Molecular Mechanism Of Quercetin And Catechin In Protecting Cell Against Oxidative Stress By Targeting FOXO3 To Synergistically Inhibit CHUK Transcription

Quercetin and catechin are two of the most abundant flavonoids in human diet and have a variety of biological functions beneficial to human health.Our previous study has found that quercetin and catechin could protect cells from oxidative stress through synergically inhibiting the transcription expression of the conserved helix-loop-helix ubiquitous kinase（CHUK）.However,their synergistic mechanism remains unclear.In this study,rat livers and HHL-5 cells were chosen to further investigate the synergistic anti-oxidative stress effect and molecular mechanism of quercetin and catechin in other hepatocytes.The specific promoter sequence of CHUK was screened by double luciferase reporter gene assay.The transcription factors（TFs）of CHUK gene were identified by yeast single hybridization technique combined with luciferase reporting assay and bioinformatics.The binding of FOXO3 to the CHUK promoter was clarified by double luciferase reporter gene assay,EMSA and Ch IP-PCR.Quercetin and catechin was found to synergistically inhibit the binding between FOXO3 and CHUK promoter.Molecular docking simulation and cellular thermal shift assay（CETSA）were used to identify the direct binding of quercetin and catechin to FOXO3 protein as well as their interaction patterns.Then the protein expression with/without the site-directed mutation,the characterization methods,such as UV-Vis absorption spectroscopy,fluorescence spectroscopy and circular dichroism were performed to study the binding sites of quercetin/catechin to FOXO3,the action properties and synergistic regulatory mechanism,elucidating ultimately the molecular mechanism of quercetin and catechin synergistically protecting cells against oxidative stress by inhibiting the CHUK transcription.There results contributes to reveal the synergistically antioxidant mechanism of quercetin and catechin,and to provide a theoretical reference for identifying the biological functions of dietary antioxidant components.The main results are summarized as follows:（1）Quercetin and catechin had the synergistic effects in protecting rat livers,Hep G2 and HHL-5 cells against ethyl alcohol or H₂O₂-induced oxidative stress injury,which was regulated by synergistically activiting Keap1/Nrf2 and inhibiting CHUK/p53/Bax signaling pathway.CHUK was the critical target in the synergistic anti-oxidation of quercetin and catechin.Quercetin and catechin synergistically inhibited CHUK gene transcription to enhance the anti-oxidative stress effects.（2）The screening and validation of CHUK transcription factor FOXO3.Different truncated CHUK promoter sequences（-2000～-1）were constructed,and the specific CHUK promoter sequence（CHUK Pro-500～-1）was screened by double luciferase reporter assay.A human c DNA library and a bait yeast vector were constructed.Eight potential transcription factors（ETV4,FOXO3,Elk1,TBP,RXRA,YY1,AP1,SP1）of CHUK were screened out by yeast single hybridization screen.The prey vectors p GADT7-TFs were constructed for yeast single hybridization rotation verification experiment.The transcription factor overexpression vectors pc DNA3.1-TFs were constructed and used to transfect cells for double luciferase verification experiment.Results combined with two verification experiments showed that transcription factor FOXO3 might be the transcription factor of CHUK.The direct binding of FOXO3 to CHUK promoter and their binding sites（-TTGTTTCC,-124～-117）were confirmed by double luciferase reporter gene assay,EMSA and Ch IP-PCR.（3）Quercetin and catechin synergically inhibited the binding of transcription factor FOXO3 to CHUK promoter by simultaneously interacting with FOXO3.The treatment of quercetin and catechin inhibited the binding between FOXO3 and CHUK promoter respectively via double luciferin reporter assay and Ch IP-PCR assay.Compared to the quercetin alone and catechin alone treatment,the half-dose combination of quercetin and catechin showed a stronger inhibitory effect.Quercetin and catechin could directly bind to FOXO3 separately and simultaneously,showing synergistic effect by cellular thermal shift assay.The molecular docking results found that quercetin and catechin inhibited the transcription of CHUK might through binding to different sites in FOXO3 DNA binding domain（DBD）,simultaneously inhibiting the binding of FOXO3 to DNA to synergistically suppress the transcription of CHUK.（4）The interactions and target confirmation of quercetin and catechin with FOXO3.The interactions between quercetin/catechin and FOXO3 protein were identified via UV-Vis absorption spectrum,fluorescence spectrum and circular dichroism with/without site-directed mutation.Quercetin and catechin promoted the complex formation through static quenching.Their interaction reduced the hydrophobicity of the tryptophan microenvironment,significantly reduced the content ofα-helix structure,increasedβ-sheet structure of FOXO3 protein,promoting the stretching of the peptide chain.Notably,this interaction reflected a significantly synergistic effect.The key binding sites of quercetin and catechin to FOXO3 may be Ser218,Asn237,Asp239,Gly244,Arg248 and Leu214,Phe220,Pro247,Arg249,respectively.（5）The molecular mechanism of quercetin and catechin in protecting cell against oxidative stress by targeting FOXO3 to synergistically inhibit CHUK transcription may be as follows:quercetin and catechin simultaneously bound to transcription factor FOXO3 DBD different sites,competing for DNA binding sites of target genes and destroying the stability of protein-DNA complex to restrain the interaction of FOXO3 to CHUK promoter,thus suppressing the CHUK transcription and its downstream gene expression to protect Hep G2 cells from H₂O₂-induced oxidative stress.