天然抗氧化剂抗氧化损伤作用及机制
|
摘要
自本世纪二十年代自由基学说创立以来,对于自由基损伤的研究就不断深入。大量研究表明,有机体的多种疾病都与自由基对机体的氧化损伤有关,自由基对生命与健康的影响和重要性已毋庸置疑,因此寻找有效的抗氧化剂就显得十分重要。目前,由于合成抗氧化剂如二丁基羟基甲苯(BHT)、丁基羟基茴香醚(BHA)等多数技术复杂,成本较高,且有较大的毒性甚至致癌作用,使人们对合成抗氧化剂产生疑虑和排斥心理,临床应用有较大的局限性,而天然抗氧化剂安全,低毒,符合新世纪人们对健康的需求,因此,从资源丰富的天然药物中寻找高效、低毒、价廉的抗氧化剂已成为该领域研究的一个重要方向,其抗氧化作用机制的研究,对进一步天然抗氧化药物的研究开发具有重要意义。
第一部分葡萄籽原花青素抗心肌细胞氧化损伤作用及机制
原花青素(Proanthocyanidins或Procyanidins,简称PC)是植物中广泛存在的一大类多酚化合物的总称。起初统归于缩合鞣质或黄烷醇类。随着分离鉴定技术的提高和对此类物质的深入研究与深刻认识,现已成为独树一帜的一大类物质并称之为原花青素。40多年来人们对从众多不同植物中提取出的PC进行了大量研究,作为PC的理想来源,葡萄籽原花青素提取物(Grape seed proanthocyanidinsextract,GSP)得到了最为深入和广泛的关注。GSP是由不同数量的儿茶素或表儿茶素聚合而成。最简单的原花青素是儿茶素与表儿茶素形成的二聚体,此外还有三聚体、四聚体等直至十聚体。按聚合度的大小,通常将二～四聚体称为低聚体,将五聚体上的称为高聚体。GSP以B型为主,二聚体中,因两个单体的构象或键合位置的不同,可有多种异构体,已分离鉴定的8种结构形式分别命名为B1～B8。GSP具有多种重要生物活性,成为近年研究的热点。
心血管疾病是世界范围内导致人类死亡的一个主要病因。它包括其自身的一套病理现象,主要有动脉硬化,高血压,充血性心脏损伤,心肌病,冠心病,心肌肥大,心律不齐,心室纤维颤动,心动过速,心肌梗塞等。大量动物实验以及人体实验表明氧化压力在心脏损伤的发病机理中发挥重要作用。当自由基产生和细胞内抗氧化防御的平衡被过多的自由基打破时,将会产生氧化压力从而诱导细胞损伤。自由基和氧化压力可以诱导细胞凋亡,主要是通过脂质过氧化反应以及形成脂质过氧化氢物。自由基清除物和抗氧化剂对多种心血管疾病具有保护作用。
第一节葡萄籽原花青素通过诱导细胞内的抗氧化酶保护心肌细胞凋亡
大量研究表明葡萄籽提取物(Grape seed extract,GSE)对多种心脏疾病有保护作用。GSE的心血管保护作用主要归功于其抗氧化的特性。一系列研究表明多酚是GSE抗氧化功能的主要成分。本研究探讨从GSE中提取分离的两种主要的多酚化合物(儿茶素和原花青素B4)能否增加心肌细胞内的抗氧化酶,且这种增强的细胞内抗氧化防御能否保护氧化心肌细胞损伤,取得了以下结果:
1.儿茶素或原花青素B4可以显著诱导心肌细胞H9C2内的抗氧化酶,该作用成浓度依赖的趋势。
2.儿茶素或原花青素B4预处理可以显著降低黄嘌呤氧化酶/黄嘌呤诱导的H9C2细胞内ROS积聚和细胞凋亡。
以上研究结果表明葡萄籽原花青素可以通过诱导细胞内的抗氧化酶保护心肌细胞凋亡。这个可能是GSE保护多种形式心血管疾病的一个重要机制。
第二节葡萄籽原花青素对阿霉素诱导的心肌细胞毒性的保护作用及机制
阿霉素是一种高效的抗癌药,但其严重的心肌损伤的副作用限制了其临床应用。据报道表明GSE对阿霉素诱导的心肌细胞毒性有保护作用。本研究探讨从GSE中提取分离的两种主要的多酚化合物(儿茶素和原花青素B4)对阿霉素诱导的心肌细胞损伤的保护作用以及其相关机制,取得了以下结果:
1.儿茶素和原花青素B4可以减少阿霉素诱导的心肌细胞内ROS的积聚。
2.儿茶素和原花青素B4可以减少阿霉素诱导的心肌细胞凋亡并防止DNA断裂。
3.儿茶素和原花青素B4可以调节心肌细胞内促凋亡蛋白Bax-α和抗凋亡蛋白Bcl-2的表达。
4.儿茶素和原花青素B4可以抑制心肌细胞内的凋亡信号通路。
由于葡萄籽原花青素是GSE的主要活性成分,以上研究结果可能是GSE对阿霉素诱导的心脏毒性保护作用的重要机制,因此,为临床应用GSE和葡萄籽原花青素治疗由阿霉素引起的心脏毒性副作用提供了理论依据。
第二部分冬凌草甲素对砷诱导的细胞毒性和细胞凋亡的保护作用及机制
砷(Arsenic,As)是环境中一种主要的污染物,它存在与泥土和矿石中最终进入地下水系统,从而污染饮用水资源,含砷的地下水对人类的健康是一个巨大的威胁。在不同的地区地下水砷污染的浓度有很大的差别,在东亚地区砷污染的浓度最高,其中包括孟加拉国,印度和中国。国际健康组织和地方政府为降低由饮用水砷污染引起的损失做了很多努力,然而,大量人群还是暴露在砷污染的危险中,承受着砷污染可能带来的副作用,例如高血压,动脉硬化,糖尿以及多种癌症。最好的方法当然是降低砷的摄入,然而这并不是很现实,因为很多人无法选择只有依靠含有砷污染的饮用水和粮食生活,因为那是他们唯一的食物来源。所以另一种重要的解决方法就是通过调节机体的防御机制来抵抗砷的有害作用。转录因子Nrf2是细胞内抗氧化应答和外来物质代谢的一个关键调控子,它通过上调ARE相关的基因,包括那些编码细胞内抗氧化剂,二相酶和转录子的基因来决定细胞的命运。Nrf2依赖的蛋白表达对维持细胞内氧化还原动态平衡起到关键作用,因此,天然化合物或合成化合物通过激活Nrf2途径可以保护细胞有毒性物质或致癌物引起的损伤,用天然化合物或合成化合物来激活Nrf2依赖的适应性应答从而抵抗环境中的毒性是癌症预防的一个重要策略。近期研究表明,激活Nrf2信号通路对砷诱导的细胞毒性有保护作用,应用天然Nrf2激活剂对抗砷的毒性具有重要意义。
冬凌草甲素(Oridonin)是从唇形科香茶菜属植物中分离出的一种贝壳杉烯二萜类天然化合物,主要植物来源有:香茶菜、显脉叶香茶菜、毛叶香茶菜、道孚香茶菜、冬凌草、延命草等。目前关于冬凌草甲素的研究主要集中在其抗肿瘤的作用,其抗癌活性被认为主要是依赖其抑制肿瘤细胞增长,减少血管生成以及增加肿瘤细胞凋亡。本研究探讨了冬凌草甲素对砷诱导的细胞毒性和凋亡的保护作用,我们证明了冬凌草甲素是个新的Nrf2激活剂并阐明了Nrf2途径被激活的机制。进一步,我们证明了用针对Nrf2的天然化合物作为一种治疗手段的可行性,从而保护人类免于日常生活中的各种环境因素的伤害。
第一节冬凌草甲素保护由砷诱导的UROtsa细胞毒性和细胞凋亡
为了明确冬凌草甲素对砷诱导的细胞毒性的保护作用,我们采用了UROtsa细胞(膀胱尿路上皮细胞)模型,该模型为研究砷毒性的经典模型。取得了以下结果:
1.1.4μM冬凌草甲素可以显著提高细胞内谷胱甘肽水平。
2.30μM亚砷酸钠[As(Ⅲ)]作用24h可以显著提高细胞内的ROS水平,用一系列不同浓度的冬凌草甲素可以显著地降低ROS水平,5.6μM冬凌草甲素的作用最为明显。
3.用Annexin V-FITC流式细胞检测法和Hoechst染色法均表明1.4μM冬凌草甲素可以降低由砷诱导的细胞凋亡。
4.冬凌草甲素可以诱导UROtsa细胞中的Nrf2蛋白,在浓度为1.4μM到14μM间呈现剂量依赖关系,当剂量高于14μM,会对细胞产生毒性,对Nrf2的诱导降低。
5.用MTT法和克隆形成检测法均表明1.4μM冬凌草甲素预处理UROtsa24h可以显著提高用砷处理48h的细胞的存活率,用Nrf2-siRNA转染UROtsa细胞后,MTT结果表明冬凌草甲素失去了其对砷诱导的细胞毒性的保护作用。
以上研究结果表明低剂量的冬凌草甲素可以通过提高细胞内的氧化还原能力,抑制由砷诱导的氧化压力,提高细胞的存活率以及降低细胞凋亡从而抵抗砷诱导的细胞损伤,且冬凌草甲素的保护作用需要通过激活Nrf2途径。
第二节冬凌草甲素是一个Nrf2转录因子激活剂
研究表明ARE-荧光素酶报告基因的活性与Nrf2的蛋白表达水平联系紧密,因此检测冬凌草甲素对ARE-荧光素酶报告基因的活性的影响,可以反应其对Nrf2的作用。
1.用高通量荧光素酶报告系统检测,结果显示冬凌草甲素剂量依赖性的提高荧光素酶的活性。
2.为了进一步确证高通量荧光素酶报告系统的检测结果,采用双荧光素酶报告系统检测,结果也显示冬凌草甲素剂量依赖性的提高萤火虫荧光素酶的活性。
以上研究结果证明冬凌草甲素是一个Nrf2转录因子激活剂。
第三节冬凌草甲素通过上调Nrf2蛋白水平激活ARE依赖的抗氧化应答
本章围绕冬凌草甲素对Nrf2蛋白水平和对相关基因mRNA水平的作用取得了以下结果:
1.将双荧光素酶实验的细胞裂解液用于Nrf2,Keapl andβ-actin的免疫印迹检测,结果表明,冬凌草甲素素可以剂量依赖性的提高Nrf2的蛋白水平,14μM时作用最强。
2.检测高浓度冬凌草甲素作用是对Nrf2蛋白水平的影响,结果表明当冬凌草甲素浓度高于28μM时剂量依赖性地降低Nrf2蛋白水平,且Keap1和β-actin的表达没有明显变化。
3.用real time RT-PCR检测了冬凌草甲素处理后Nrf2以及其下游基因NQO1和HO-1 mRNA的表达水平。结果表明Nrf2 mRNA水平有较弱的提高,呈剂量依赖关系。NQO1和HO-1的mRNA水平都有显著的提高,且呈剂量关系。
以上研究结果表明冬凌草甲素主要通过上调Nrf2的蛋白水平来诱导Nrf2信号通路。
第四节冬凌草甲素阻断Nrf2泛素化并增强Keap1泛素化
tBHQ通过干扰keap-1依赖的泛素结合过程从而提高Nrf2蛋白水平,因此,我们也检测了冬凌草甲素调节Nrf2泛素化的能力,取得了以下结果:
1.冬凌草甲素抑制MDA-MB-231细胞和UROtsa细胞中Nrf2的泛素化。
2.冬凌草甲素增加MDA-MB-231细胞和UROtsa细胞中keap1的泛素化。
3.冬凌草甲素延长MDA-MB-231细胞中内源性Nrf2蛋白的半衰期,从19分钟到51分钟;冬凌草甲素延长UROtsa细胞中内源性Nrf2蛋白的半衰期,从10分钟到16分钟。
以上研究结果表明冬凌草甲素诱导泛素化由Nrf2转移到Keap1,它通过抑制Nrf2的泛素化和降解使Nrf2的蛋白水平提高,从而激活Nrf2依赖的抗氧化应答。
From the foundation of free radical theory in 1920s,the research about free radicals injury continuously deepened.A large number of researches indicate that many diseases are related with oxidative injury,there is no doubt about the impacts and importance of free radicals to life and health,so it is vital to look for potent antioxidants.At present,synthetic antioxidants such as BHT,BHA et al,they always have complicated synthetic technology with high cost,moreover,synthetic antioxidants have relatively higher toxicity even carcinogenic effects,so people nowadays disbelieve and refuse to use synthetic antioxidants,the clinical use of synthetic antioxidants is greatly limited.Natural antioxidants are safer and with lower toxicity which meet the health needs of people in new century,so finding antioxidants with high effect,low toxicity and low price from natural compounds has become a important research direction of this field,studies about the mechanisms underlying their antioxidant effects are of great significance in the further development of natural antioxidants.
