Study On Mechanisms And Metabonomics Of Interactions Between AhR Exogenous Ligand And Endogenous Ligand

Aryl hydrocarbon receptor(AhR)is a ligand-activated transcription factor,which mediates the transcription of drugs or toxic metabolic enzymes and plays an essential role in cell proliferation,regulation,tumorigenesis,homeostasis and immune response.Its ligands mainly come from environmental pollutants,endogenous metabolites,plant derived flavonoids,drugs and so on.Ligands from different sources cause different physiological reactions through the AhR pathway.Most of exogenous ligands have constitutive activation ability and often induce some diseases,while most of endogenous ligands are nontoxic and eliminated by metabolism.To reveal the underlying mechanisms of the interaction of AhR endogenous and exogenous ligands,our study has been conducted and the results were as follows:Firstly,the luciferase reporter system Hepa1-6-XRE-Luc was constructed and the interactions of various exogenous ligands and endogenous ligands were investigated.The activation effect of FICZ(10 nM)on the AhR pathway reached to the peak at 4 h,which was weaker than B[a]P(1 ?M).Daidzein(DAI)and apigenin(API)can not directly activate the AhR pathway,while both of them significantly improved the activation abilities and prolonged activation time of FICZ(P<0.01),it is same to resveratrol(RES),Curcumin(CUR)and quercetin(QUE).And as an AhR antagonist,synthetic compound 3,4-DMF significantly inhibited the AhR activation induced by FICZ.According to the MTT assays,proliferation rate of cells treated by FICZ significantly reduced by FICZ(P<0.01).DAI,API,QUE,RES and 3,4-DMF have the similar effects,while CUR has no effect on the regulation of FICZ to cell viability.Therefore,different exogenous compounds have different effects on the biological functions of endogenous compounds.Secondly,we investigated the effect on cell migration and genes levels.Hepa1-6 cells were exposed to FICZ(10 nM),B[a]P(1 ?M)and FICZ(10 nM)+B[a]P(1 ?M)for 24 h.As the wound assay shown,though FICZ made no effect on cell migration,it inhibited the promotion of B[a]P.Both of the two compounds stimulated the up-regulation expression of cyplal gene;FICZ reduced the down-regulation expression of bcl2 induced by B[a]P;exposure of B[a]P up-regulated p53 gene,and FICZ eliminated the up-regulation.And we investigated the effects of B[a]P to FICZ on extracellular levels of FICZ,as the data of LC-MS shown,most of the addition of FICZ was degraded in less than 12 h(degradation rate 97.5%),and B[a]P significantly prevented the degradation of FICZ(P<0.01).Accordingly,the two compounds may regulate the related physiological processes through the competition of binding to AhR protein.Thirdly,the regulation effects and mechanism under the interaction between the two compounds in apoptosis were explored.According to the TUNNEL assay and flow cytometry results,FICZ inhibited the apoptosis and efficiently prevented apoptosis induced by B[a]P.Furtherly,after the co-treatment of these two compounds,mitochondrial membrane potentials and reduced glutathione levels were significantly declined,and reactive oxygen levels were increased and caspase-3 was activated.All of them proved that FICZ inhibited apoptosis B[a]P induced in a mitochondrial dependent manner.Finally,combined with non-targeted metabolomics,the regulatory mechanisms of physiologically process induced by these two compounds were explored.There are 26 kinds metabolic differentials in the positive ion mode and 49 kinds in the negative ion mode in the cells treated by B[a]P,and 40 different metabolites were screened out between the FICZ group and the FICZ+B[a]P group under negative ion mode.The interactions between FICZ and B[a]P had a significant impact on the four metabolic pathways:tryptophan metabolism,indole alkaloid biosynthesis,glutathione metabolism and oxidative phosphorylation.In conclusion,the AhR endogenous ligand FICZ competitively bind to AHR protein against B[a]P,which interferes with the occurrence of apoptosis in a mitochondrial dependent manner through affecting tryptophan metabolism,oxidative phosphorylation process,glutathione metabolism and so on.