Study Of Induction Of Phase â…¡ Enzymes By Sulforaphane And Relationship Between Phase â…¡ Enzymes And Nuclear Transcription Factors Nrf1Nrf2and Nrf3

Oxidative stress plays an important part in the pathogenesis of a varietyof diseases. The ability to mount an efficient response against the continuousthreat posed by exogenous and endogenous oxidants is essential for cellularhomeostasis and survival. In the central nervous system, transcription factornuclear factor-erythroid2-related factor2(Nrf2) plays an important role inastrocyte mediated protection of neurons against oxidative stress response,Nrf2promotes transcription of the antioxidant gene in promoter sequencegenes of antioxidant response element(ARE), such genes include thoseencoding the glutamate–cysteine ligase catalytic and modifier subunits(GclcGclm), glutathione transferase (GST), NAD(P)H:quinine oxidoreductase1(Nqo1), haem oxygenase1(Ho1). Nrf2belongs to CNC (cap’n’collar) bZIP(basic-region leucine zipper) family of regulatory proteins that also includesNrf1, Nrf3, and p45NFE2. Nrf1and Nrf2positively regulate ARE-mediatedgene expression, c-Fos and Fra1negatively regulate this process. Sulforaphane(SFN) has been found to have the chemical protective function since1992, itgradually became a research focus at home and broad. SFN haspharmacological effects in anti-tumor, anti-oxidative damage, immuneregulation and antibacterial. SFN induces phase Ⅱ enzyme through splittingthe connection between Nrf2and Keap1, McWalter found that Nqo1, GST1/2,GSTA3and GSTM1/2expression was increased in the stomach, smallintestine, liver in Nrf2knockout mice when fed with SFN for7days, althoughit can not compared with the normal group, but indicated that there might beother mechanisms. Some people think that Nrf3negatively regulatesARE-mediated gene expression with certain physiological functions.Therefore, our group intend to use sulforaphane to interfere Nrf2+/+, Nrf2-/- primary mouse astrocytes models,explore the relationgship between phaseⅡenzymes and Nrf1, Nrf2, Nrf3.Part1Establishment of cell model of astrocytesObjective: Study the molecular mechanisms of oxidative stress pathway,culture primary astrocytes in vitro.Methods:1Culture of primary astrocytesThe cerebral cortex of Nrf2+/+Nrf2-/-newborn mice was disinfected,decapitated, removed and put in D-Hank’s solution, meninges were strippedunder a dissecting microscope. Tissue was cut into pieces, digested by trypsin,centrifuged after termination. Discarded the Supernatant, and resuspended thecell suspension to the bottom of25cm~2glass cultrure bottles, put the bottles at37℃，5%CO~2culture incubator. The medium was changed after24-36hours,then changed the medium every4-5days, cell growth was observed byfluorescence microscope.2Purification and identification of primary astrocytes.More than90%of integration astrocytes was placed in incubation, shakedat200rpm37℃for3hours, the medium was changed and put back to cellincubator. The cells was digested by trypsin, seeded in24-well culture plate,when cells confluenced, they were fixed by paraformaldehyde, punched andclosed by horse serum. GFAP mouse monoclonal antibody was added to cellsand shaked overnight at4℃.The next day fluorescent secondary antibodyanti-mouse CY5and hochest were added in darkness, removed the small glassslides, anti-fluorescent decay agent was added, observed by laser scaningconfocal microscopy and took photographs.Results:Cell growth and identification: cerebral cortex cells wereadherented to the bottom of bottle after cultured for4-6hours, the culturemedium was changed after24-48hours. Then the cells grew large, axonsbecame longer, a small number of cells were merged into sheets. Cells were confluenced by95%after cultured for12-14days, they were separated into2layers, the bottom layer was lager astrocytes confluenced into sheets, thecytoplasm was abundant, partial nuclear position, more and longer axons, thecells was crossed into nets with each other, the boundaries between cells werenot obvious. The upper layer was spot light smaller microglia, the cytoplasmwas inadequate with slender axons, the boundaries between cells were obvious.After purified by shaker, the upper cells were fell off to obtain a single celltype, dyed with astrocytes marker GFAP, more than95%were positive.Conclusion:This study succeed in estabolishing method of primaryastrocyte culture with more than95%purification, it was a favorable cellmodel to study oxidative-stress pathway in vitro.Part2Phase Ⅱenzymes induced by sulforaphane and therelationship between phase Ⅱe