PART ONE Protective effects of grape seed proanthocyanidins against cardiac cells oxidative damage and the mechanisms underlying it
Proanthocyanidins or Procyanidins(PC) is a group of polyphenols which occur widely in plant.At first,they are put into the category of condensation tannins or flavanols.With the development of isolation and identification technology,at the same time,with deeper researches and understanding of these kinds of compounds, now they have become a large group of compounds called proanthocyanidins.For many years,people have done a lots of studies on PC extracted from different plants. As a ideal source of PC,Grape seed proanthocyanidins extract(GSP) has received the deepest and widest attention.GSP is polymerized by different amounts of catechin or epicatehin.The simplest proanthocyanidins is the dimer formed by catechin and epicatehin,besides this,there are trimer,tetramer till decamer.Sized by polymerization level,dimer to tetramer are called oligomer,more than 5 are called high polymer.A predominant proportion of GSP is type B,because the differences in conformation and biding sites in dimmers,there are several isomers,8 have been isolated and identified,called B1～B8.GSP have many important bioactivities,it has become a hot research topic.
Cardiovascular disease remains a leading cause of death worldwide.It comprises its own set of pathologies,chief among which are atherosclerosis,hypertension, congestive heart failure,cardiomyopathy,coronary heart disease,hypertrophy, arrhythmias,ventricular fibrillation(VF),ventricular tachycardia(VT),myocardial infarction,and stroke.Strong evidence for the role of oxidative stress in the pathogenesis of heart failure has been provided by studies on experimental animals as well as humans.When the equilibrium between flee-radical production and cellular antioxidant defenses is disturbed in favour of more flee radicals,it causes oxidative stress which can promote cellular injury.Free radicals and oxidative stress also appear to be a common mediator of apoptosis,directly or via the formation of lipid peroxidation and lipid hydroperoxides.Free radical scavengers and antioxidants have been found to be cardioprotective against various ischemic heart diseases.
SectionⅠGrape seed proanthocyanidins protect cardiac cells from apoptosis via induction of endogenous antioxidant
Grape seed extract(GSE) has been reported to exert protective effects on various forms of cardiac disorders.The cardiovascular protective effects of GSE are believed to be ascribed to its antioxidative properties.A series of studies have demonstrated that polyphenols are instrumental for the antioxidative properties of GSE.This study was undertaken to investigate whether two major polyphenols isolated from GSE(catechin and proanthocyanidin B_4) could increase the endogenous antioxidant enzymes in cardiomycytes,and whether such increased cellular defenses could provide protection against oxidative cardiac cell injury.The results are showed below:
1.Incubation of cardiac H9C2 cells with catechin or proanthocyanidin B4 resulted in a significant induction of cellular antioxidant enzymes in a concentration-dependent fashion.
2.Catechin or proanthocyanidin B4 pretreatment led to a marked reduction in xanthine oxidase(XO)/xanthine -induced intracellular reactive oxygen species (ROS) accumulation and cardiac cell apoptosis.
These results indicated that grape seed proanthocyanidins could protect against cardiac cell apoptosis via the induction of endogenous antioxidant enzymes.This may be an important mechanism underlying the protective effects of GSE observed with various forms of cardiovascular disorders
SectionⅡProtection effects of grape seed proanthocyanidins against doxorubicin-induced toxicity in cardiomyocytes and the mechanisms underlying it
The clinical use of doxorubicin,a highly active anticancer drug,is limited by its severe cardiotoxic side effects.Grape seed extract(GSE) has been reported to exert protective effects on doxorubicin cardiotoxicity.This study was designed to investigate whether two major polyphenols isolated from GSE(catechin and proanthocyanidin B4(pc B_4)) had protective effects against doxorubicin-induced cardiotoxicity and the proper mechanisms underlying it.The results are showed below:
1.Catechin and pc B_4 pretreatment would decrease doxorubicin-induced ROS generation in cardiac cells.
