Characterization of the Antioxidant Effects of γ-Oryzanol: Involvement of the Nrf2 Pathway

Abstract

γ-Oryzanol (ORY) is well known for its antioxidant potential. However, the mechanism by which ORY exerts its antioxidant effect is still unclear. In this paper, the antioxidant properties of ORY were investigated for its potential effects as a reactive oxygen and nitrogen species (ROS/RNS) scavenger and in activating antioxidant-promoting intracellular pathways utilizing the human embryonic kidney cells (HEK-293). The 24 h ORY exposure significantly prevented hydrogen peroxide- (H₂O₂-) induced ROS/RNS production at 3 h, and this effect was sustained for at least 24 h. ORY pretreatment also enhanced the activity of antioxidant enzymes: superoxide dismutase (SOD) and glutathione peroxidase (GPX). Interestingly, ORY induced the nuclear factor (erythroid-derived 2)-like 2 (Nrf2) nuclear translocation and upregulation of Nrf2-dependent defensive genes such as NAD(P)H quinone reductase (NQO1), heme oxygenase-1 (HO-1), and glutathione synthetase (GSS) at mRNA and protein levels in both basal condition and after H₂O₂ insult. Thus, this study suggested an intriguing effect of ORY in modulating the Nrf2 pathway, which is also involved in regulating longevity as well as age-related diseases.

Figure 1

Oryzanol protects from cell death and decreases H₂O₂-induced ROS/RNS generation. (a) HEK-293 cells were pretreated for 24 h with ORY (ORY_24h) at different concentrations and then stressed by the addition of 100 μM H₂O₂ for 24 h. Cell viability was evaluated with MTT assay. Data are shown as percentage of cell viability compared with untreated cells; ^∗∗∗p ≤ 0.001 versus untreated cells and ^##p ≤ 0.01 and ^#p ≤ 0.05 versus H₂O₂ group. (b) Effects of 5 μg/mL ORY_24h on H₂O₂-induced ROS/RNS production were determined by H₂DCF-DA oxidation using a fluorescence microplate reader. Fluorescence intensity of ORY_24h (open square symbol) after oxidative insult significantly decreased over time with ^∗∗p ≤ 0.01 at 3 h and ^∗∗∗p ≤ 0.001 at 24 h versus the corresponding untreated control group (open circle symbol). Bonferroni's multiple comparison test was used.

Figure 2

Oryzanol effect on antioxidant enzyme activities and MnSOD and Cu-ZnSOD expression. HEK-293 cells were pretreated with 5 μg/mL ORY for 24 h followed by 100 μM H₂O₂ for 3 h or 24 h. (a, b) Effect of ORY_24h on the expression of MnSOD (SOD2) and Cu-ZnSOD (SOD1) in H₂O₂-induced oxidative stress. The protein expression was assessed by Western blotting. Tubulin expression was used as loading control. Data are presented as mean ± SEM; ^∗∗∗p ≤ 0.001 and ^∗∗p ≤ 0.01 versus untreated cells. The activity of total SOD (c) and GPx (d) enzymes was assessed, as reported in the Material and Methods, before and after 3 h and 24 h of H₂O₂ oxidative insult. The results are represented as mean ± SEM; ^∗∗∗p ≤ 0.001, ^∗∗p ≤ 0.01, and ^∗p ≤ 0.05 versus untreated cells and ^#p < 0.05 versus the corresponding H₂O₂ control group. Bonferroni's multiple comparison test was used.

Figure 3

Nrf2 nuclear translocation induced by oryzanol. HEK-293 cells were pretreated with ORY for 3, 6, and 24 h and then stressed with 100 μM H₂O₂ for 3 h. Nuclear and cytoplasmic fractions were isolated as described in the Material and Methods. (a) Nuclear expression of Nrf2 was assessed by Western blotting and lamin A/C expression was used as loading control. Data are represented as mean ± SD; ^∗∗∗p ≤ 0.001, ^∗∗p ≤ 0.01, and ^∗p ≤ 0.05 versus untreated cells, Dunnett's multiple comparison test. (b) Cytoplasmic expression of Nrf2 was assessed by Western blotting, and tubulin expression was used as loading control. Data are represented as mean ± SD.

Figure 4

Oryzanol activates the transcription of Nrf2 target genes. HEK-293 cells were pretreated with 5 μg/mL ORY for 24 h followed by a time course of 100 μM H₂O₂ (1, 3, 6, and 12 h). Cells were then processed for measuring Nrf2 (a), Keap1 (b), HO-1 (c), and NQO1 (d) mRNA levels by real-time PCR. GAPDH was used to normalize the results. Data are shown as mean ± SEM. Statistically significant differences were represented as follows: ^∗∗∗∗p ≤ 0.0001, ^∗∗∗p ≤ 0.001, ^∗∗p ≤ 0.01, and ^∗p ≤ 0.05 versus untreated cells and ^###p ≤ 0.001 and ^#p ≤ 0.05 versus the corresponding H₂O₂ control group, Bonferroni's multiple comparison test.

Figure 5

Oryzanol induces an increase in Nrf2 target gene protein levels. HEK-293 cells were pretreated with 5 μg/mL ORY for 24 h followed by 24 h of 100 μM H₂O₂. Cells were then processed for measuring Nrf2 (a), HO-1 (b), GSS (c), and NQO1 (d) protein levels by Western blotting. Tubulin and actin were used as loading control according with the molecular weight of the above investigated proteins. Data are shown as mean ± SD; ^∗∗p ≤ 0.01 and ^∗p ≤ 0.05 versus untreated cells, Dunnett's multiple comparison test.

Figure 6

Molecular structure of oryzanol. The structure of ORY consists of two moieties: ferulic acid and phytosterols (National Center for Biotechnology Information; PubChem Compound Database, CID = 6450219.)