【作者】 张婷；

【导师】 糜漫天；

【作者基本信息】 第三军医大学 ， 营养与食品卫生学， 2012， 博士

【摘要】 动脉粥样硬化（Atherosclerosis, AS）是全球头号致死病因，且其流行趋势不断加剧，我国AS的发病率也呈逐年升高的态势。流行病学证据显示，心血管疾病（Cardiovasculardiseases，CVD）的患病率通常为男性高于同龄的绝经前女性，但女性绝经后由于体内雌激素水平急剧下降，其CVD患病率却远远高于男性。大量研究表明，雌激素替代疗法（Hormone replacement therapy，HRT）能够降在一定程度上降低绝经后女性心血管疾病的发病率，却会显著增加此类人群中风和恶性肿瘤的罹患风险。在寻找HRT的替代疗法时，许多研究都围绕选择性雌激素受体调节剂（Selected estrogen receptormodulators，SERMs）而开展，如化学合成药物雷洛昔芬（Raloxifene）和抗雌激素他莫昔芬（Tamoxifen）。一项名为RUTH（Raloxifene Use for the Heart）的大规模临床实验对SERMs用于预防冠心病的长效性和安全性以及对罹患乳腺癌的风险进行了评估。这项为期5.6年的跟踪干预实验结果表明，雷洛昔芬对绝经后妇女的心血管系统并无保护作用，反而进一步警告其可能带来的血栓形成和中风的风险。因此，寻找天然无毒副作用的SERMs成为研究者们关注的焦点。流行病学调查发现，膳食中富含大豆或大豆制品的亚洲人中，其CVD的发病率显著低于没有大豆或大豆制品饮食习惯的欧美人。大豆异黄酮（Soy isoflavone，SI）是主要存在于豆科植物中的一类植物雌激素，SI具有类雌激素活性，能双向调节雌激素受体（Estrogen receptor，ER）活性，是一类天然的SERMs。近年来，大量的研究表明，SI的抗氧化效应介导了其广泛的生物学效应，包括防治CVD、更年期综合症、骨质疏松、乳腺癌和前列腺癌等疾病，是一类优良的天然抗氧化剂。因此，研究和开发安全有效的天然SERMs或抗氧化剂成为AS早期防治的有效途径。而且作为天然存在的营养素成分，植物雌激素较药物而言具有更高的安全性，患者更易接纳，因此，对SI抗氧化剂机制的研究对开发防治AS的天然抗氧化药物具有重要意义。第一部分染料木素对氧化应激诱导内皮细胞损伤的保护作用及机制研究染料木素（Genistein），又称染料木黄酮、金雀异黄酮，或5,7,4’-三羟异黄酮，属于异黄酮类，是主要存在于豆科植物中的一类重要的植物雌激素。染料木素分子结构与雌二醇相似，能竞争性结合雌激素受体，具有弱雌激素活性。由于其分子结构含有三个酚羟基，故染料木素又具有一定的抗氧化效应。染料木素和大豆苷元（Daidzein）是近十年来被研究得最多的异黄酮类化合物。大量的体内外实验表明，SI的抗氧化活性和雌激素受体结合活性介导了其广泛的生物学效应，包括防治AS、妇女更年期症状、骨质疏松、前列腺癌和乳腺癌等。因此，特别是在营养学领域，人们对SI的研究兴趣方兴未艾。Nrf2（NF-E2-related factor2）是调控细胞内对氧化应激应答和外源物质代谢的一个关键转录因子，它通过与抗氧化元件（Antioxiant response element, ARE）结合，上调ARE相关抗氧化基因，包括编码细胞内内源性抗氧化剂、Ⅱ相解毒酶和转录子的基因类决定细胞生存或死亡。受Nrf2调控的蛋白的持续表达对细胞氧化还原内环境的稳定起到非常关键的作用，业已证实，许多天然或合成的小分子化合物可通过激活Nrf2/ARE途径保护细胞免受有毒物或致癌物的伤害，因此，利用天然化合物或合成化合物激活Nrf2依赖的细胞对外源性毒剂的适应性应答，从而抵抗各种病理条件下的应激反应，成为各种慢性病或癌症预防的一个重要策略。本研究证实，染料木素对氧化应激诱导的细胞活力下降和凋亡有保护作用，并探讨了Nrf2在染料木素介导的抗氧化作用中的分子机制。