2.Catechin and pc B_4 pretreatment would decrease doxorubicin-induced decrease the number of apoptotic cells and prevent DNA fragmentation in cardiac cells.
3.Catechin and pc B_4 pretreatment would regulate the levels of expression of the pro-apoptotic protein Bax-αand the anti-apoptotic protein Bcl-2 in cardiac cells.
4.Catechin and pc B_4 pretreatment would inhibit apoptotic signaling pathways in cardiac cells.
Because grape seed proanthocyanidins are instrumental for the bioactive properties of GSE,above results may be important mechanisms underlying the protective effects of GSE observed in doxorubicin cardiotoxicity.Thereby,we provide the mechanical basis of GSE and grape seed proanthocyanidins in clinical application to protect patients from doxorubicin induced cardiotoxicity.
PART TWO Protection effects of oridonin against arsenic-induced cell toxicity and apoptosis and the mechanisms underlying it
Arsenic is one of the major environmental pollutants.It exists in soil and minerals and readily enters the ground water system,contaminating drinking water.The concentration of arsenic in the ground water varies significantly in different geographic areas.Arsenic concentrations are highest in East Asia,including Bangladesh,West Bengal,India,and China.Many efforts have been made in an attempt to reduce arsenic damage as exemplified by the guideline for arsenic in drinking water set by the World Health Organization and by local governments. Nevertheless,a large number of populations are still at risk of arsenic exposure and are suffering from arsenic-induced adverse effects,such as hypertension, arteriosclerosis,diabetes,hyperkeratosis,neuropathy,and cancer in the skin,liver, bladder,and lung.Clearly,the best way to protect humans from arsenic-induced damage is to reduce arsenic intake.However,it is not always practical,because many people have no choice but to live off from drinking water and rice that are heavily contaminated with arsenic,as these are their only sources of food and water. Therefore,an alternative choice,of equal importance,is to subvert the detrimental effects of arsenic by modulating the body's defense system.Nrf2 is a critical transcription factor that regulates a cytoprotective response.Many of its downstream target genes are important in maintaining the cellular antioxidant response as well as xenobiotic metabolism.Nrf2 controls the fate of cells through transcriptional upregulation of antioxidant response dement(ARE)-bearing genes,includingthose encoding endogenous antioxidants,phaseⅡdetoxifying enzymes,and transporters. Expression of the Nrf2-dependent proteins is critical for ameliorating or eliminating toxicants/carcinogens to maintain cellular redox homeostasis.As a result,activation of the Nrf2 pathway,by naturally-occurring compounds or synthetic chemicals at sub-toxic doses,confers protection against subsequent toxic/carcinogenic exposure. Thus,the use of dietary compounds or synthetic chemicals to boost the Nrf2-dependent adaptive response to counteract environmental insults has emerged to be a promising strategy for cancer prevention.Recently,upregulation of the Nrf2-dependent defense response has proved to be beneficial in reducing arsenic-induced toxicity in a cell culture model.So using Nrf2 activators is of great significance to resist arsenic-induced toxicity.Oridonin is a kind of ent - kaurene diterpenoid purified from the Chinese medicinal herb Rabdosia rubescens.The main plant sources of oridinin are:R.am ethystoides,R.nervosa,R.rabdosia,R.downsis, R.rubescens,Isodon japonicus et al.Currently the major research focus on oridonin is in its antiproliferation and antitumor activities.The anticancer activity of oridonin is thought to rely on its ability to inhibit cell growth,reduce angiogenesis,and enhance apoptosis.This study was designed to test the protection effects of oridonin against arsenic-induced cell toxicity and apoptotic.We identified oridonin as representing a novel class of Nrf2 activators and illustrated the mechanism by which the Nrf2 pathway is activated.Furthermore,we demonstrated the feasibility of using natural compounds targeting Nrf2 as a therapeutic approach to protect humans from various environmental insults that may occur daily.
SectionⅠEfficacy of oridonin in protecting against Arsenic-induced Urosta cells toxicity and apoptosis
The chemopreventive activity of oridonin was demonstrated using a previously established arsenic-UROtsa cell model.The results are showed below:
1.Oridonin treatment resulted in a significant increase in the glutathione level
2.Treatment with 30μM As(Ⅲ) for 24 hr increased the level of ROS significantly, whereas 5.6μM oridonin itself had no effect.Pretreatment of cells with several doses of oridonin for 24 hr and further cotreatment with As(Ⅲ) for an additional 24 hr resulted in a significant reduction of ROS levels,especially with 5.6μM oridonin.
3.By using Annexin V-FITC flow cytometry and Hoechst staining,we demonstrated that 1.4μM oridonin can inhibit As(Ⅲ)-induced cell apoptosis.
4.Oridonin can induce Nrf2 in a dose-dependent fashion,from 1.4 to 14μM. Doses more than 14μM appeared to be toxic,and induction of Nrf2 was decreased
5.By using both MTT and colony formation assays,we have demonstrated that pretreatment with1.4μM oridonin in UROtsa for 24h can significantly improved cell survival which were treated with As(Ⅲ) for 48 hr first.When using Nrf2-siRNA transfected UROtsa cells,oridonin lost its protection against As(Ⅲ) toxicity.
Together,these results demonstrate that a low dose of oridonin is able to protect cells from As(Ⅲ)-induced damage,as illustrated by increased cellular antioxidant ability, inhibited As(Ⅲ)-induced oxidative stress,increased cell survival in response to As(Ⅲ) and decreased cell apoptosis,moreover,the protection effect was attributed to the activation of Nrf2 by oridonin.
SectionⅡOridonin is an Nrf2 activator.
Studies have showed that the activity of ARE-luciferase reporter gene is closely related with Nrf2 protein level.So testing effect of oridonin on ARE-luciferase reporter gene's activity can show us its effect on Nrf2.
1.Using highthroughput stable ARE luciferase reporter system established in our laboratory,we found out that oridonin dose-dependently increased the activity of luciferase.
2.To confirm oridonin activation of Nrf2 using the high-throughput screening method,we also performed a dual luciferase reporter gene assay.Consistent with the data obtained from the high-throughput screening,oridonin induced the ARE-dependent luciferase activity in a dose-dependent manner Above results demonstrated that oridonin is an Nrf2activator.
SectionⅢOridonin activated the ARE-dependent response primarily through up-regulation of the Nrf2 protein level.
This section is about oridonin's effects on Nrf2 protein level and the mRNA level of Nrf2 downstream genes,the results are showed below:
1.We used the same cell lysates from the dual luciferase reporter gene assay for immunoblot analysis for detection of Nrf2,Keap1,andβ-actin.Oridonin enhanced the levels of Nrf2 protein in a dose-dependent manner,with the highest induction at 14μM.
2.We tested Nrf2 protein levels in response to high doses of oridonin.At doses more than 28μM,Nrf2 protein levels decreased in a dose-dependent manner, whereas the expression of Keap1 andβ-actin had no significant change.
3.We measured mRNA expression of Nrf2 and its target genes,NQO1 and HO-1, in response to oridonin using real-time RT-PCR.Nrf2 mRNA increased slightly in a dosedependent manner in response to oridonin,mRNA of NQO1 and HO-1 were induced significantly by oridonin in a dose-dependent manner.
These results demonstrate that oridonin is able to induce the Nrf2 signaling pathway mainly through upregulation of Nrf2 at the protein level.
SectionⅣOridonin blocked Nrf2 ubiquitination and enhanced Keap1 ubiquitination
tBHQ enhances the Nrf2 protein level by interfering with the Keap1-dependent ubiquitin conjugation process.Therefore,we tested the ability of oridonin in modulating Nrf2 ubiquitination.The results are showed below:
1.Oridonin suppressed Nrf2 ubiquitination in MDA-MB-231cells and UROtsa cells.
2.Oridonin treatment was also effective in enhancing ubiquitination of Keap1 MDA-MB-231cells and UROtsa cells.
