Protective Effects And Mechanisms Of Hyperoside On Hepatic Cell L02against Oxidative Stress Damage

Objective：Oxidative injury is commonly induced by drugs, chemicals, heavy metals andionizing radiation and other harmful conditions. Free radicals, such as reactive oxygenspecies (ROS), reactive nitrogen radicals (RNS) could disturb the balance between freeradicals and anti-oxidant system. These pathological processes result in damage ofintracellular proteins, lipids, nucleic acids and other biological macromolecules, as wellas the damage on structures and functions of multi-organs. Nrf2/Keap1-ARE antioxidantsystem signaling pathway is currently considered as one of the most importantmechanisms of cellular resistance to oxidative injury.NF-E2-related factor-2(Nrf2), a member of the CNC family, is a gene transcriptionfactor which sensitive to intracellular oxidative stress response. Nrf2can induce a varietyof antioxidant protein synthesis. In the physiological condition, Nrf2and the Kelch likeECH associated protein-1(Keap1) are combined to form a complex and remain latentwith low transcriptional activity through the transfer of degradation by the26Sproteasome. During the oxidative injury, the cells become oxidized. During the oxidativeinjury, the cells become oxidized, Keap1mercapto group within the molecule is oxidizedto disulfide. Nrf2is released from the complex and transferred into the nucleus todimerize with the small Maf proteins (including MafG, MafK and MafF) and bind to theantioxidant response element (ARE) to regulate the expression of the genes of phase IIdetoxification enzymes. Antioxidant enzymes increased intracellular antioxidant activity,reducing oxidative stress damage to cells.Exogenous BTB-CNC homolog1(Bach1) and Nrf2belong to one of the members ofthe CNC transcription factor family, and is a heme binding protein. Bach1and Nrf2arefunctional different. During oxidative stress, Bach1can be combined with the small Mafproteins to generate heterologous dimers and bind to Maf recognition element (MARE).It inhibits the antioxidant gene transcription. In recent years, it has been found that Nrf2 and Bach1can regulate antioxidant gene heme oxygenase (HO-1) expression. These tworoles are mutually antagonistic.We found that natural flavonoids of Hyperoside (Hyp) showed an obvious protectiveeffect in liver via increasing the expression of cellular protection protein. This proteincould strengthen the protection of cells from protease attack. Our studies intend toinvestigate the Hyp-induced expression of HO-1and the oxidation resistance of liver celldamage basing on further clarifying the protective effect of Hyp on hepatic cells, whichaim at competitive Regulation of Nrf2/Keap1-Bach1with HO-1gene promoter region ofARE.Our study will contribute to the indepth exploration of the mechanism of Hyp, andprovide a theoretical basis for the development of antioxidant of drug-induced liverinjury.Methods：(1) Oxidative stress damage model of Hepatic L02Cells, the model was establishedby the treatment of H2O2. Cell viability was detected by MTT method.(2) MTT was employed to assess the cell viability. MDA and ROS assay wereperformed to reflect the cellular redox state and oxidative stress.The time-dependent anddose-dependent relationship of cytoprotection that Hyp to against oxidative stress.(3) After Hyp treatment on H2O2-induced Hepatic L02cells, HO-1mRNA levelswere analyzed by RT-PCR and Real-time RT-PCR. HO-1transcriptional activity wasevaluated by luciferase reporter assay (hHO4.9luc transfected plasmid).(4) Nrf2, Bach1and Keap1expression were analyzed by western blot.(5) Using fluorescent immunocytochemistry analysis of key regulatory factor Nrf2and Bach1expression distributed in the cytoplasm and the nucleus (nuclear translocation)(6) Construction of expression vector and transfected pEGFP-C1-Bach1,Overexpression Bach1in Hepatic L02cells to study the effect on Nrf2and HO-1expression.(7) After Hyp treatment, chromatin immunoprecitation analysis Bach1and Nrf2binding to the ARE of HO-1promoter region.Results：(1) H2O2decreased Hepatic L02cell activity, and the effect is in a dose dependentmanner, when H2O2concentration reached to2.5M,1hour the growth of Hepatic L02 cells can be completely inhibited. IC50of H2O2which inhibited the growth of HepaticL02cells is1hours for0.126M. When hepatic L02cells were pretreated with Hyp(12.5-800μM) for24h, the H2O21H0.1M, compared with the normal control group, theconcentration of Hyp was200,400and800μM, cell viability changes. At the same time,Hyp reduced H2O2induced increase of intracellular ROS and MDA levels significantly.(2) Hepatic L02cells pretreatmented with200μM Hyp for24h, and then inducedby H2O2for1h. Compared with the normal control group, Hyp could induce Nrf2mRNAlevels and protein levels rised, and the effect is in a dose-dependent and time-dependentmanner. The effects of Hyp on Keap1and Bach1level are not obvious. Hyp alsoincreased Nrf2and Bach1in the nuclear accumulation, but the increased effect of Nrf2ismore apparently.(3) Dual luciferase report gene showed that, compared with the control group, Hypsignificantly enhance the transcription activity of HO-1gene promoter, is about2.7timeshigher than that of control group.(4) Transfection of pGFP-C1-Bach1expression vector, overexpression of Bach1,Nrf2and HO-1expression decreased significantly.(5) CHIP analysis proved that, both Bach1and Nrf2are competitive with AREprocess, Hyp enhances the levels of Nrf2and ARE combination, but has no obviouseffect on Bach1.Conclusions：1．Hyp has a protective effect against H2O2-induced oxidative damage of HepaticL02Cell.2．Hyp promotes HO-1gene promoter transcriptional activity and gene expression.3．Indirect change nuclear Nrf2and Bach1ratio, and the combination of the two onthe antioxidant response element (ARE) ratio, may be the main mechanism of Hyppromoting Nrf2competition ability.