Mechanisms Of Activation Of Antioxidant Genes In Soy Isoflavone-mediated Attenuaton In Oxidative Stress-induced Cell Injury In EA.hy926Cells

Atherosclerosis (AS) is the leading cause worldwide presently, and the incidence ofAS is arising year by year. Epidemiological evidence shows that the prevalence ofcardiovascular diseases (CVD) is usually higher than that for men the same age inpremenopausal women, however an significant increase in CVD prevalence in menopausalwomen than men for a sharp decline in estrogen levels in those menopausal women. A largenumber of studies have shown that Hormone replacement therapy (HRT) can reduce theincidence of CVD in postmenopausal women, but it will significantly increase the risk ofstroke and malignant tumors of this people. For seeking of alternative therapies for HRT,many studies focus on carrying out with the selective estrogen receptor modulators(SERMs), such as synthetic drugs Raloxifene and anti-estrogen Tamoxifen. One calledRUTH (Raloxifene Use for the Heart), a large-scale clinical trails on SERMs for thelong-term prevention and security of coronary heart disease as well as the risk of breastcancer were assessed. This5.6years-lasting period follow-intervention experiment showsthat Raloxifene has no protective effect on the cardiovascular system of postmenopausalwomen, but further warning of its potential risk of thrombosis and stroke. Therefore,looking for a natural non-toxic side effect of SERMs attracts interests of many researchers.Epidemiological survey found that the incidence rate of CVD in Asian whose diet rich insoy or soy products was significantly lower than the Westerner those whose eating habitswithout soy or soy products. Soy isoflavones (SI) is mainly present in the legumes as aphytoestrogen, SI has estrogenic activity with bidirectional regulation of estrogen receptor(ER), it is a kind of natural SERMs. In recent years, a large number of studies show thatmany kinds of biological avtivity including prevention and treatment of CVD, menopausalsyndrome, osteoporosis, breast and prostate cancer and other diseases, attribute to itsantioxidant effect, SI is an excellent natural anti oxidants. Therefore, the research and development of a safe and effective natural SERMs or antioxidants become an effectiveway for early prevention and treatment of AS. Nutrient composition as a naturally occurringplant estrogen than the drugs, with better security, makes it more likely to be accepted forpatients. Therefore, the study on the antioxidant mechanisms of the SI as an natural nutrientin prevention and treatment for AS is of great significance.Part Ⅰ Mechanisms of attenuation of genistein on oxidativestress-induced EA.hy926cell injuryGenistein,as an isoflavone, is an important phytoestrogen mainly present in legumes.For its molecular structure is similar to estradiol, genistein could competitively binding toestrogen receptors and has weak estrogenic activity. Due to its molecular structure containsthree phenolic hydroxyl groups, genistein also has some antioxidant effects. Over the pastdecade, genistein and daidzein has been the most studied isoflavones compounds. A largenumber of in vivo and in vitro experiments show that the SI antioxidant activity andestrogen receptor binding activity mediated its wide range of biological effects, includingprevention AS, women's menopausal symptoms, osteoporosis, prostate cancer and breastcancer. Therefore, especially in the research field of nutrition, people still interested in SI'shealthy effect.Nrf2(NF-E2-related factor2) is a key transcription factor that regulate theintracellular metabolism of endogenous and exogenous substances on the oxidative stressresponse, it binds to antioxidant response element (ARE), up-regulates ARE-related anti-oxidation genes, including endogenous antioxidants, phase II detoxification enzymes andtranscripts encoding intracellular gene to determine cell survival or death. The sustainedexpression of Nrf2-regulated protein in cell plays a crucial role in restoring homeostasis incells. it has confirmed that many natural or synthetic small molecule compounds canactivate Nrf2/ARE signaling pathway to protect the cells from the toxic or carcinogenicmaterial-induced cell damage. Therefore, the use of natural compounds