3.We measured the half-life of Nrf2 in the absence or presence of oridonin. Half-life of the endogenous Nrf2 protein in MDA-MB-231 cells was 19 min, whereas treatment with oridonin increased the half-life to 51 min.The half life of Nrf2 in UROtsa was increased from 10 min in the untreated condition to 16 min in response to oridonin treatment.
Above results indicate that oridonin activates the Nrf2 pathway by inhibiting ubiquitination and degradation of Nrf2,leading to an increase in Nrf2 protein level and activation of the Nrf2-dependent response.
第一部分葡萄籽原花青素抗心肌细胞氧化损伤作用及机制
原花青素(Proanthocyanidins或Procyanidins,简称PC)是植物中广泛存在的一大类多酚化合物的总称。起初统归于缩合鞣质或黄烷醇类。随着分离鉴定技术的提高和对此类物质的深入研究与深刻认识,现已成为独树一帜的一大类物质并称之为原花青素。40多年来人们对从众多不同植物中提取出的PC进行了大量研究,作为PC的理想来源,葡萄籽原花青素提取物(Grape seed proanthocyanidinsextract,GSP)得到了最为深入和广泛的关注。GSP是由不同数量的儿茶素或表儿茶素聚合而成。最简单的原花青素是儿茶素与表儿茶素形成的二聚体,此外还有三聚体、四聚体等直至十聚体。按聚合度的大小,通常将二～四聚体称为低聚体,将五聚体上的称为高聚体。GSP以B型为主,二聚体中,因两个单体的构象或键合位置的不同,可有多种异构体,已分离鉴定的8种结构形式分别命名为B1～B8。GSP具有多种重要生物活性,成为近年研究的热点。
心血管疾病是世界范围内导致人类死亡的一个主要病因。它包括其自身的一套病理现象,主要有动脉硬化,高血压,充血性心脏损伤,心肌病,冠心病,心肌肥大,心律不齐,心室纤维颤动,心动过速,心肌梗塞等。大量动物实验以及人体实验表明氧化压力在心脏损伤的发病机理中发挥重要作用。当自由基产生和细胞内抗氧化防御的平衡被过多的自由基打破时,将会产生氧化压力从而诱导细胞损伤。自由基和氧化压力可以诱导细胞凋亡,主要是通过脂质过氧化反应以及形成脂质过氧化氢物。自由基清除物和抗氧化剂对多种心血管疾病具有保护作用。
第一节葡萄籽原花青素通过诱导细胞内的抗氧化酶保护心肌细胞凋亡
大量研究表明葡萄籽提取物(Grape seed extract,GSE)对多种心脏疾病有保护作用。GSE的心血管保护作用主要归功于其抗氧化的特性。一系列研究表明多酚是GSE抗氧化功能的主要成分。本研究探讨从GSE中提取分离的两种主要的多酚化合物(儿茶素和原花青素B4)能否增加心肌细胞内的抗氧化酶,且这种增强的细胞内抗氧化防御能否保护氧化心肌细胞损伤,取得了以下结果:
1.儿茶素或原花青素B4可以显著诱导心肌细胞H9C2内的抗氧化酶,该作用成浓度依赖的趋势。
2.儿茶素或原花青素B4预处理可以显著降低黄嘌呤氧化酶/黄嘌呤诱导的H9C2细胞内ROS积聚和细胞凋亡。
以上研究结果表明葡萄籽原花青素可以通过诱导细胞内的抗氧化酶保护心肌细胞凋亡。这个可能是GSE保护多种形式心血管疾病的一个重要机制。
第二节葡萄籽原花青素对阿霉素诱导的心肌细胞毒性的保护作用及机制
阿霉素是一种高效的抗癌药,但其严重的心肌损伤的副作用限制了其临床应用。据报道表明GSE对阿霉素诱导的心肌细胞毒性有保护作用。本研究探讨从GSE中提取分离的两种主要的多酚化合物(儿茶素和原花青素B4)对阿霉素诱导的心肌细胞损伤的保护作用以及其相关机制,取得了以下结果:
1.儿茶素和原花青素B4可以减少阿霉素诱导的心肌细胞内ROS的积聚。
2.儿茶素和原花青素B4可以减少阿霉素诱导的心肌细胞凋亡并防止DNA断裂。
3.儿茶素和原花青素B4可以调节心肌细胞内促凋亡蛋白Bax-α和抗凋亡蛋白Bcl-2的表达。
4.儿茶素和原花青素B4可以抑制心肌细胞内的凋亡信号通路。
由于葡萄籽原花青素是GSE的主要活性成分,以上研究结果可能是GSE对阿霉素诱导的心脏毒性保护作用的重要机制,因此,为临床应用GSE和葡萄籽原花青素治疗由阿霉素引起的心脏毒性副作用提供了理论依据。
第二部分冬凌草甲素对砷诱导的细胞毒性和细胞凋亡的保护作用及机制
砷(Arsenic,As)是环境中一种主要的污染物,它存在与泥土和矿石中最终进入地下水系统,从而污染饮用水资源,含砷的地下水对人类的健康是一个巨大的威胁。在不同的地区地下水砷污染的浓度有很大的差别,在东亚地区砷污染的浓度最高,其中包括孟加拉国,印度和中国。国际健康组织和地方政府为降低由饮用水砷污染引起的损失做了很多努力,然而,大量人群还是暴露在砷污染的危险中,承受着砷污染可能带来的副作用,例如高血压,动脉硬化,糖尿以及多种癌症。最好的方法当然是降低砷的摄入,然而这并不是很现实,因为很多人无法选择只有依靠含有砷污染的饮用水和粮食生活,因为那是他们唯一的食物来源。所以另一种重要的解决方法就是通过调节机体的防御机制来抵抗砷的有害作用。转录因子Nrf2是细胞内抗氧化应答和外来物质代谢的一个关键调控子,它通过上调ARE相关的基因,包括那些编码细胞内抗氧化剂,二相酶和转录子的基因来决定细胞的命运。Nrf2依赖的蛋白表达对维持细胞内氧化还原动态平衡起到关键作用,因此,天然化合物或合成化合物通过激活Nrf2途径可以保护细胞有毒性物质或致癌物引起的损伤,用天然化合物或合成化合物来激活Nrf2依赖的适应性应答从而抵抗环境中的毒性是癌症预防的一个重要策略。近期研究表明,激活Nrf2信号通路对砷诱导的细胞毒性有保护作用,应用天然Nrf2激活剂对抗砷的毒性具有重要意义。
冬凌草甲素(Oridonin)是从唇形科香茶菜属植物中分离出的一种贝壳杉烯二萜类天然化合物,主要植物来源有:香茶菜、显脉叶香茶菜、毛叶香茶菜、道孚香茶菜、冬凌草、延命草等。目前关于冬凌草甲素的研究主要集中在其抗肿瘤的作用,其抗癌活性被认为主要是依赖其抑制肿瘤细胞增长,减少血管生成以及增加肿瘤细胞凋亡。本研究探讨了冬凌草甲素对砷诱导的细胞毒性和凋亡的保护作用,我们证明了冬凌草甲素是个新的Nrf2激活剂并阐明了Nrf2途径被激活的机制。进一步,我们证明了用针对Nrf2的天然化合物作为一种治疗手段的可行性,从而保护人类免于日常生活中的各种环境因素的伤害。
第一节冬凌草甲素保护由砷诱导的UROtsa细胞毒性和细胞凋亡
为了明确冬凌草甲素对砷诱导的细胞毒性的保护作用,我们采用了UROtsa细胞(膀胱尿路上皮细胞)模型,该模型为研究砷毒性的经典模型。取得了以下结果:
1.1.4μM冬凌草甲素可以显著提高细胞内谷胱甘肽水平。
2.30μM亚砷酸钠[As(Ⅲ)]作用24h可以显著提高细胞内的ROS水平,用一系列不同浓度的冬凌草甲素可以显著地降低ROS水平,5.6μM冬凌草甲素的作用最为明显。
3.用Annexin V-FITC流式细胞检测法和Hoechst染色法均表明1.4μM冬凌草甲素可以降低由砷诱导的细胞凋亡。
4.冬凌草甲素可以诱导UROtsa细胞中的Nrf2蛋白,在浓度为1.4μM到14μM间呈现剂量依赖关系,当剂量高于14μM,会对细胞产生毒性,对Nrf2的诱导降低。
5.用MTT法和克隆形成检测法均表明1.4μM冬凌草甲素预处理UROtsa24h可以显著提高用砷处理48h的细胞的存活率,用Nrf2-siRNA转染UROtsa细胞后,MTT结果表明冬凌草甲素失去了其对砷诱导的细胞毒性的保护作用。
以上研究结果表明低剂量的冬凌草甲素可以通过提高细胞内的氧化还原能力,抑制由砷诱导的氧化压力,提高细胞的存活率以及降低细胞凋亡从而抵抗砷诱导的细胞损伤,且冬凌草甲素的保护作用需要通过激活Nrf2途径。
第二节冬凌草甲素是一个Nrf2转录因子激活剂
研究表明ARE-荧光素酶报告基因的活性与Nrf2的蛋白表达水平联系紧密,因此检测冬凌草甲素对ARE-荧光素酶报告基因的活性的影响,可以反应其对Nrf2的作用。
1.用高通量荧光素酶报告系统检测,结果显示冬凌草甲素剂量依赖性的提高荧光素酶的活性。
2.为了进一步确证高通量荧光素酶报告系统的检测结果,采用双荧光素酶报告系统检测,结果也显示冬凌草甲素剂量依赖性的提高萤火虫荧光素酶的活性。
以上研究结果证明冬凌草甲素是一个Nrf2转录因子激活剂。
第三节冬凌草甲素通过上调Nrf2蛋白水平激活ARE依赖的抗氧化应答
本章围绕冬凌草甲素对Nrf2蛋白水平和对相关基因mRNA水平的作用取得了以下结果:
1.将双荧光素酶实验的细胞裂解液用于Nrf2,Keapl andβ-actin的免疫印迹检测,结果表明,冬凌草甲素素可以剂量依赖性的提高Nrf2的蛋白水平,14μM时作用最强。
2.检测高浓度冬凌草甲素作用是对Nrf2蛋白水平的影响,结果表明当冬凌草甲素浓度高于28μM时剂量依赖性地降低Nrf2蛋白水平,且Keap1和β-actin的表达没有明显变化。
3.用real time RT-PCR检测了冬凌草甲素处理后Nrf2以及其下游基因NQO1和HO-1 mRNA的表达水平。结果表明Nrf2 mRNA水平有较弱的提高,呈剂量依赖关系。NQO1和HO-1的mRNA水平都有显著的提高,且呈剂量关系。
以上研究结果表明冬凌草甲素主要通过上调Nrf2的蛋白水平来诱导Nrf2信号通路。
第四节冬凌草甲素阻断Nrf2泛素化并增强Keap1泛素化
tBHQ通过干扰keap-1依赖的泛素结合过程从而提高Nrf2蛋白水平,因此,我们也检测了冬凌草甲素调节Nrf2泛素化的能力,取得了以下结果:
1.冬凌草甲素抑制MDA-MB-231细胞和UROtsa细胞中Nrf2的泛素化。
2.冬凌草甲素增加MDA-MB-231细胞和UROtsa细胞中keap1的泛素化。
3.冬凌草甲素延长MDA-MB-231细胞中内源性Nrf2蛋白的半衰期,从19分钟到51分钟;冬凌草甲素延长UROtsa细胞中内源性Nrf2蛋白的半衰期,从10分钟到16分钟。
以上研究结果表明冬凌草甲素诱导泛素化由Nrf2转移到Keap1,它通过抑制Nrf2的泛素化和降解使Nrf2的蛋白水平提高,从而激活Nrf2依赖的抗氧化应答。
From the foundation of free radical theory in 1920s,the research about free radicals injury continuously deepened.A large number of researches indicate that many diseases are related with oxidative injury,there is no doubt about the impacts and importance of free radicals to life and health,so it is vital to look for potent antioxidants.At present,synthetic antioxidants such as BHT,BHA et al,they always have complicated synthetic technology with high cost,moreover,synthetic antioxidants have relatively higher toxicity even carcinogenic effects,so people nowadays disbelieve and refuse to use synthetic antioxidants,the clinical use of synthetic antioxidants is greatly limited.Natural antioxidants are safer and with lower toxicity which meet the health needs of people in new century,so finding antioxidants with high effect,low toxicity and low price from natural compounds has become a important research direction of this field,studies about the mechanisms underlying their antioxidant effects are of great significance in the further development of natural antioxidants.
