MicroRNAs-mediated Molecular Mechanisms Underlying The Synergistic Antioxidant Action Of Quercetin And Catechin

Oxidative stress reflects an abnormal physiological state of redox imbalance which is directly or indirectly associated with human health.Flavonoids have been found to play significant roles in cytoprotective action against oxidative stress-mediated damage,which have been attributed largely to their direct antioxidant activities.The diversity of flavonoids ingested depends on naturally widespread flavonoids and multifarious human dietary structure,and the concept of food synergy believes that the individual food components are coordinated to exert health benefits.Therefore,exploring the synergistic mechanism between flavonoids indicates stronger physiological effects and lower effective concentrations than single components,and it can also reveal the actual situation of these substances in physiological functions in vivo.In this study,the model of oxidative stress was established using HepG2cells injured by H₂O₂,focusing herein on the synergism of quercetin and catechin which are widely distributed and rich in flavonoids.Synergistic antioxidant actions between quercetin and catechin were detected in the cellular biological behavior experiments.In order to analyze its internal molecular mechanism,we initially used high-throughput sequencing of whole transcriptome platform combined with bioinformatics analysis to make preliminary predictions of potential molecular targets（including miRNA and mRNA）for the synergistic antioxidant effect of two active ingredients,and furthered to optimize the related targets by gene function,pathway enrichment and differentially expressed miRNA-mRNA interaction analysis.Subsequently,we selected the molecular targets and signaling pathways with significant difference and higher research value for further verification to clarify the molecular mechanism of synergy between quercetin and catechin from the levels of miRNA,gene,and signaling pathway.The main results are as follows:（1）The oxidative stress injury model of HepG2 cells induced by H₂O₂（500μM）was constructed,and the synergistic protective effect of quercetin and catechin was explored from the level of cellular biological behavior.The results showed that the combined use of quercetin and catechin（12.5μM+12.5μM）could significantly reverse the decrease in cell survival,excessive accumulation of reactive oxygen species（ROS）,and the increase of the lipid peroxidation malondialdehyde（MDA）content in cells caused by H₂O₂ oxidative damage,and these effects were significantly stronger than that of quercetin（25μM）or catechin（25μM）alone,indicating that the combination of quercetin and catechin could synergistically enhance the defensive abilities against oxidative stress of HepG2 cells damaged by H₂O₂.（2）The whole transcriptome analysis was conducted based on the illumina high-throughput sequencing platform,and the expression difference between different treatment groups were analyzed by bioinformatics:control group（Control）,hydrogen peroxide treatment group（H₂O₂）,quercetin 25μM treatment group（Quer）,catechin 25μM treatment group（Cat）,quercetin and catechin combined 12.5μM+12.5μM treatment group（QC）.The differences of miRNA and mRNA between Quer,Cat,and QC were emphasized to reveal the multiple-target interactions and multiple-pathway regulation of the antioxidant synergism between quercetin and catechin.The GO function annotation and KEGG pathway enrichment analysis in the comparison group of Quer vs Cat vs QC showed that differentially expressed genes and differentially expressed miRNAs were mainly involved in the regulation of genetic material,signaling pathways,and cell life processes,and were concentrated in oxidative damage-related disease pathways such as cancer,inflammation and cardiovascular diseases,cell self-repair and regulation processes such as cell autophagy,apoptosis,and the signal transduction pathways mostly related to inflammation,cell redox homeostasis regulation,cell proliferation,differentiation,apoptosis and tumor generation.According to differentially expressed miRNA,gene cluster analysis,and miRNA-mRNA interaction network analysis in the comparison group,we finally selected BACH1 and let-7 family as potential targets of synergistic effect due to their significantly differential expression among Quer vs Cat vs QC groups and great participation in the miRNA-mRNA interaction network.（3）BACH1 expression characteristics were verified in the co-presence of quercetin and catechin and its function in synergistic antioxidant effect was analyzed.It was revealed that quercetin and catechin could synergistically down-regulate BACH1 mRNA and protein expression levels,which was consistent with the expression characteristics and regulatory pattern presented by high-throughput sequencing results.The results showed that the combined use of quercetin and catechin could promote the expression of Nrf2 and nuclear translocation,thereby initiating high expression of phase II detoxifying enzymes HO-1 and NQO1.From the investigation of biological function and specific mechanism of BACH1,we noticed that suppression of BACH1 not only led to significant increase of cell viability and markedly decreased ROS level,but also countermanded the synergies between quercetin and catechin.These findings confirmed that BACH1 served as an indispensable target gene of synergism of quercetin and catechin,and acted as a negative regulator of the Nrf2 signaling pathway which was activated under stimulation of the co-existing components.（4）Screening,validation and functional analysis of miRNAs targeting BACH1.Based on the results of bioinformatics prediction,overexpression/inhibition miRNA transfection and dual luciferase assays,it seemed that BACH1 was a target gene of miR-25-3p and let-7a-5p.Further verification demonstrated that the transcription and translation of BACH1 was suppressed to a greater degree in HepG2 cells（in which the two miRNAs were overexpressed together）compared with only one miRNA.This result indicated the presence of synergistic effects between miR-25-3p and let-7a-5p on targeting BACH1.Simultaneously inhibiting the expression of let-7a-5p and miR-25-3p significantly reduced the cell survival rate and induced the accumulation of ROS in the cells.The co-inhibition of let-7a-5p and miR-25-3p did not influence significantly the synergistic action of quercetin and catechin on cell viability and ROS level,even though the two miRNAs could partially weaken the synergistic effects of quercetin and catechin.Accordingly,we speculated that other miRNAs are involved in regulating BACH1 expression level.Moreover,up-regulation of BACH1 could significantly decrease cell viability and enhance ROS accumulation,and these cellular physiological alternations were largely abolished when let-7a-5p and miR-25-3p mimics were simultaneously transfected,thus confirming that the two miRNAs played a positive regulatory role in cellular antioxidant function via directly regulating the expression and function of BACH1.（5）Based on the results of high-throughput sequencing,we selected let-7 family as potential targets for synergistic mechanism and investigated its downstream targeting genes and related signaling pathways.Bioinformatics analysis was performed and the obtained results showed that CHUK contained predicted target sites for let-7a-5p.Combined with the previous research from our laboratory,we further inhibited CHUK by RNAi and it was found that suppression of CHUK exhibited promoting effect on both cell survival and ROS scavenging ability,as well as countermanding the synergies between quercetin and catechin.Subsequent the results of overexpression/inhibition miRNA and dual luciferase assays confirmed that CHUK is a target gene for let-7a-5p.（6）We predicted the potential target miRNAs of the key regulatory factors in the two verified pathways for synergism（Keap1-Nrf2 and NF-κB signaling）using bioinformatics and screened out the possible connection between them by intersection analysis,in order to explore the intermodulation of the two signaling pathways from the post-transcriptional level and constructing a miRNA-mediated regulatory network of quercetin and catechin protecting cells from oxidative stress.The obtained results indicated that besides let-7a-5p,miR-155-5p,miR-23-3p,miR-9-5p,miR-125-5p,miR-128-1-5p,miR-15-5p/16-5p/195-5p/424-5p/497-5p could simultaneously participate in the regulation of the main signal genes of the two pathways,and Maf,MafK,BACH1,IKKβand CHUK contained two or more common miRNA targets with the other pathway.These miRNAs and genes were likely to be key nodes in the interaction network of Keap1-Nrf2 and NF-κB signaling pathways.They interacted and participated in the construction of regulatory networks for quercetin and catechin protecting cells against oxidative stress and played important roles in maintaining cell redox homeostasis.