or syntheticcompounds that activate of Nrf2in response to exogenous agents, so as to resist the stressin various pathological conditions, becomes an important strategy in a variety of chronicdiseases or cancer prevention. Present study demonstrated that genistein can inhibit the decrease of cell viability and cell apoptosis induced by oxidative stress, and clarified themolecular mechanisms of Nrf2in the genistein-mediated antioxidant effect.SectionⅠ Effect of genistein on cell toxicity under oxidative stressIn human body, even if in the population of long-term intake of SI, the concentrationof genistein or daidzein in plasma is difficult to achieve μM, and therefore to investigate thebiological effects of physiological concentrations of SI is particularly important and morepractical. To investigate the protective effect of low concentration (physiologicalconcentration, nM) of genistein on oxidative stress induced cell toxicity, human umbilicalvein endothelial cell strains-EA.hy926cell was adopted. An oxidative stress model ofEA.hy926cells was established by hydrogen peroxide (H2O2)or tert-butyl hydrogenperoxide (t-BHP). The results are as follows:1. A dose-dependent decrease in cell viability was observed in response to H2O2ort-BHP, with IC50were650μM and75μM seperatly.2. Genistein (1nM-500nM) could dose-dependently inhibite the decrease in cellbiability induced by oxidative stress, with significant differences in100nM-500nMpretreatement group compared with the control group.3. AnnexinⅤ-FITC flow cytometry and TUNEL assays showed that500nMgenistein can reduce oxidative stress-induced apoptosis. And genistein inhibited oxidativestress-induced upregulation of caspase-3protein expression and decrease of Bcl-2proteinexpression.The above results show that low doses (nM) of genistein can reduce oxidativestress-induced cytotoxicity, including inhibition of cell viability decrease, cell apoptosis,upregulation of caspase-3protein level and inhancement of Bcl-2protein expression.Section II Effect of genistein on endogenous antioxidantsSuperoxide dismutase (SOD) and catalase (CAT) are the most important endogenousantioxidant enzyme in body. Glutathione (GSH) is the most important non-enzymaticantioxidants. The results in this section are as follows:1. Genistein (20nM-500nM) dose-dependently upregulated SOD level, andsignificantly inhibitd oxidative stress-induced reduction in SOD level.2. Genistein (20nM-500nM) dose-dependently upregulated CAT level, andsignificantly inhibitd oxidative stress-induced reduction in CAT level. 3. Genistein (20nM-500nM) dose-dependently upregulated GSH level, andsignificantly inhibitd oxidative stress-induced reduction in GSH level.The results show that the elevation of endogenous antioxidants plays a part ingenistein's antioxidant effect.Section III Detection of genistein as a Nrf2transcription factor agonistDetection of genistein on the impact of the ARE-luciferase reporter gene activity, itcan reflect a direct response to Nrf2by genistein. Meanwhile, Nrf2protein and mRNAlevels and nuclear translocation were also tested. The results are as follows:1. Dual luciferase reporter gene system assay was adopted, the results showed thatgenistein (20nM-500nM) dose-dependently increased the firefly luciferase activity.2. Genistein increased Nrf2mRNA levels, and activated Nrf2protein expression atonly1h, and keep Nrf2protein level within1h-10h, but not in a time-dependent manner.3. Genistein (20nM-500nM) dose-dependently up-regulated Nrf2nuclear proteinexpression level.The above results demonstrated that genistein as a Nrf2activator.Section IV Effect of genistein on PPARγ transcription factorActivation of genistein on PPREx3-luciferase reporter gene directly reflects the effectof genistein on activation of the transcription factor-PPARγ. Effect of genistein on PPARγprotein and mRNA levels and nuclear translocation were also studied. The results are asfollows:1. It has been demonstrated that genistein (20nM-500nM) dose-dependentlyincreased the firefly luciferase activity, but the effect is relatively weak.2. Genistein enhanced PPARγ mRNA level, and time-dependently increased PPARγprotein levels (1h-10h).3. Genistein promoted the expression of PPARγ nuclear protein level.The above results show that genistein