PART ONE Protective effects of grape seed proanthocyanidins against cardiac cells oxidative damage and the mechanisms underlying it
Proanthocyanidins or Procyanidins(PC) is a group of polyphenols which occur widely in plant.At first,they are put into the category of condensation tannins or flavanols.With the development of isolation and identification technology,at the same time,with deeper researches and understanding of these kinds of compounds, now they have become a large group of compounds called proanthocyanidins.For many years,people have done a lots of studies on PC extracted from different plants. As a ideal source of PC,Grape seed proanthocyanidins extract(GSP) has received the deepest and widest attention.GSP is polymerized by different amounts of catechin or epicatehin.The simplest proanthocyanidins is the dimer formed by catechin and epicatehin,besides this,there are trimer,tetramer till decamer.Sized by polymerization level,dimer to tetramer are called oligomer,more than 5 are called high polymer.A predominant proportion of GSP is type B,because the differences in conformation and biding sites in dimmers,there are several isomers,8 have been isolated and identified,called B1～B8.GSP have many important bioactivities,it has become a hot research topic.
Cardiovascular disease remains a leading cause of death worldwide.It comprises its own set of pathologies,chief among which are atherosclerosis,hypertension, congestive heart failure,cardiomyopathy,coronary heart disease,hypertrophy, arrhythmias,ventricular fibrillation(VF),ventricular tachycardia(VT),myocardial infarction,and stroke.Strong evidence for the role of oxidative stress in the pathogenesis of heart failure has been provided by studies on experimental animals as well as humans.When the equilibrium between flee-radical production and cellular antioxidant defenses is disturbed in favour of more flee radicals,it causes oxidative stress which can promote cellular injury.Free radicals and oxidative stress also appear to be a common mediator of apoptosis,directly or via the formation of lipid peroxidation and lipid hydroperoxides.Free radical scavengers and antioxidants have been found to be cardioprotective against various ischemic heart diseases.
SectionⅠGrape seed proanthocyanidins protect cardiac cells from apoptosis via induction of endogenous antioxidant
Grape seed extract(GSE) has been reported to exert protective effects on various forms of cardiac disorders.The cardiovascular protective effects of GSE are believed to be ascribed to its antioxidative properties.A series of studies have demonstrated that polyphenols are instrumental for the antioxidative properties of GSE.This study was undertaken to investigate whether two major polyphenols isolated from GSE(catechin and proanthocyanidin B_4) could increase the endogenous antioxidant enzymes in cardiomycytes,and whether such increased cellular defenses could provide protection against oxidative cardiac cell injury.The results are showed below:
1.Incubation of cardiac H9C2 cells with catechin or proanthocyanidin B4 resulted in a significant induction of cellular antioxidant enzymes in a concentration-dependent fashion.
2.Catechin or proanthocyanidin B4 pretreatment led to a marked reduction in xanthine oxidase(XO)/xanthine -induced intracellular reactive oxygen species (ROS) accumulation and cardiac cell apoptosis.
These results indicated that grape seed proanthocyanidins could protect against cardiac cell apoptosis via the induction of endogenous antioxidant enzymes.This may be an important mechanism underlying the protective effects of GSE observed with various forms of cardiovascular disorders
SectionⅡProtection effects of grape seed proanthocyanidins against doxorubicin-induced toxicity in cardiomyocytes and the mechanisms underlying it
The clinical use of doxorubicin,a highly active anticancer drug,is limited by its severe cardiotoxic side effects.Grape seed extract(GSE) has been reported to exert protective effects on doxorubicin cardiotoxicity.This study was designed to investigate whether two major polyphenols isolated from GSE(catechin and proanthocyanidin B4(pc B_4)) had protective effects against doxorubicin-induced cardiotoxicity and the proper mechanisms underlying it.The results are showed below:
1.Catechin and pc B_4 pretreatment would decrease doxorubicin-induced ROS generation in cardiac cells.
2.Catechin and pc B_4 pretreatment would decrease doxorubicin-induced decrease the number of apoptotic cells and prevent DNA fragmentation in cardiac cells.
