| Objective:Farrerol is the major bioactive component isolated from the leaves of traditional Chinese herb Rhododendron dauricum L..It has been documented that farrerol possesses many biological activities,such as antioxidative,anti-inflammatory,antibacterial,anticancer,and inhibition of VSMC proliferation.In our previous study,farrerol has been reported to show protective effects against oxidative stress-induced apoptosis and endothelial tight junction disruption in human endothelium-derived EA.hy926 cells.However,the underlying antioxidant molecular mechanisms and targets of farrerol implicated in the observed endothelial protection effects remain to be elucidated.Nuclear factor erythroid 2-related factor 2（Nrf2）is an important regulator of endogenous antioxidant systems.As a key transcription factor,Nrf2regulates the basal and inducible expression of a battery of antioxidant genes and other cytoprotective Phase II detoxifying enzymes and therefore is critical for the maintenance of cellular redox homeostasis.The evidence from recent studies suggests that glycogen synthase kinase 3β（GSK-3β）,a ubiquitously expressed serine/threonine protein kinase,is a Keap1（Kelch-like epichlorohydrin-associated protein1）-independent Nrf2 regulator.In the first part of this paper,we explored and found that there exists a direct and specific binding between GSK-3βand farrerol,and further proved that farrerol is a direct inhibitor of GSK-3βin vitro.Also,we clarified the binding affinity and inhibition mechanism.In the second part of the paper,the regulation effect of farrerol on Akt/GSK-3β/Nrf2/ARE signaling pathway in EA.hy926 cells was investigated in order to elucidate the possible role of GSK-3βin farrerol-mediated protective effect against H2O2-induced oxidative stress.In the third part of the paper,enzyme kinetic analysis,molecular docking and molecular dynamics were employed to illustrate the binding mode between farrerol and GSK-3βat the molecular level.Based on the above-mentioned three parts of research,this paper was designed to reveal the underlying antioxidant molecular mechanisms and targets of farrerol in human endothelium-derived EA.hy926 cells.Methods:1.Drug affinity reaction target stability（DARTS）assay combined with LC-MS/MS mass spectrometry analysis was used to identify the specific affinity proteins of farrerol in EA.hy926 cells.Briefly,EA.hy926 cells were lysed and the lysates were then incubated with farrerol or DMSO control.Proteolysis was performed by adding pronase solution.Then,samples were resolved by SDS-PAGE and stained with Coomassie blue.Finally,the differential bands were excised from the gel and subjected to LC-MS/MS analysis.2.DARTS assay combined with Western blot assay was employed to verify interaction between farrerol and GSK-3β.Furthermore,cellular thermal shift assay（CETSA）combined with Western blot assay was performed to further confirm the binding affinity between farrerol and GSK-3β.Briefly,EA.hy926 cells were lysed and the lysates were then incubated with farrerol or DMSO control.The cell lysates were heated to different temperatures,cooled,and cleared by centrifugation.Finally,the levels of soluble target protein were measured by Western blot assay.3.Biolayer interferometry（BLI）assay was adopted to detect the binding specificity between farrerol and GSK-3β.ForteBio Data Analysis software was used to obtain the quantitative binding kinetic parameters of farrerol to GSK-3β.4.The optimized Kinase-Glo Luminescent Kinase assay was employed to evaluate the inhibitory activity of farrerol on GSK-3β.The IC50 value was further determined by nonlinear regression analysis of GSK-3βinhibition versus different concentrations of farrerol.5.Western blot assay was used to examine the effects of farrerol on the expressions of the nuclear translocation of Nrf2 as well as the protein expression of its downstream target genes heme oxygenase-1（HO-1）and NAD（P）H:quinone oxidoreductase 1（NQO1）in EA.hy926 cells.Quantitative real-time polymerase chain reaction（RT-PCR）assay was used to determine the mRNA expression of antioxidant genes Nrf2,HO-1 and NQO1.6.MTT quantitative colorimetric assay was performed to estimate the effect of specific GSK-3βinhibitor lithium chloride（LiCl）on farrerol-mediated endothelial protection.Immunofluorescence combined with DAPI staining for nuclei was applied to detect the effect of specific GSK-3βinhibitor LiCl on Nrf2 nuclear accumulation in EA.hy926 cells.7.Western blot assay was employed to examine the effects of farrerol on the expressions of GSK-3β,p-GSK-3β（Ser9）,p-GSK-3β（Thr390）,Akt and p-Akt in EA.hy926 cells.8.Small interfering RNA（siRNA）transfection was performed to knockdown Nrf2 in EA.hy926 cells.Nrf2 knockdown efficiency at protein level was detected by Western blot.Firstly,the effect of Nrf2 silencing on farrerol-mediated endothelial protective effect against oxidative damage was detected by MTT assay.Then,after being transfected with Nrf2 siRNA,the regulation of farrerol on H2O2-induced cellular reactive oxygen species（ROS）generation was measured using flow cytometry.Finally,Western blot was employed to further examine the impact of Nrf2silencing on farrerol-mediated induction of p-GSK-3β（Ser9）,HO-1 and NQO1 in EA.hy926 cells.9.siRNA transfection was performed to knockdown GSK-3βin EA.hy926 cells.GSK-3βknockdown efficiency at protein level was detected by Western blot.Firstly,the effect of GSK-3βsilencing on farrerol-mediated endothelial protective effect against oxidative damage was detected by MTT assay.Then,after being transfected with GSK-3βsiRNA,the regulation of farrerol on H2O2-induced ROS generation was measured using flow cytometry.Then,Western blot was performed to further examine the impact of GSK-3βsilencing on farrerol-mediated nuclear translocation of Nrf2and induction of HO-1 and NQO1 in EA.hy926 cells.10.ADP-Glo