activates PPARγ transcription factor.Section V Effect of genistein on heme oxygenase-1Heme oxygenase-1(HO-1) plays an important role in protection of cardiovascularsystem. It has been confirmed that HO-1was regulated by Nrf2and PPARγ. This sectionstudies the impact of genistein on HO-1, the results are as follows: 1. Western blot analysis showed that genistein dose-dependently (20nM-500nM)and time-dependently (1h-10h) up-regulated HO-1protein levels.2. Real-time PCR analysis showed that genistein (1h-10h) promoted HO-1mRNAlevels in a time-dependent manner.3. Genistein (20nM-500nM) dose-dependently enhanced HO-1activity.The above results demonstrated the activation induced by genistein.Section VI Effect of inhibition of Nrf2, PPARγ and HO-1on protective effect bygenistein under oxidative stressCells were pretreated with Nrf2-siRNA, specific antagonist of PPARγ-GW9662andspecific antagonist of HO-1-Znpp, and cell viability induced by genistein against oxidativestress was tested. The results are as follows:1. Cells were transfected with FITC-siRNA, and analyzed by laser scanning confocalmicroscopy, it reflected a higher transfection efficiency by siRNA on cell.2. Western blot analysis showed that Nrf2-siRNA silenced the Nrf2proteinexpression with or without genistein treatment, while Control-siRNA had no effect on Nrf2expression.3. Nrf2-siRNA treatment relieved the inhibition of cell viability induced by genisteinagainst oxidative stress, while Control-siRNA treatment had no effect.4. GW9662and Znpp pretreatment attenuated the cell protection by genistein underoxidative stress.The above results show that Nrf2plays an important role in antioxidant effect bygenistein, and PPARγ and HO-1also to some extent mediated the protection induced bugenistein.Part II Mechanisms of Nrf2in inhibition of oxidativestress-induced cell toxicity by equol in EA.hy926cellEquol, which produced as a final metabolite by human intestinal bacteria with daidzeinas a synthetic substrate specificity. About30%to50%of adults can not produce equol, andthis individual difference continuously existed in "Equol producers, Eq+" and"Non-equol-producers, Eq-". Investigations found that the proportion Eq+in Asians is apparently higher than in Westerners, suggesting that the Eq+compared with the Eq-mayhave a lower risk affecting cardiovascular disease risk. Due to the special molecularstructure, the antioxidant activity of equol, gene transcriptional activity and estrogenreceptor binding capacity are stronger than its predecessor-daidzein. In human body,49.7%of the circulating equol exists in free rather in combined form, the proportion or free form issignificantly higher than that of daidzein (18.7%) or estradiol (4.6%), and equol also has alonger half-life in plasma, these specialties makes equol effectively play itspharmacological effects. Therefore, the excellent antioxidant properties and a wide range ofbiological effects by equol prompt to it being used for the prevention and treatment incardiovascular disease.Section I Detection of equol as a Nrf2transcription factor agonistBased on the established cell-based ARE-luciferase reporter system screening modelsfor seeking of Nrf2small molecule agonists, effect of equol on the ARE-luciferase activitydirectly reflects the activation of Nrf2by genistein. The results are as follows:1. Using the dual luciferase reporter system assay, the results showed that equoldose-dependently (10nM-250nM) and time-dependent (0.5h-2h) enhancedARE-luciferase activity, which demonstrated that equol is a Nrf2activator.Section II Equol activates ARE-dependent antioxidant response by up-regulatingNrf2protein expressionThis section focus on the study of equol on Nrf2and its downstream antioxidant genesquinone oxidoreductase (NQO1) and HO-1protein and mRNA levels. The results are asfollows:2. Western blot analysis showed that equol (10nM-250nM) dose-dependentlyincreased Nrf2, HO-1and NQO1protein levels, and similarly,500nM genistein anddaidzein also had the same effect as equol.3. Cells were transfected with HA-Nrf2expression plasmid, then analyzed byWestern blotting, the results showed that equol (10nM-250nM) dose-dependentlyincreased HA, Nrf2, HO-1and NQO1protein levels.4. Real-time PCR analysis demonstrated that equol had no effect on