3.Catechin and pc B_4 pretreatment would regulate the levels of expression of the pro-apoptotic protein Bax-αand the anti-apoptotic protein Bcl-2 in cardiac cells.
4.Catechin and pc B_4 pretreatment would inhibit apoptotic signaling pathways in cardiac cells.
Because grape seed proanthocyanidins are instrumental for the bioactive properties of GSE,above results may be important mechanisms underlying the protective effects of GSE observed in doxorubicin cardiotoxicity.Thereby,we provide the mechanical basis of GSE and grape seed proanthocyanidins in clinical application to protect patients from doxorubicin induced cardiotoxicity.
PART TWO Protection effects of oridonin against arsenic-induced cell toxicity and apoptosis and the mechanisms underlying it
Arsenic is one of the major environmental pollutants.It exists in soil and minerals and readily enters the ground water system,contaminating drinking water.The concentration of arsenic in the ground water varies significantly in different geographic areas.Arsenic concentrations are highest in East Asia,including Bangladesh,West Bengal,India,and China.Many efforts have been made in an attempt to reduce arsenic damage as exemplified by the guideline for arsenic in drinking water set by the World Health Organization and by local governments. Nevertheless,a large number of populations are still at risk of arsenic exposure and are suffering from arsenic-induced adverse effects,such as hypertension, arteriosclerosis,diabetes,hyperkeratosis,neuropathy,and cancer in the skin,liver, bladder,and lung.Clearly,the best way to protect humans from arsenic-induced damage is to reduce arsenic intake.However,it is not always practical,because many people have no choice but to live off from drinking water and rice that are heavily contaminated with arsenic,as these are their only sources of food and water. Therefore,an alternative choice,of equal importance,is to subvert the detrimental effects of arsenic by modulating the body's defense system.Nrf2 is a critical transcription factor that regulates a cytoprotective response.Many of its downstream target genes are important in maintaining the cellular antioxidant response as well as xenobiotic metabolism.Nrf2 controls the fate of cells through transcriptional upregulation of antioxidant response dement(ARE)-bearing genes,includingthose encoding endogenous antioxidants,phaseⅡdetoxifying enzymes,and transporters. Expression of the Nrf2-dependent proteins is critical for ameliorating or eliminating toxicants/carcinogens to maintain cellular redox homeostasis.As a result,activation of the Nrf2 pathway,by naturally-occurring compounds or synthetic chemicals at sub-toxic doses,confers protection against subsequent toxic/carcinogenic exposure. Thus,the use of dietary compounds or synthetic chemicals to boost the Nrf2-dependent adaptive response to counteract environmental insults has emerged to be a promising strategy for cancer prevention.Recently,upregulation of the Nrf2-dependent defense response has proved to be beneficial in reducing arsenic-induced toxicity in a cell culture model.So using Nrf2 activators is of great significance to resist arsenic-induced toxicity.Oridonin is a kind of ent - kaurene diterpenoid purified from the Chinese medicinal herb Rabdosia rubescens.The main plant sources of oridinin are:R.am ethystoides,R.nervosa,R.rabdosia,R.downsis, R.rubescens,Isodon japonicus et al.Currently the major research focus on oridonin is in its antiproliferation and antitumor activities.The anticancer activity of oridonin is thought to rely on its ability to inhibit cell growth,reduce angiogenesis,and enhance apoptosis.This study was designed to test the protection effects of oridonin against arsenic-induced cell toxicity and apoptotic.We identified oridonin as representing a novel class of Nrf2 activators and illustrated the mechanism by which the Nrf2 pathway is activated.Furthermore,we demonstrated the feasibility of using natural compounds targeting Nrf2 as a therapeutic approach to protect humans from various environmental insults that may occur daily.
SectionⅠEfficacy of oridonin in protecting against Arsenic-induced Urosta cells toxicity and apoptosis
The chemopreventive activity of oridonin was demonstrated using a previously established arsenic-UROtsa cell model.The results are showed below:
1.Oridonin treatment resulted in a significant increase in the glutathione level
2.Treatment with 30μM As(Ⅲ) for 24 hr increased the level of ROS significantly, whereas 5.6μM oridonin itself had no effect.Pretreatment of cells with several doses of oridonin for 24 hr and further cotreatment with As(Ⅲ) for an additional 24 hr resulted in a significant reduction of ROS levels,especially with 5.6μM oridonin.
3.By using Annexin V-FITC flow cytometry and Hoechst staining,we demonstrated that 1.4μM oridonin can inhibit As(Ⅲ)-induced cell apoptosis.
4.Oridonin can induce Nrf2 in a dose-dependent fashion,from 1.4 to 14μM. Doses more than 14μM appeared to be toxic,and induction of Nrf2 was decreased
5.By using both MTT and colony formation assays,we have demonstrated that pretreatment with1.4μM oridonin in UROtsa for 24h can significantly improved cell survival which were treated with As(Ⅲ) for 48 hr first.When using Nrf2-siRNA transfected UROtsa cells,oridonin lost its protection against As(Ⅲ) toxicity.
Together,these results demonstrate that a low dose of oridonin is able to protect cells from As(Ⅲ)-induced damage,as illustrated by increased cellular antioxidant ability, inhibited As(Ⅲ)-induced oxidative stress,increased cell survival in response to As(Ⅲ) and decreased cell apoptosis,moreover,the protection effect was attributed to the activation of Nrf2 by oridonin.
SectionⅡOridonin is an Nrf2 activator.
Studies have showed that the activity of ARE-luciferase reporter gene is closely related with Nrf2 protein level.So testing effect of oridonin on ARE-luciferase reporter gene's activity can show us its effect on Nrf2.
1.Using highthroughput stable ARE luciferase reporter system established in our laboratory,we found out that oridonin dose-dependently increased the activity of luciferase.
2.To confirm oridonin activation of Nrf2 using the high-throughput screening method,we also performed a dual luciferase reporter gene assay.Consistent with the data obtained from the high-throughput screening,oridonin induced the ARE-dependent luciferase activity in a dose-dependent manner Above results demonstrated that oridonin is an Nrf2activator.
SectionⅢOridonin activated the ARE-dependent response primarily through up-regulation of the Nrf2 protein level.
This section is about oridonin's effects on Nrf2 protein level and the mRNA level of Nrf2 downstream genes,the results are showed below:
1.We used the same cell lysates from the dual luciferase reporter gene assay for immunoblot analysis for detection of Nrf2,Keap1,andβ-actin.Oridonin enhanced the levels of Nrf2 protein in a dose-dependent manner,with the highest induction at 14μM.
2.We tested Nrf2 protein levels in response to high doses of oridonin.At doses more than 28μM,Nrf2 protein levels decreased in a dose-dependent manner, whereas the expression of Keap1 andβ-actin had no significant change.
3.We measured mRNA expression of Nrf2 and its target genes,NQO1 and HO-1, in response to oridonin using real-time RT-PCR.Nrf2 mRNA increased slightly in a dosedependent manner in response to oridonin,mRNA of NQO1 and HO-1 were induced significantly by oridonin in a dose-dependent manner.
These results demonstrate that oridonin is able to induce the Nrf2 signaling pathway mainly through upregulation of Nrf2 at the protein level.
SectionⅣOridonin blocked Nrf2 ubiquitination and enhanced Keap1 ubiquitination
tBHQ enhances the Nrf2 protein level by interfering with the Keap1-dependent ubiquitin conjugation process.Therefore,we tested the ability of oridonin in modulating Nrf2 ubiquitination.The results are showed below:
1.Oridonin suppressed Nrf2 ubiquitination in MDA-MB-231cells and UROtsa cells.
2.Oridonin treatment was also effective in enhancing ubiquitination of Keap1 MDA-MB-231cells and UROtsa cells.
3.We measured the half-life of Nrf2 in the absence or presence of oridonin. Half-life of the endogenous Nrf2 protein in MDA-MB-231 cells was 19 min, whereas treatment with oridonin increased the half-life to 51 min.The half life of Nrf2 in UROtsa was increased from 10 min in the untreated condition to 16 min in response to oridonin treatment.
Above results indicate that oridonin activates the Nrf2 pathway by inhibiting ubiquitination and degradation of Nrf2,leading to an increase in Nrf2 protein level and activation of the Nrf2-dependent response.
引文
1.Bagchi,D.;Bagchi,M.;Stohs,S.;Ray,S.D.;Sen,C.K.;Preuss,H.G.,Cellular protection with proanthocyanidins derived from grape seeds.Ann N Y Acad Sci 2002,957,260-70.