第一节染料木素对氧化应激诱导EA.hy926细胞毒性的影响人体中即便是长期摄入SI的人群中，血浆中的染料木素或大豆苷元的浓度很难达到μM，因此探讨生理浓度的SI的生物学效应显得尤为重要且更加实际。为明确低浓度（生理浓度，nM）的染料木素对氧化应激诱导的细胞毒性的保护作用，采用人脐带静脉内皮细胞株EA.hy926，围绕以过氧化氢（Hydrogen peroxide，H₂O₂）或叔丁基过氧化氢（tert-butyl hydroperoxide，t-BHP）建立的EA.hy926细胞氧化应激模型开展研究，结果如下：1.H₂O₂或t-BHP均能计量依赖的诱导细胞活力下降，其IC50分别为650μM和75μM。2.染料木素（1nM⁵00nM）可浓度依赖的抑制氧化应激诱导的细胞活力下降，其中100nM⁵00nM剂量组与对照组相比有显著性差异。3.Annexin Ⅴ-FITC流式细胞法和TUNEL荧光染色法均表明500nM染料木素能降低氧化应激诱导的细胞凋亡。且染料木素可抑制氧化应激诱导的Caspase-3蛋白表达升高和Bcl-2蛋白表达的降低。以上研究结果表明，低剂量（nM）的染料木素可减轻氧化应激诱导的细胞毒性，并可通过抑制Caspase-3和上调Bcl-2蛋白表达抑制细胞凋亡。第二节染料木素对EA.hy926细胞内源性抗氧化剂水平的影响超氧化物歧化酶（Superoxide dismutase，SOD）和过氧化氢酶（Catalase，CAT）是机体最重要的内源性抗氧化酶，还原型谷胱甘肽（Glutathione，GSH）是机体内最重要的非酶性抗氧化剂。本节研究结果如下：1.染料木素（20nM⁵00nM）可浓度依赖的上调SOD的水平，并抑制氧化应激诱导的SOD水平的降低。2.染料木素（20nM⁵00nM）可浓度依赖的上调CAT的水平，并抑制氧化应激诱导的CAT水平的降低3.染料木素（20nM⁵00nM）可浓度依赖的上调GSH的水平，并抑制氧化应激诱导的GSH水平的降低。结果表明，染料木素对内源性抗氧化剂的上调是其抗氧化作用的重要机制之一。第三节染料木素对NQO1-ARE-Luc荧光素酶的活性的影响检测染料木素对ARE-荧光素酶报告基因活性的影响，可以反应直接反应染料木素对Nrf2的激活作用。同样对Nrf2的蛋白和mRNA水平以及核转位进行了检测，结果如下：1.采用双荧光素酶报告基因系统检测，结果显示染料木素（20nM⁵00nM）能浓度依赖的提高萤火虫荧光素酶的活性。2.染料木素可上调Nrf2mRNA表达水平，且在1h时就可激活Nrf2蛋白的表达，并在1h¹0h持续上调Nrf2的蛋白表达水平，但并不呈时间依赖性。3.染料木素（20nM⁵00nM）可浓度依赖的上调Nrf核蛋白的表达水平。以上研究结果表明，染料木素对Nrf2转录因子有激活作用。第四节染料木素对PPARγ转录因子的激活作用检测染料木素对PPREx3-荧光素酶报告基因的活性的影响直接反映了染料木素对PPARγ转录因子的激活作用。同样围绕染料木素对PPARγ蛋白和mRNA表达水平以及核转位进行了研究。结果如下：1.采用双荧光素酶报告基因系统检测，结果显示染料木素（20nM⁵00nM）可浓度依赖的提高萤火虫荧光素酶的活性，但作用比较微弱。2.染料木素可增强PPARγ mRNA的表达，且可时间依赖（1h¹0h）的上调PPARγ的蛋白表达水平。3.染料木素可促进PPARγ的核蛋白水平的表达。以上研究结果表明，染料木素对PPARγ转录因子有激活作用。第五节染料木素对血红素加氧酶-1的激活效应血红素加氧酶-1（Heme oxygenase-1，HO-1）对心血管系统有重要的保护作用，它同时受到Nrf2和PPARγ的调节，是这两个转录因子下游关键的抗氧化酶。本节研究染料木素对HO-1的影响，结果如下：1.免疫印迹结果显示，染料木素（20nM⁵00nM）可剂量依赖和时间依赖的（1h¹0h）的上调HO-1的蛋白表达水平。