Nrf2mRNA, butincreased HO-1and NQO1mRNA levels in a dose-dependent (10nM-250nM) manner. The above results suggested that equol activats Nrf2/ARE signaling pathway mainlythrough increasing Nrf2protein level.Section Ⅲ Mechanisms of ER, ERK1/2and PI3K/Akt in activation of Nrf2induced by equol under oxidative stressCells were pretreated with specific antagonist of ER, ERK1/2and PI3K/Akt,ICI182,780, PD98059and LY294002seperately, and following treatment of equol,daidzein and genistein, Nrf2, HO-1and NQO1protein level and Nrf2nuclear translocationwere tested. The results are as follows:1. Western blot analysis showed that no significant effect on Nrf2protein levelsattenuation by pretreatment of ICI182,780, PD98059and LY294002induced byequol,while ICI182,780and LY294002treatment inhibited the up-regulation of HO-1andNQO1by equol, and PD98059pretreatment had no significant effect on these two gene;ICI182,780, PD98059and LY294002treatment inhibited daidzein-induced Nrf2and HO-1protein levels increase, and ICI182,780and LY294002pretreatment attenuated theup-regulation of NQO1induced by daidzein, however PD98059treatment had nosignificant effect on it; PD98059pretreatment genistein did not affect Nrf2protein levelup-regulated by genistein, while ICI182,780and LY294002pretreatment inhibited theup-regulation of Nrf2protein level induced by genistein, and PD98059and LY294002processing attenuated HO-1and NQO1level induced by genistein, and ICI182,780pretreatment had no effect on HO-1and NQO1expression.2. Cells were transfected with HA-Nrf2plasmid, immunofluorescence and confocallaser microscopy were adopted. The results showed that HA expression was enhanced whentransfected with HA-Nrf2plasmid, and mainly expressed in the cytoplasm. Equol treatmentsignificantly induced HA nuclear translocation. PD98059pretreatment attenuated HAexpression in cytoplasm, but still HA expressed mainly in nucleus. LY294002and ICI182,780pretreatment respectively inhibited the expression of HA in nucleus.3. Immunofluorescence assay results showed that equol, daidzein and genisteintreatment induced Nrf2nuclear translocation. LY294002or ICI182,780pretreatmentinhibited equol-induced Nrf2nuclear translocation, While PD98059treatment had nosignificant effect on Nrf2nuclear translocation and fluorescence intensity. LY294002andICI182,780pretreatment separately inhibited daidzein or genistein-induced Nrf2nuclear translocation. PD98059treatment inhibited daidzein-induced Nrf2expression but notnuclear translocation. Pretreatment with PD98059inhibited genistein-induced Nrf2nucleartranslocation.4. Cells were transfected with ARE-luciferase reporter gene plasmid, and teste bydual luciferase reporter gene system. It showed that LY294002and ICI182,780pretreatment separately significantly inhibited equol-induced luciferase activity increase,while PD98059pretreatment had no effect.The above results show that equol may through ER and PI3K/Akt pathway but not viaERK1/2activating Nrf2/ARE signaling pathway.Section IV Assessment of antioxidant efficacy of equolThis section focused on the antioxidant effects of equol, the results are as follows:1.250nM equol pretreatment showed different degrees of protection in cells againstseries of concentrations of H2O2(100μM-800μM) or t-BHP (20μM-80μM).2. Oxidative stress treatment significantly increased ROS levels, equol pretreatmentsignificantly inhibited the elevated level of ROS induced by oxidative stress in cell, whileequol treatment alone had no effect on ROS levels..3. Annexin the Ⅴ-FITC/PI flow cytometry and TUNEL analysis showed that H2O2treatment induced cell apoptosis, and equol pretreatment inhibited H2O2-induced apoptosis.4. Western blot analysis showed that Control-siRNA pretreatment had no effect onup-regulation of Nrf2protein level induced by equol, while Nrf2-siRNA pretreatmenteffectively silenced Nrf2protein expression with or without equol treatment.5. Control-siRNA interference has no effect on cell survival by equol under oxidativestress, and Nrf2-siRNA made equol lost the protection in cell viability decline.These results suggested that equol have significant protective effect in endothelialcells againt oxidative stress-induced cell toxicity. And Nrf2transcription factor plays animportant role in equol's antioxidant efficacy.