2.Bagchi,D.;Bagchi,M.;Stohs,S.J.;Das,D.K.;Ray,S.D.;Kuszynski,C.A.;Joshi,S.S.;Pruess,H.G.,Free radicals and grape seed proanthocyanidin extract:importance in human health and disease prevention.Toxicology 2000,148,(2-3),187-97.
3.Vinson,J.A.;Mandarano,M.A.;Shuta,D.L.;Bagchi,M.;Bagchi,D.,Beneficial effects of a novel IH636 grape seed proanthocyanidin extract and a niacin-bound chromium in a hamster atherosclerosis model.Mol Cell Biochem 2002,240,(1-2),99-103.
4.马亚兵;高海青;伊永亮.,葡萄籽原花青素降低动脉粥样硬化兔血清C反应蛋白水平.中国动脉硬化杂志 2004,12,(5),549-552.
5.Pataki,T.;Bak,I.;Kovacs,P.;Bagchi,D.;Das,D.K.;Tosaki,A.,Grape seed proanthocyanidins improved cardiac recovery during reperfusion after ischemia in isolated rat hearts.Am J Clin Nutr 2002,75,(5),894-9.
6.Peng,N.;Clark,J.T.;Prasain,J.;Kim,H.;White,C.R.;Wyss,J.M.,Antihypertensive and cognitive effects of grape polyphenols in estrogen-depleted,female,spontaneously hypertensive rats.Am J Physiol Regul Integr Comp Physiol 2005,289,(3),R771-5.
7.Shao,Z.H.;Becker,L.B.;Vanden Hoek,T.L.;Schumacker,P.T.;Li,C.Q.;Zhao,D.;Wojcik,K.;Anderson,T.;Qin,Y.;Dey,L.;Yuan,C.S.,Grape seed proanthocyanidin extract attenuates oxidant injury in cardiomyocytes.Pharmacol Res 2003,47,(6),463-9.
8.Kensler,T.W.;Chen,J.G.;Egner,P.A.;Fahey,J.W.;Jacobson,L.P.;Stephenson,K.K.;Ye,L.;Coady,J.L.;Wang,J.B.;Wu,Y.;Sun,Y.;Zhang,Q.N.;Zhang,B.C.;Zhu,Y.R.;Qian,G.S.;Carmella,S.G.;Hecht,S.S.;Benning, L.;Gange,S.J.;Groopman,J.D.;Talalay,P.,Effects of glucosinolate-rich broccoli sprouts on urinary levels of aflatoxin-DNA adducts and phenanthrene tetraols in a randomized clinical trial in He Zuo township,Qidong,People's Republic of China.Cancer Epidemiol Biomarkers Prev 2005,14,(11 Pt 1),2605-13.
9.E1-Alfy,A.T.;Ahmed,A.A.;Fatani,A.J.,Protective effect of red grape seeds proanthocyanidins against induction of diabetes by alloxan in rats.Pharmacol Res 2005,52,(3),264-70.
10.Ray,S.D.;Parikh,H.;Bagchi,D.,Proanthocyanidin exposure to B6C3F1 mice significantly attenuates dimethylnitrosamine-induced liver tumor induction and mortality by differentially modulating programmed and unprogrammed cell deaths.Mutat Res 2005,579,(1-2),81-106.
11.Roy,A.M.;Baliga,M.S.;Elmets,C.A.;Katiyar,S.K.,Grape seed proanthocyanidins induce apoptosis through p53,Bax,and caspase 3 pathways.Neoplasia 2005,7,(1),24-36.
12.Joshi,S.S.;Kuszynski,C.A.;Bagchi,D.,The cellular and molecular basis of health benefits of grape seed proanthocyanidin extract.Curr Pharm Biotechnol 2001,2,(2),187-200.
13.Sangeetha,P.;Balu,M.;Haripriya,D.;Panneerselvam,C.,Age associated changes in erythrocyte membrane surface charge:Modulatory role of grape seed proanthocyanidins.Exp Gerontol 2005,40,(10),820-8.
14.Sharma,S.D.;Katiyar,S.K.,Dietary grape-seed proanthocyanidin inhibition of ultraviolet B-induced immune suppression is associated with induction of IL-12.Carcinogenesis 2006,27,(1),95-102.
15.Yahara,N.;Tofani,I.;Maki,K.;Kojima,K.;Kojima,Y.;Kimura,M.,Mechanical assessment of effects of grape seed proanthocyanidins extract on tibial bone diaphysis in rats.J Musculoskelet Neuronal Interact 2005,5,(2),162-9.
16.Tseng,C.H.,An overview on peripheral vascular disease in blackfoot disease-hyperendemic villages in Taiwan.Angiology 2002,53,(5),529-37.
17.Khanna,S.;Roy,S.;Bagchi,D.;Bagchi,M.;Sen,C.K.,Upregulation of oxidant-induced VEGF expression in cultured keratinocytes by a grape seed proanthocyanidin extract.Free Radic Biol Med 2001,31,(1),38-42.
18.Khanna,S.;Venojarvi,M.;Roy,S.;Sharma,N.;Trikha,P.;Bagchi,D.;Bagchi,M.;Sen,C.K.,Dermal wound healing properties of redox-active grape seed proanthocyanidins.Free Radic Biol Med 2002,33,(8),1089-96.
19.Yamakoshi,J.;Saito,M.;Kataoka,S.;Kikuchi,M.,Safety evaluation of proanthocyanidin-rich extract from grape seeds.Food Chem Toxicol 2002,40,(5),599-607.
20.Bentivegna,S.S.;Whitney,K.M.,Subchronic 3-month oral toxicity study of grape seed and grape skin extracts.Food Chem Toxicol 2002,40,(12),1731-43.
21.Shao,Z.H.;Vanden Hoek,T.L.;Xie,J.;Wojcik,K.;Chan,K.C.;Li,C.Q.;Hamann,K.;Qin,Y.;Schumacker,P.T.;Becker,L.B.;Yuan,C.S.,Grape seed proanthocyanidins induce pro-oxidant toxicity in cardiomyocytes.Cardiovasc Toxicol 2003,3,(4),331-9.
22.Shao,Z.H.;Vanden Hoek,T.L.;Li,C.Q.;Schumacker,P.T.;Becker,L.B.;Chan,K.C.;Qin,Y.;Yin,J.J.;Yuan,c.S.,Synergistic effect of Scutellaria baicalensis and grape seed proanthocyanidins on scavenging reactive oxygen species in vitro.Am J Chin Med 2004,32,(1),89-95.
23.Corbe,C.;Boissin,J.P.;Siou,A.,[Light vision and chorioretinal circulation.Study of the effect of procyanidolic oligomers(Endotelon)].J Fr Ophtalmol 1988,11,(5),453-60.
24.Yamakoshi,J.;Sano,A.;Tokutake,S.;Saito,M.;Kikuchi,M.;Kubota,Y.;Kawachi,Y.;Otsuka,F.,Oral intake of proanthocyanidin-rich extract from grape seeds improves chloasma.Phytother Res 2004,18,(11),895-9.
25.Pieur,C.;Rigaud,J.;Cheynier,V.;Moutounet,M.,Oligomeric and polymeric procyanidins from grape seeds.Phytochemistry 1994,36,781-784.
26.de Vries,J.H.;Hollman,P.C.;Meyboom,S.;Buysman,M.N.;Zock,P.L.;van Staveren,W.A.;Katan,M.B.,Plasma concentrations and urinary excretion of the antioxidant flavonols quercetin and kaempferol as biomarkers for dietary intake..Am.J..Clin.Nutr.1998,68,(60 - 65).
27.Fan,P.;Lou,H.,Effects of polyphenols from grape seeds on oxidative damage to cellular DNA.Moll.Cell Biochem.2004,267,(1-2),67-74.
28.Budihardjo,I.;Oliver,H.;Lutter,M.;Luo,X.;Wang,X.,Biochemical pathways of caspase activation during apoptosis.Annu.Rev.Cell Dev.Biol.1999,15,269-90.
29.Griendling,K.K.;FitzGerald,G.A.,Oxidative stress and cardiovascular injury:Part Ⅱ:animal and human studies.Circulation 2003,108,(17),2034-40.
