Farrerol Directly Targets GSK-3 β to Activate Nrf2-ARE Pathway and Protect EA.hy926 Cells against Oxidative Stress-Induced Injuries

Abstract

Oxidative stress-mediated endothelial injury is considered to be involved in the pathogenesis of various cardiovascular diseases. Farrerol, a typical natural flavanone from the medicinal plant Rhododendron dauricum L., has been reported to show protective effects against oxidative stress-induced endothelial injuries in our previous study. However, its action molecular mechanisms and targets are still unclear. In the present study, we determined whether farrerol can interact with glycogen synthase kinase 3β- (GSK-3β-) nuclear factor erythroid 2-related factor 2- (Nrf2-) antioxidant response element (ARE) signaling, which is critical in defense against oxidative stress. Our results demonstrated that farrerol could specifically target Nrf2 negative regulator GSK-3β and inhibit its kinase activity. Mechanistic studies proved that farrerol could induce an inhibitory phosphorylation of GSK-3β at Ser9 without affecting the expression level of total GSK-3β protein and promote the nuclear translocation of Nrf2 as well as the mRNA and protein expression of its downstream target genes heme oxygenase-1 (HO-1) and NAD(P)H: quinone oxidoreductase 1 (NQO1) in EA.hy926 cells. Further studies performed with GSK-3β siRNA and specific inhibitor lithium chloride (LiCl) confirmed that GSK-3β inhibition was involved in farrerol-mediated endothelial protection and Nrf2 signaling activation. Moreover, molecular docking and molecular dynamics studies revealed that farrerol could bind to the ATP pocket of GSK-3β, which is consistent with the ATP-competitive kinetic behavior. Collectively, our results firstly demonstrate that farrerol could attenuate endothelial oxidative stress by specifically targeting GSK-3β and further activating the Nrf2-ARE signaling pathway.

Figure 1

Effects of farrerol on the cell viability of EA.hy926 cells. (a) Chemical structure of farrerol. (b) The cytotoxic effects of farrerol on EA.hy926 cells were determined at various concentrations for 24 h using a MTT assay. Data are presented as means ± SD (n = 5). ^∗∗p < 0.01 versus control group.

Figure 2

Effects of farrerol on Nrf2-associated antioxidant enzyme expression and Nrf2 nuclear translocation in EA.hy926 cells. (a, b) The protein expression levels of HO-1 and NQO1 were measured by Western blot assay. The β-actin protein level was considered as an internal control. (c) The nuclear extracts, cytosolic extracts, and total cell lysates were prepared, and the protein expression of Nrf2 was examined by Western blot. Lamin B1 and β-actin were used as loading controls for nuclear extracts and cytosolic extracts, respectively. (d–f) Total RNA was extracted from EA.hy926 cells treated as indicated. The mRNA expression of HO-1, NQO1, and Nrf2 was determined by real-time PCR. The values shown represent the means ± SD obtained for three independent experiments. ^∗p < 0.05 and ^∗∗p < 0.01 compared to the control group.

Figure 3

Nrf2 silencing attenuated farrerol-mediated cytoprotective effect and induction of HO-1 and NQO1. (a) Nrf2 knockdown efficiency at protein level was detected by Western blot. Data are presented as means ± SD (n = 3). ^∗p < 0.05 compared to the control group. (b) Nrf2 silencing reduced the cytoprotective effects of 40 μmol/L farrerol on H₂O₂-induced cell damage. Data are presented as means ± SD (n = 3). ^∗∗p < 0.01 compared to the indicated group. (c) The protein expression of HO-1 and NQO1 was examined by Western blot. Data are presented as means ± SD (n = 3). ^#p < 0.05 compared to the indicated group, ^∗p < 0.05 compared to the NC siRNA-treated group.

Figure 4

Effects of farrerol on GSK-3β phosphorylation in EA.hy926 cells. (a, b) Farrerol induced GSK-3β phosphorylation at Ser9 without affecting GSK-3β expression in a dose- and time-dependent manner. (c) Farrerol increased the phosphorylation level of Akt without significantly altering the levels of total Akt in a dose-dependent manner. The data in the figures represent the means ± SD (n = 3). ^∗p < 0.05 compared to the control group.

Figure 5

GSK-3β inhibitor LiCl enhances farrerol-mediated cytoprotective effect and Nrf2 nuclear accumulation in EA.hy926 cells. (a) LiCl enhanced the cytoprotective effects of 40 μmol/L farrerol on H₂O₂-induced cell damage. ^∗p < 0.05 compared to the indicated group. ^#p < 0.05 compared to the H₂O₂-treated group. (b) The nuclear translocation of Nrf2 was detected by immunofluorescence combined with DAPI staining for nuclei. Scale bar = 50 μm. (c) The nuclear translocation of Nrf2 was assessed as a ratio of nuclear to cytoplasmic fluorescence using the ImageJ software (n = 10). ^∗∗p < 0.01 compared to the control group. ^#p < 0.05 compared to the indicated group.

Figure 6

Effects of GSK-3β siRNA-mediated GSK-3β inhibition on farrerol-induced activation of the Nrf2-ARE pathway in EA.hy926 cells. (a) GSK-3β knockdown efficiency at protein level was detected by Western blot. (b) Cytoplasmic and nuclear levels of Nrf2 were detected by Western blotting to analyze the translocation of Nrf2. Lamin B1 and β-actin were used as loading controls for nuclear and cytosolic protein fractions, respectively. (c) The protein expression of HO-1 and NQO1 was examined by Western blot. Data are presented as means ± SD (n = 3); ^∗p < 0.05.

Figure 7

GSK-3β is a direct target protein for farrerol. (a) Farrerol protects GSK-3β against proteolysis in DARTS assays. (b) BLI analysis of farrerol binding to GSK-3β. (c) The kinase activity of GSK-3β in different concentrations of farrerol. (d) Lineweaver-Burk plots. ATP concentration varied from 1 to 8 μmol/L; phospho-glycogen synthase peptide-2 concentration was kept constant at 25 μmol/L; farrerol concentrations were depicted in the plot.

Figure 8

Docking binding mode of farrerol in the ATP-binding site of GSK-3β.

Figure 9

MD simulation trajectory analysis. (a) RMSF of native structure of GSK-3β (complex with the cocrystal inhibitor GR9) as well as GSK-3β in complex with farrerol. (b, c) RMSD of protein backbone and farrerol obtained during the 20 ns MD simulation. (d) Rg of the GSK-3β-farrerol complex obtained during 20 ns of MD simulation. (e) Time evolution plot of the intermolecular hydrogen bond numbers between GSK-3β and farrerol. (f) Hydrogen bond existence map for the GSK-3β-farrerol complex during the 20 ns MD simulation. (g) The distance between the H (HZ2) atom of Lys85 and the center of the benzene group (ring B of farrerol) in the 20 ns simulations.

Figure 10

Schematic presentation of probable protective mechanism of farrerol against oxidative stress injury in EA.hy926 cells. Farrerol directly inhibited the kinase activity of GSK-3β to activate the Nrf2-ARE pathway and further attenuate oxidative stress damage in EA.hy926 cells.