2.Realtime PCR结果显示，染料木素可时间依赖（1h¹0h）的促进HO-1mRNA的表达水平。3.染料木素（20nM⁵00nM）可剂量依赖的增强HO-1的活性。以上研究结果表明了染料木素对HO-1的激活作用。第六节Nrf2、PPARγ和HO-1在染料木素介导EA.hy926细胞保护效应中的作用采用Nrf2-siRNA干扰、PPARγ的特异性拮抗剂GW9662和HO-1的特异性拮抗剂Znpp预处理细胞，研究染料木素对氧化应激诱导细胞毒性的影响，结果如下：1.用FITC-siRNA转染细胞，激光共聚焦显微镜观察显示，siRNA对细胞有较高的转染效率。2.免疫印迹检测结果显示，对照组或染料木素处理组中，Nrf2-siRNA沉默了Nrf2的蛋白表达，而Control-siRNA对Nrf2蛋白表达没有影响，说明Nrf2-siRNA转染能有效沉默Nrf2基因的表达。3.Nrf2-siRNA处理消除了染料木素对氧化应激诱导的细胞活力下降的抑制作用，Control-siRNA处理则无此作用。4.GW9662和Znpp特异性拮抗剂的分别处理细胞削弱了染料木素对氧化应激诱导的细胞活力下降的保护作用。以上研究结果表明，Nrf2在染料木素抵抗细胞氧化应激损伤中发挥了重要的作用，PPARγ和HO-1也在一定程度上介导了染料木素的抗氧化效应。第二部分Nrf2在雌马酚抑制氧化应激诱导EA.hy926细胞毒性中的作用机制研究雌马酚（Equol）是人体肠道细菌以大豆苷元为底物合成的专一性产物和最终代谢产物，人群中有30～50%的成年人不能代谢生成雌马酚，且这种个体差异持续的存在于“雌马酚产生者”（Equol producers, Eq+）和“非雌马酚产生者”（Equol non-producers, Eq-）中，调查发现亚洲人中Eq+的比例明显高于西方人，提示Eq+较Eq-罹患心血管疾病的风险更低。由于具有特殊的分子结构，雌马酚的抗氧化活性、雌激素受体结合能力和启动基因转录活性比其前体更强。而在体内，49.7%的循环雌马酚以游离或非结合形式存在，显著高于游离大豆苷元（18.7%）或游离雌二醇（4.6%），且在血浆中有更长的半衰期，令雌马酚能够有效的发挥其药理作用。因此，雌马酚优良的抗氧化特性和广泛的生物学效应提示其被用于防治心血管疾病中的可能。第一节雌马酚对NQO1-ARE-Luc荧光素酶的调节利用业已建立的基于ARE-荧光素酶报告系统Nrf2小分子激动剂筛选细胞模型，检测雌马酚对ARE-荧光素酶报告系统的影响，直接反映其对Nrf2的激活作用。研究结果如下：1.采用双荧光素酶报告系统检测，结果显示，雌马酚可剂量依赖（10nM²50nM）和时间依赖（0.5h²h）的增强ARE-荧光素酶的活性。以上研究结果证明，雌马酚是一个Nrf2转录因子激动剂。第二节雌马酚对Nrf2及其下游分子HO-1和NQO1蛋白和mRNA水平的影响本节围绕雌马酚对Nrf2及受其调控的抗氧化基因醌氧化还原酶（NAD（P）H quinineoxidoreductase1，NQO1）和HO-1的蛋白和mRNA水平的影响进行研究。结果如下：1.免疫印迹结果显示，雌马酚（10nM²50nM）可剂量依赖的提高Nrf2、HO-1和NQO1的蛋白表达水平，同样，500nM染料木素和大豆苷元处理均能提高Nrf2、HO-1和NQO1的蛋白表达水平。2.用HA-Nrf2表达型质粒转染细胞后，免疫印迹检测结果显示，雌马酚（10nM²50nM）浓度依赖的上调HA、Nrf2、HO-1和NQO1蛋白水平的表达。3.Real-time PCR检测结果显示，雌马酚对Nrf2mRNA影响不显著，但可浓度依赖（10nM²50nM）的上调HO-1和NQO1mRNA的表达水平。以上研究结果表明，雌马酚主要通过上调Nrf2蛋白水平激活Nrf2/ARE信号通路。第三节ER、ERK1/2和PI3K/Akt在雌马酚激活Nrf2转录因子中的作用机制研究分别采用ER、ERK1/2和PI3K/AktI的特异性拮抗剂CI182,780、PD98059和LY294002预处理细胞，研究雌马酚、大豆苷元和染料木素对Nrf2转录因子的影响，结果如下：1.