kinase assay and Lineweaver-Burk double reciprocal plots analysis were used to determine the kinetic mode of inhibition of farrerol on GSK-3β.11.Molecular docking,molecular dynamics simulation and MM-PBSA calculations were carried out to further clarify the binding mode between farrerol and GSK-3βat the molecular level.Results:1.3 possible binding proteins were obtained by LC-MS/MS mass spectrometry analysis.GSK-3βwas identified as specific affinity protein of farrerol in EA.hy926cells and chosen for further research.2.The results from DARTS assay showed that farrerol could concentration-dependently inhibit GSK-3βdegradation induced by pronase,when proteolysis was performed at the ratio of 1 mg pronase to 100 mg lysate and 1 mg pronase to 200 mg of lysate,respectively.Moreover,the results from CETSA assay showed that farrerol could protect GSK-3βfrom temperature dependent degradation and concentration-dependently inhibit GSK-3βdegradation induced by heating.3.The results from BLI assay showed that farrerol could bind to GSK-3βin a concentration-dependent manner,with an equilibrium dissociation constant（KD）value of 6.338×10-4 mol/L.4.The results from the optimized Kinase-Glo Luminescent Kinase assay showed that farrerol inhibited the kinase activity of GSK-3βin a dose-dependent manner.The concentration of farrerol that leading to a loss of 50%enzyme activity（IC50）was determined to be 27.14±1.08μmol/L.5.Mechanistic studies proved that farrerol could promote the nuclear translocation and mRNA expression of Nrf2 in a dose-dependent manner and increased the mRNA and protein expression of its downstream target genes HO-1 and NQO1 in a dose-and time-dependent manner in EA.hy926 cells.6.The results from MTT assay demonstrated that the cytoprotective effects of farrerol were enhanced by the addition of LiCl.In addition,immunofluorescence analysis revealed that enhanced nuclear Nrf2 protein levels were observed in the cells treated with farrerol and LiCl in combination compared with individual effects of each agent alone.7.Western blot analysis showed that farrerol dose-and time-dependently enhanced the phosphorylation level of GSK-3βat Ser9 without affecting the protein expression level of GSK-3βand the phosphorylation level of GSK-3βat Thr390.Further mechanistic studies suggested that treatment with farrerol dose-dependently increased the phosphorylation level of Akt without significantly altering the levels of total Akt.8.The results from MTT assay revealed that the cytoprotective effect of farrerol were significantly reduced by siRNA-induced knockdown of Nrf2.Further flow cytometry analysis showed that Nrf2 knockdown increased H2O2-induced cellular ROS generation and attenuated farrerol-induced suppression of cellular ROS generation in H2O2 treated EA.hy926 cells.Western blot analysis showed that Nrf2silencing significantly suppressed farrerol-induced upregulation of HO-1 and NQO1and did not affect the protein expression level of GSK-3βand the phosphorylation levels of GSK-3βat Ser9 and Thr390.9.The results from MTT assay revealed that the cytoprotective effect of farrerol were significantly enhanced by siRNA-induced knockdown of GSK-3β.Further flow cytometry analysis showed that GSK-3βknockdown decreased H2O2-induced cellular ROS generation and suppressed the cellular ROS generation in H2O2 treated EA.hy926 cells.Western blot analysis showed that GSK-3βsilencing significantly promoted Nrf2 nuclear translocation and upregulated HO-1 and NQO1 protein expression.Moreover,GSK-3βsilencing also facilitated farrerol-induced nuclear accumulation of Nrf2 and upregulation of HO-1 protein expression.10.The results from the ADP-Glo Kinase assay showed that farrerol inhibited the kinase activity of GSK-3βin an ATP-competitive manner.11.Molecular docking simulation suggested that farrerol could bind to the ATP pocket of GSK-3βand formed hydrogen bonds with Ile62,Glu97,Val135 and Asp200and pi-cation interaction with Lys85 of the active site.Molecular dynamics simulation was performed to assess whether the molecular docking simulation results are reasonable and reliable and demonstrated that farrerol formed stable hydrogen bonds with Ile62,Val135 and Asp200 and weak pi-cation interaction with Lys85.MM-PBSA calculations showed that farrerol-GSK-3βcomplex possessed a negative binding free energy of-65.236±10.563 kJ/mol.In terms of components making up binding energy,van der Waals,electrostatic,and SASA energy contributed to the binding interaction.Conclusion:1.Farrerol could specifically bind to GSK-3βand inhibit the kinase activity of GSK-3βin an ATP competitive manner.2.Farrerol could promote the nuclear translocation of Nrf2 as well as the mRNA and protein expression of its downstream target genes HO-1 and NQO1 in EA.hy926cells.Furthermore,the cytoprotective effect of farrerol were significantly reduced by siRNA-induced knockdown of Nrf2.All these results indicated that farrerol protected EA.hy926 cells against oxidative stress-induced injuries through Nrf2-ARE signaling pathway.3.In addition to directly interacting with GSK-3βand inhibiting its activity,farrerol also induced inhibitory phosphorylation of the Nrf2 negative regulator GSK-3βat Ser9 through Akt activation.Besides,LiCl,a GSK-3βinhibitor,increased farrerol-mediated cytoprotective effect and Nrf2 nuclear accumulation.Moreover,siRNA-mediated inhibition of GSK-3βenhanced farrerol-induced activation of Nrf2-ARE signaling pathway and the cytoprotective effect of farrerol.All these results indicated that the inhibition of GSK-3βactivity was involved in farrerol-mediated endothelial cytoprotective effects and upregulation of Nrf2-ARE signaling pathway.4.Farrerol could form a stable binding with GSK-3βand the intermolecular binding interaction was a spontaneous process. |
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