Eriodictyol Protects Endothelial Cells against Oxidative Stress-Induced Cell Death through Modulating ERK/Nrf2/ARE-Dependent Heme Oxygenase-1 Expression

Abstract

The pathophysiology of cardiovascular diseases is complex and may involve oxidative stress-related pathways. Eriodictyol is a flavonoid present in citrus fruits that demonstrates anti-inflammatory, anti-cancer, neurotrophic, and antioxidant effects in a range of pathophysiological conditions including vascular diseases. Because oxidative stress plays a key role in the pathogenesis of cardiovascular disease, the present study was designed to verify whether eriodictyol has therapeutic potential. Upregulation of heme oxygenase-1 (HO-1), a phase II detoxifying enzyme, in endothelial cells is considered to be helpful in cardiovascular disease. In this study, human umbilical vein endothelial cells (HUVECs) treated with eriodictyol showed the upregulation of HO-1 through extracellular-regulated kinase (ERK)/nuclear factor erythroid 2-related factor 2 (Nrf2)/antioxidant response element (ARE) signaling pathways. Further, eriodictyol treatment provided protection against hydrogen peroxide-provoked cell death. This protective effect was eliminated by treatment with a specific inhibitor of HO-1 and RNA interference-mediated knockdown of HO-1 expression. These data demonstrate that eriodictyol induces ERK/Nrf2/ARE-mediated HO-1 upregulation in human endothelial cells, which is directly associated with its vascular protection against oxidative stress-related endothelial injury, and propose that targeting the upregulation of HO-1 is a promising approach for therapeutic intervention in cardiovascular disease.

Keywords: cell death; endothelial cells; eriodictyol; flavonoid; heme oxygenase-1; oxidative stress.

Figure 1

Chemical structures of the flavonoid eriodictyol (30,40,5,7-tetrahydroxyflavanone).

Figure 2

Upregulation of HO-1 by eriodictyol in HUVECs. Cells were treated with the indicated concentrations of eriodictyol (5, 10, and 20 μM) for 18 (A) and 1 h (B), and HO-1 levels were measured by Western blot and RT-PCR. GAPDH (glyceraldehyde 3-phosphate dehydrogenase) served as a loading control; (C,D) Cells were treated with 10 μM eriodictyol for the indicated times, and HO-1 levels were measured by Western blot and RT-PCR; and (E) Cell viability was estimated by MTT method. Data represents the mean ± SD of results in three independent experiments. * p  <  0.05 vs. control group.

Figure 2

Upregulation of HO-1 by eriodictyol in HUVECs. Cells were treated with the indicated concentrations of eriodictyol (5, 10, and 20 μM) for 18 (A) and 1 h (B), and HO-1 levels were measured by Western blot and RT-PCR. GAPDH (glyceraldehyde 3-phosphate dehydrogenase) served as a loading control; (C,D) Cells were treated with 10 μM eriodictyol for the indicated times, and HO-1 levels were measured by Western blot and RT-PCR; and (E) Cell viability was estimated by MTT method. Data represents the mean ± SD of results in three independent experiments. * p  <  0.05 vs. control group.

Figure 3

HO-1 activity in cells was measured 18 h after treatment with various concentrations of eriodictyol. Each bar represents the mean ± SD of four independent experiments. * p  <  0.05 vs. control group; ** p  <  0.05, between eriodictyol 10 μM and eriodictyol 10 μM plus ZnPP 1 μM co-treated samples.

Figure 4

Blockage of eriodictyol-induced HO-1 protein expression by an ERK inhibitor. (A) Cells were pre-treated with increasing doses of PD 98059 (an ERK inhibitor) for 1 h prior to the treatment of eriodictyol 10 μM; (B) cell lysates were immunoblotted with antibodies against the phosphorylated form of ERK1/2 and total ERK; and (C) transient transfection of cells with increasing doses of ERK siRNA (20 and 30 nM) inhibited the induction of HO-1 protein expression by eriodictyol 10 μM. Western blots representative of three independent experiments are shown: C, untreated cells; +, treated with eriodictyol only; black line, treated with 10 µM eridictyol; , dose increasing.

Figure 5

Nrf2 nuclear translocation induced by eriodictyol. (A) Cells were treated with 10 and 20 μM eriodictyol at the indicated concentrations for 4 h. Nuclear extracts were subjected to Western blot, using an anti-Nrf2 antibody and anti-lamin B antibody (a marker of nuclear protein); and (B) Transient transfection of cells with increasing doses of Nrf2-specific siRNA (10 and 20 nM) reduced HO-1 expression. Western blots representative of three independent experiments are shown: C, untreated cells; +, eriodictyol treatment only; , dose increasing.

Figure 6

Activation of the ARE-luciferase reporter by eriodictyol. Cells were transfected with an ARE-luciferase construct. After transfection, the cells were treated with the indicated concentrations of eriodictyol for 6 h and the lysates were mixed with a luciferase substrate. A luminometer was used for measurement of luciferase activity. Data are presented as mean ± SD of quintuplicate experiments. * p < 0.05 vs. control.

Figure 7

Protective effect of eriodictyol-induced HO-1 expression on H₂O₂-induced cell damage. (A) H₂O₂-exposed cells were pretreated for 1 h with or without ZnPP and then treated with eriodictyol. The inhibitory effect of eriodictyol on H₂O₂-induced intracellular ROS generation was observed by fluorescence microscopy; (B) H₂O₂-stimulated cells were pretreated for 1 h with or without ZnPP or Nrf2 or HO-1 siRNA and then treated with eriodictyol. Protective effect of HO-1 induction on cell death was determined by in situ terminal nick end-labeling (TUNEL) assay: −, untreated; +, treated. ROS and TUNEL staining was quantified in four randomly selected fields for each group: C, control. The scale bars for image (B) are the same as that in (A), 100 μm. * p < 0.05 vs. control, ** p < 0.05 vs. eriodictyol 10 μM + H₂O₂ co-treated samples.