Transcriptome Profiling Analysis Of Mice In Different Se Status And The Influence Of (-)-Epigaliocateghin-3-gallate (EGCG) On Antioxidant Defense Systems

Selenium(Se),one of trace elements,exerts various essentially physiological functions in the forms of selenoenzymes or selenoproteins containing selenocysteine(Sec),and lack of this element will reduce the synthesis of selenoenzymes including glutathione peroxidase(GPx)and thioredoxin reductase(TrxR),which can affect redox homeostasis in many organisms.Epigallocatechin-3-gallate(EGCG)is the most abundant and bioactive catechin in green tea.EGCG may function either as an antioxidant or as an inducer of antioxidant defense via its pro-oxidant action or other underlying mechanisms under different dose and biological conditions,where it is well-known that it is powerful to scavenge reactive oxygen species(ROS)and reactive nitrogen species(RNS).In addition,EGCG can also regulate antioxidants and antioxidant enzymes levels to maintain cellular redox balance,such as increases in superoxide dismutase(SOD),catalase(CAT),glutathione reductase(GR)and peroxiredoxin 6(Prx6).However,the research in indirect antioxidant activity of EGCG is still at the exploratory stage.Se participates in the antioxidant defense mainly through a class of selenoproteins including TrxR and GPx,and cellular Se status plays an important role in antioxidant defense.However,the interactions of EGCG with antioxidant defense systems by manipulating the cellular Se levels have not been reported.The thioredoxin(Trx)system consisting of TrxR and Trx,and the GSH system consisting of GSH and GR coupled with glutaredoxin(Grx),are the two well-documented NADPH-dependent disulfide reduction systems that are essential and the first line of defense to maintain cellular redox homeostasis against oxidative damage.In response to oxidative stress,Nuclear factor E2-related factor 2(Nrf2)mediates antioxidant response element(ARE)-mediated signaling pathway,which is responsible for induction of some antioxidant and phase 2 enzymes including heme oxygenase 1(HO1)and NAD(P)H:quinone oxidoreductase 1(NQO1)as the second defense line.In this study,some differently expressed genes were screened based on large amount of transcriptome sequence information obtained from different Se status mice by high-throughput RNA-seq technique.The key genes and the biological pathways related to Se deficiency were explored using Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)databases.The aim of this study is to investigate the role of Se starvation in biological metabolisms and the underlying molecular mechanisms involved in these specific biological processes.Subsequently,we investigated the interactions among these antioxidant defense systems and potential regulation mechanism after administration of EGCG to mice using Se-free nutrition as a perturbator.The results and conclusions were summarized as below:1.Levels of serum and hepatic GPx activity as well as serum TrxR activity were significantly decreased in the mice fed with Se-deficient diet,indicating that the Se deficiency model had been successfully established.2.Transcriptome analysis showed that there were 85 differentially expressed genes identified in Se-optimal and Se-deficient mice,including 47 up-regulated genes and 38 down-regulated ones.Cluster analysis showed that starvation of Se resulted in significant increases in some selenoproteins mRNA levels,but upregulated the redox-,antioxidant defense-and p53-related gene expression.Based on the analysis of differentially expressed or enriched genes by KEGG database,the most enriched pathways induced by the Se starvation include glutathione(GSH)metabolism,cytochrome P450 drug metabolic pathway and extracellular signal-regulated signal kinase(MAPK)signaling pathway.Overall,deficiency of Se may alter the antioxidant genes expression in mice.3.In Se-optimal mice administrated intraperitoneally once daily with EGCG(45 mg/kg)for 7 days,the mRNA and protein levels of hepatic TrxR1,enzymatic activity of TrxR as well as hepatic GR and Grx activities were significantly increased as compared with non-treatment group.Interestingly,melatonin(50 mg/kg;i.p.),one highly effective antioxidant,did not influence the effects of EGCG on either hepatic TrxR1 protein or TrxR and Grx activities in Se-optimal mice treated with EGCG plus melatonin once daily for 5 days.These results suggest that EGCG can activate hepatic Trx and GSH systems in Se-optimal mice,and activation of these key antioxidant enzymes by EGCG probably involves only mild rather than overt pro-oxidant action of EGCG.4.Treatment with same dose of EGCG(45 mg/kg;i.p.)for 7 days had no effects on activities of hepatic TrxR,GR or Grx in Se-deficient mice as compared with the non-treatment group.However,western blot analysis showed that EGCG significantly increased hepatic Nrf2 protein levels in the nucleus in Se-deficient mice.Also,enzymatic activity assay and qPCR analysis showed that EGCG caused significant increases in both mRNA and protein levels of HO1 and NQO1,two critical downstream effectors of Nrf2,as well as a significant increase of Trx activity in Se-deficient mice compared to other counterpart groups,indicating that EGCG is involved in the activation of Nrf2 pathway in Se-starved conditions,and this response seems to be associated with activation of p38 mitogen-activated protein kinase by western blot analysis.5.EGCG-evoked hepatotoxicity is normally associated with hepatic Nrf2 activation in vivo,whereas our data showed that EGCG at a non-toxic dose(45 mg/kg,i.p.)for 7 days neither affected body weight nor caused hepatotoxicity as indicated by the normal serum levels of alanine aminotransferase(ALT),aspartate aminotransferase(AST)and 4-hydroxynonenal(4-HNE),as well as hepatic phosphorylated histone 2A vatiant(?-H2AX)protein in the Se-optimal and Se-deficient mice.In addition,administration of EGCG(70 mg/kg,i.p.)to Se-optimal and Se-deficient mice,once daily for 3 days,resulted in the same(50%)mortality.Together,these findings suggest that Se deficiency does not enhance the toxicity of EGCG in vivo.In conclusion,EGCG could activate Trx and GSH systems but not Nrf2 pathway in the Se-optimal mouse livers,whereas hepatic Nrf2 instead of Trx or GSH pathway would be induced in the Se-deficient mice.Therefore,the Trx and GSH systems serve as the prior defense line than Nrf2 signal by EGCG in the Se-optimal mice,whereas in Se starvation conditions,EGCG is a potent inducer of the Nrf2 pathway.Taken together,the present study revealed that EGCG could exert indirect antioxidant roles through activation of different antioxidant defense pathways to maintain cellular redox homeostasis under Se-optimal and/or Se-deficient conditions.