免疫印迹结果显示，ICI182,780、PD98059和LY294002处理对雌马酚对Nrf2蛋白水平的上调作用的影响不明显，但ICI182,780和LY294002处理抑制了对雌马酚对HO-1和NQO1的上调作用，而PD98059处理对这两种基因表达影响不明显；ICI182,780、PD98059和LY294002处理抑制了大豆苷元对Nrf2和HO-1蛋白水平的上调，ICI182,780和LY294002处理抑制了大豆苷元对NQO1的上调作用，但PD98059处理对NQO1影响不明显；PD98059处理对染料木素对Nrf2的上调作用没有影响，ICI182,780和LY294002处理则抑制了染料木素对Nrf2的上调作用，PD98059和LY294002处理抑制HO-1和NQO1的上调表达，但ICI182,780处理对HO-1和NQO1表达没有影响。2.采用HA-Nrf2表达型质粒转染内皮细胞，激光共聚焦显微镜检测结果显示，未经转染的内皮细胞中无HA表达，经HA-Nrf2表达质粒转染后，细胞内HA主要荧光增强，但主要表达在胞浆内。Equol处理能明显诱导HA的核转位。PD98059预处理使HA在胞浆中的表达减弱，但仍然主要表达与胞核。LY294002和ICI182,780分别处理抑制HA在胞核中的表达。3.免疫荧光化学法检测结果显示，雌马酚、大豆苷元和染料木处理可诱导Nrf2的核转位。LY294002或ICI182,780预处理抑制雌马酚诱导的Nrf2核转位。PD98059处理对Nrf2核转位或荧光强度均没有显著影响。LY294002或ICI182,780预处理可抑制大豆苷元或染料木素诱导的Nrf2核转位。PD98059处理可抑制大豆苷元诱导的Nrf2的表达但对核转位没有影响。PD98059处理可抑制染料木素诱导的的Nrf2核转位。4.用ARE荧光素酶报告基因质粒转染内皮细胞，采用双荧光素酶报告基因检测结果显示，LY294002或ICI182,780预处理显著抑制了雌马酚对荧光素酶的活性的上调，PD98059预处理对雌马酚诱导的荧光素酶的活性无影响。以上研究结果表明，雌马酚可能通过ER和PI3K/Akt但不通过ERK1/2途径激活Nrf2/ARE信号通路，大豆苷元和染料木素对Nrf2的激活可能通过ER和PI3K/Akt途径，大豆苷元对Nrf2的激活不通过ERK1/2，而染料木素对Nrf2的激活可能通过ERK1/2。第四节雌马酚的抗氧化效应评估本节围绕雌马酚的抗氧化作用开展研究，结果如下：1.250nM雌马酚预处理对不同浓度H2O2（100μM⁸00μM）和t-BHP（20μM⁸0μM）处理组细胞活力的下降均有不同程度的抑制作用。2.氧化应激处理使内皮细胞中ROS水平明显升高，雌马酚单独处理对ROS水平没有显著影响。雌马酚预处理可明显抑制氧化应激诱导的细胞中的ROS水平的升高。3.Annexin Ⅴ-FITC/PI流式细胞和TUNEL检测结果显示，H2O2处理使内皮细胞凋亡数量明显增多，Equol预处理可抑制H2O2诱导的细胞凋亡。4.免疫印迹检测结果显示，Control-siRNA处理对雌马酚对Nrf2蛋白的上调作用没有影响，Nrf2-siRNA处理则沉默了雌马酚对Nrf2蛋白的上调作用。5.对细胞的活力检测结果显示，Control-siRNA干扰对雌马酚对氧化应激诱导的内皮细胞损伤的保护作用没有影响，而Nrf2-siRNA干扰则令雌马酚失去了对内皮细胞活力下降的保护作用。以上结果表明，雌马酚对氧化应激诱导的内皮细胞毒性有明显的抑制作用，Nrf2转录因子在雌马酚介导的内皮细胞保护作用中有重要的作用。更多还原

【Abstract】 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.更多还原