nzymes andNrf1, Nrf2, Nrf3.Objective:To observe induction of phase Ⅱe nzymes by sulforaphaneinprimary astrocytes and the relationship between phaseⅡ enzymes andtranscription factor Nrf1, Nrf3.Methods:1Identificaton of Nrf2+/+Nrf2-/-transgenic miceThe DNA of Nrf2+/+Nrf2-/-transgenic mice tails were extracted, targetgene was amplified according to Nrf2+/+Nrf2-/-primers, the amplifiedresults were checked by agarose gel electrophpresis.2Experimental groups and collections of cellsCells were divided into2groups which were Nrf2+/+and Nrf2-/-, eachgroup was devided into solvent control group and intervention group. At first,Nrf2+/+group was treated with different concentrations of sulforaphane,which were DMSO,2.5umol/l,5umol/l,10umol/l,15umol/l,the concentrationwas chosen by measuring levels of phase Ⅱe nzymes. ThenNrf2+/+Nrf2-/-groups were treated by DMSO and optimal concentration of sulforaphane, themedium was discarded after24hours, cells were collected, stored in-80℃ refrigerator.3Measuring phase Ⅱe nzymesexpressions: At first the protein wasextracted by lysis buffer and quantified by BCA assay kit, calculated thevolume that20μg protein was needed, then protein was carried on10%SDS-PAGE electrophoresis, transferred to PVDF membrane, the membranewas closed and added with primary antibody: rabbit anti-Gclc Ab(1:500),rabbit anti-Ho1Ab(1:500), rabbit anti-Gclm Ab(1:200), goat anti-Nqo1（1:3000) or mice anti-Actin Ab(1:500) overnight at4℃. The next day,rinsed the transfers then added anti-rabbit, anti-goat or anti-miceFluorescence-labeled secondary antibodies(1:10000) and incubated at roomtemperature, Odyssey Infrared Imaging System（LI-COR company USA) wasused to test the band, analyzed the results.4Measuring mRNA levels of Nrf1Nrf2and Nrf3: Nrf2+/+Nrf2-/-administration group and control group cells were taken out, RNA wasextracted by EasyPure RNA Kit, RNA concentration was measured, cDNAwas reversed according to reverse transcription system by gradient PCRequipment. Nrf1Nrf3forward and reverse primer, TransStartGreenqPCRSuperMix, cDNA, Passive Reference Dye were added to establish theamplification system, gene content was determined by quantitive PCR.5Statistical analysis: Statistical analysis was used by SPSS13.0software,statistical method was single factor analysis of variance and wilcoxon, resultswere presented by mean±standard deviztion(x±s), statistical results wereremarkable different when P<0.05(α=0.05).Results:1PCR products of DNA in Nrf2transgenic mice were shown: PCRproducts of Nrf2+/+mice were located at700bp bands, PCR products ofNrf2-/-mice were located at400bp bands.2According to the expression of phase Ⅱe nzymesin Nrf2+/+primaryastrocytes induced by sulforaphane, protein levels of phase Ⅱ enzymes Ho1,Nqo1and Gclm highly expressed in10μmol/l sulforaphane, therefore10μmol/l was chosen as optimal drug concentration. 3Basal expression of Gss was obvious different in Nrf2+/+and Nrf2-/-group, the expression of Ho1, Nqo1, Gclc, Gclm were not different. Theexpression of Ho1was increased in Nrf2+/+and Nrf2-/-cells, and differentafter administration in Nrf2+/+and Nrf2-/-cells. Nqo1and Gclm wereobviously increased in Nrf2+/+cells, not increased in Nrf2-/-cells, expressionof Nqo1and Gclm were different after administration in Nrf2+/+and Nrf2-/-cells. For Gss and Gclc, they were not different after administration inNrf2+/+and Nrf2-/-cells.However, the expression of Gss in Nrf2+/+wasmore than Nrf2-/-, Gclc was not obviously different.4The amplication and dissociation curve of Nrf1, Nrf3, GAPDH inqPCR were good. In Nrf2+/+cells, Nrf1mRNA and Nrf3mRNA expressionwere both decreased, which were statistical different from untreated group. InNrf2-/-cells, declination of Nrf1and Nrf3were not different after treated. InNrf2-/-group Nrf3mRNA expression was higher than Nrf2+/+cells aftertreated, for Nrf1mRNA it was not different between these two group.Conclution:1Primary asrocytes culture was a good model for studing oxidative stresspathway in vitro.2Induction of Ho1was not completely dependent on Nrf2transcriptionfactor by10umol/l sulforaphane, induction of Nqo1and Gclm were largelydependent on Nrf2transcription factor by sulforaphane however Gss and Gclcdid not change when treated with sulforaphane.3In primary astrocytes, when Nrf2existed to up-regulate phaseⅡe nzymesby Nrf2-ARE pathway inducer sulforaphane, Nrf1and Nrf3mRNA expression were inhibited. Nrf3was a compensatory to up-regulatephase Ⅱenzymes in primary astrocytes knockout Nrf2when treated withphase Ⅱe nzymes inducer, but Nrf1did not have sucheffect.