the Sprague-Dawley rats. For the first time, Zea is presented as a phase II enzymes inducer instead of being an antioxidant. By activating Nrf2-mediated phase II enzymes, Zea could enhance anti-oxidative capacity and prevent cell death both in vivo and in vitro.

Figure 1

Effects of Zea on phase II enzyme expression. ARPE-19 cells were treated with Zea at the indicated concentrations (1, 10, 50 μM) for 6 or 24 h. The mRNA levels of the phase II enzymes HO-1 (a), NQO-1 (b), GCLc (c), and GCLm (d) were analyzed. The protein levels of HO-1 and NQO-1 were analyzed by western blotting, both after 24 h of treatment with Zea at different doses (e, left: western blot image, right: statistical analysis) and after treatment with 10 μM Zea for different time (f, left: western blot image, right: statistical analysis). All data are shown as mean±S.E.M. The symbol ‘*' indicates statistical significance, as determined by one-way ANOVA (*P<0.05, **P<0.01, n=3)

Figure 2

Zea activates phase II enzymes through Nrf2 nuclear translocation. ARPE-19 cells were transiently transfected with Nrf2 siRNA at 100 pmol per well in six-well plates for 24 h, followed by treatment with 10 μM Zea for 6 or 24 h. The mRNA levels of Nrf2 (a), HO-1 (b), and NQO-1 (c) were analyzed after 6 h of Zea treatment, and the protein levels of HO-1 and NQO-1 were analyzed after 24 h of Zea treatment (d). Protein levels of Nrf2 and Keap1 response to Zea dose-dependent and time-dependent effects were analyzed by western blot (e and f). Cyto- and nuclear Nrf2 levels after treatment with 10 μM Zea for 0.5, 2, or 6 h (g). Co-immunoprecipitation assays after 6 h of treatment with 10 μM Zea, antibodies against Nrf2 (h) and Keap1 (i) were used for immunoprecipitation, and the antibodies employed in the western blot analysis are indicated on the left side of the panel. All data are shown as mean±S.E.M. The symbol ‘*' indicates statistical significance, as determined by one-way ANOVA (*P<0.05, **P<0.01, n=3)

Figure 3

Zea confers protection against t-BHP-induced oxidative stress and mitochondrial dysfunction. ARPE-19 cells were treated with 10 μM Zea for 24 h, followed by 6 h of treatment with 300 μM t-BHP. Cu-Zn SOD activity (a), GPx activity (b), GST activity (c), the mitochondrial membrane potential (d), cell viability (e), and cellular ATP levels (f) were analyzed. All data are shown as mean±S.E.M. The symbol ‘*' indicates statistical significance, as determined by one-way ANOVA (*P<0.05, **P<0.01, n=3)

Figure 4

Zea inhibits the t-BHP-induced activation of apoptosis. ARPE-19 cells were treated with 10 μM Zea for 24 h, followed by 6 h of treatment with 300 μM t-BHP. Hoechst 33342 staining was performed to observe apoptotic cells (a). PARP cleavage was detected by western blotting (c), and caspase 9 activity was analyzed in live cells (b). All data are shown as mean±S.E.M. The symbol ‘*' indicates statistical significance, as determined by one-way ANOVA (*P<0.05, **P<0.01, n=3)

Figure 5

GSH is the critical effector of the protective effects of Zea. Total GSH levels were measured following the Zea treatment of ARPE-19 cells: (a) dose response—cells were treated with 1, 10, or 50 μM Zea for 24 h; (b) time response—cells were treated with 10 μM Zea for 6, 12, 24 or 48 h; (c) Nrf2 knockdown response—cells were transfected with Nrf2 siRNA for 24 h, followed by treatment with 10 μM Zea for an additional 24 h; (d) t-BHP response—cells were treated with 10 μM Zea for 24 h, followed by challenge with 300 μM t-BHP for an additional 6 h. In the presence of 50 μM BSO, cells were treated with 10 μM Zea for 24 h and then challenged with 300 μM t-BHP for an additional 6 h, after which cell viability (e), the mitochondrial membrane potential (f), and apoptosis activation (g) were analyzed. All data are shown as mean±S.E.M. The symbol ‘*' indicates statistical significance, as determined by one-way ANOVA (*P<0.05, **P<0.01, n=3)

Figure 6

Zea activates the Nrf2/Keap1 pathway through PI3/Akt activation. ARPE-19 cells were treated with 10 μM Zea for the indicated time, and kinase activation was analyzed by western blotting (a). Cells were treated with 10 μM Zea in the presence of LY294002 or U0126 for 24 h, and the protein levels of HO-1 and NQO-1 were analyzed (b: western blot image; c: statistical analysis of HO-1 expression; d: statistical analysis of NQO-1 expression), GSH levels were also determined (e). Following co-treatment with Zea, LY294002, and U0126 for 24 h, cells were challenged with t-BHP for an additional 6 h, after which cell viability (f) and the mitochondrial membrane potential (g) were analyzed. All data are shown as mean±S.E.M. The symbol ‘*' indicates statistical significance, as determined by one-way ANOVA (*P<0.05, **P<0.01, n=3)

Figure 7

Zea activates phase II enzymes in the rat retina. SD rats were supplemented with Zea for 4 weeks, and their retinas were then isolated. Phase II enzyme protein expression (a: western blot image, b: statistical analysis), retina GSH contents (c), Akt activation (d: western blot image, e: statistical analysis), retina 4-HNE contents (f), and carbonyl protein levels (g: western blot image, h: statistical analysis) were analyzed. All data are shown as mean±S.E.M. The symbol ‘*' indicates statistical significance, as determined by one-way ANOVA (*P<0.05, **P<0.01, n=8)

Figure 8

Effects of Zea in the rat liver, heart, and serum. SD rats were supplemented with Zea for 4 weeks, and their liver, heart, and serum were then collected. Liver GSH (a), liver 4-HNE (b), heart GSH (c), heart 4-HNE (d), serum GSH (e), serum 4-HNE (f), and serum carbonyl levels (g: Western blot image, h: statistical analysis) were analyzed. All data are shown as mean±S.E.M. The symbol ‘*' indicates statistical significance, as determined by one-way ANOVA (*P<0.05, **P<0.01, n=8)

Figure 9

Possible mechanism of Zea protection against t-BHP-induced cell apoptosis. Zea could time and dose dependently induce expression of phase II enzymes through promoting Nrf2 nuclear translocation. GSH, the production of γ-GCL, was thereby increased, and accounted for anti-apoptotic effect of Zea. Meanwhile, the activation of PI3K/Akt pathway was found to work as upstream kinase regulating phase II enzymes expression and GSH production