30.Bagchi,D.;Sen,C.K.;Ray,S.D.;Das,D.K.;Bagchi,M.;Preuss,H.G.;Vinson,J.A.,Molecular mechanisms of cardioprotection by a novel grape seed proanthocyanidin extract.Mutat.Res.2003,523-524,87-97.
31.Oldenburg,O.;Cohen,M.V.;Yellon,D.M.;Downey,J.M.,Mitochondrial K(ATP) channels:role in cardioprotection.Cardiovasc Res 2002,55,(3),429-37.
32.Tang,X.L.;Takano,H.;Rizvi,A.;Turrens,J.F.;Qiu,Y.;Wu,W.J.;Zhang,Q.;Bolli,R.,Oxidant species trigger late preconditioning against myocardial stunning in conscious rabbits.Am J Physiol Heart Circ Physiol 2002,282,(1),H281-91.
33.Kehrer,J.P.,Free radicals as mediators of tissue injury and disease.Crit Rev Toxicol 1993,23,(1),21-48.
34.Formigli,L.;Ibba-Manneschi,L.;Perna,A.M.;Nediani,C.;Liguori,P.;Tani,A.;Zecchi-Orlandini,S.,Ischemia--reperfusion-induced apoptosis and p53expression in the course of rat heterotopic heart transplantation.Microvasc Res 1998,56,(3),277-81.
35.Greenlund,L.J.;Deckwerth,T.L.;Johnson,E.M.,Jr.,Superoxide dismutase delays neuronal apoptosis:a role for reactive oxygen species in programmed neuronal death.Neuron 1995,14,(2),303-15.
36.Wattanapitayakul,S.K.;Bauer,J.A.,Oxidative pathways in cardiovascular disease:roles,mechanisms,and therapeutic implications.Pharmacol Ther 2001,89,(2),187-206.
37.Vollrath,V.;Wielandt,A.M.;Iruretagoyena,M.;Chianale,J.,Role of Nrf2 in the regulation of the Mrp2(ABCC2) gene.Biochem J 2006,395,(3),599-609.
38.Wakabayashi,N.;Dinkova-Kostova,A.T.;Holtzclaw,W.D.;Kang,M.I.;Kobayashi,A.;Yamamoto,M.;Kensler,T.W.;Talalay,P.,Protection against electrophile and oxidant stress by induction of the phase 2 response:fate of cysteines of the Keap1 sensor modified by inducers.Proc Natl Acad Sci U S A 2004,101,(7),2040-5.
39.Hsueh,Y.M.;Huang,Y.L.;Huang,C.C.;Wu,W.L.;Chen,H.M.;Yang,M.H.;Lue,L.C.;Chen,C.J.,Urinary levels of inorganic and organic arsenic metabolites among residents in an arseniasis-hyperendemic area in Taiwan.J Toxicol Environ Health A 1998,54,(6),431-44.
40.Orhan,B.,Doxorubicin cardiotoxicity:growing importance.J.Clin.Oncol.1999,17,(7),2294-6.
41.Silber,J.H.;Barber,G.,Doxorubicin-induced cardiotoxicity.N.Engl.J.Med.1995,333,(20),1359-60.
42.De Beer,E.L.;Bottone,A.E.;Voest,E.E.,Doxorubicin and mechanical performance of cardiac trabeculae after acute and chronic treatment:a review.Eur.J.Pharmacol.2001,415,(1),1-11.
43.Balogun,E.;Hoque,M.;Gong,P.;Killeen,E.;Green,C.J.;Foresti,R.;Alam,J.;Motterlini,R.,Curcumin activates the haem oxygenase-1 gene via regulation of Nrf2 and the antioxidant-responsive element.Biochem J 2003,371,(Pt 3),887-95.
44.Antonsson,B.;Martinou,J.C.,The Bcl-2 protein family.Exp.Cell Res.2000,256,(1),50-7.
45.Crompton,M.,Bax,Bid and the permeabilization of the mitochondrial outer membrane in apoptosis.Curr.Opin.Cell Biol.2000,12,(4),414-9.
46.Shihab,F.S.;Andoh,T.F.;Tanner,A.M.;Yi,H.;Bennett,W.M.,Expression of apoptosis regulatory genes in chronic cyclosporine nephrotoxicity favors apoptosis.Kidney Int.1999,56,(6),2147-59.
47.Wang,J.S.;Shen,X.;He,X.;Zhu,Y.R.;Zhang,B.C.;Wang,J.B.;Qian,G.S.; Kuang,S.Y.;Zarba,A.;Egner,P.A.;Jacobson,L.P.;Munoz,A.;Helzlsouer,K.J.;Groopman,J.D.;Kensler,T.W.,Protective alterations in phase 1 and 2metabolism of aflatoxin B1 by oltipraz in residents of Qidong,People's Republic of China.J Natl Cancer Inst 1999,91,(4),347-54.
48.Kumar,D.;Kirshenbaum,L.;Li,T.;Danelisen,I.;Singal,P.,Apoptosis in isolated adult cardiomyocytes exposed to adriamycin.Ann.N.Y.Acad.Sci.1999,874,156-68.
49.Kumagai,Y.;Sumi,D.,Arsenic:signal transduction,transcription factor,and biotransformation involved in cellular response and toxicity.Annu Rev Pharmacol Toxicol 2007,47,243-62.
50.Smith,A.H.;Lingas,E.O.;Rahman,M.,Contamination of drinking-water by arsenic in Bangladesh:a public health emergency.Bull World Health Organ 2000,78,(9),1093-103.
51.Tchounwou,P.B.;Patlolla,A.K.;Centeno,J.A.,Carcinogenic and systemic health effects associated with arsenic exposure--a critical review.Toxicol Pathol 2003,31,(6),575-88.
52.Smith,A.H.;Marshall,G.;Yuan,Y.;Ferreccio,C.;Liaw,J.;von Ehrenstein,O.;Steinmaus,C.;Bates,M.N.;Selvin,S.,Increased mortality from lung cancer and bronchiectasis in young adults after exposure to arsenic in utero and in early childhood.Environ Health Perspect 2006,114,(8),1293-6.
53.Steinmaus,C.;Moore,L.;Hopenhayn-Rich,C.;Biggs,M.L.;Smith,A.H.,Arsenic in drinking water and bladder cancer.Cancer Invest 2000,18,(2),174-82.
54.申家英;杨波.,冬凌草甲素对HL-60细胞的生长抑制作用及其对细胞端粒酶活性的调节.白血病·淋巴瘤 2003,12(4),224-226.
55.Ikezoe,T.;Yang,Y.;Bandobashi,K.;Saito,T.;Takemoto,S.;Machida,H.;Togitani,K.;Koeffler,H.P.;Taguchi,H.,Oridonin,a diterpenoid purified from Rabdosia rubescens,inhibits the proliferation of cells from lymphoid malignancies in association with blockade of the NF-kappa B signal pathways.Mol Cancer Ther 2005,4,(4),578-86.
56.吴婕;袁守军;杨德宣.,冬凌草甲素抑制BGC823细胞的生长及MMP22M MP29的表达解放军药学学报 2007,23,(5),3442-3471.
57.刘明月;范魁生;樊青霞.,冬凌草甲素诱导结肠癌细胞凋亡的实验研究.河南肿瘤学杂志 2004,17,(3),167-168.
58.Maher,J.M.;Cheng,X.;Slitt,A.L.;Dieter,M.Z.;Klaassen,C.D.,Induction of the multidrug resistance-associated protein family of transporters by chemical activators of receptor-mediated pathways in mouse liver.Drug Metab Dispos 2005,33,(7),956-62.
59.张守伟;刘加军,复方冬凌草甲素对肺癌SPCA21细胞的端粒酶活性的影响 临床肺科杂志 2005,8,(3),2042-2061.
60.朱国臣;肖大江;张亚男,冬凌草甲素对人鼻咽癌细胞CNE-2的抑制作用.时珍国医国药 2007,18,(4),804-805 of Nrf-2 to an electrophile responsive element.Biochem Biophys Res Commun 1999,261,(3),661-8.
68.McMahon,M.;Itoh,K.;Yamamoto,M.;Hayes,J.D.,Keap1-dependent proteasomal degradation of transcription factor Nrf2 contributes to the negative regulation of antioxidant response element-driven gene expression.J Biol Chem 2003,278,(24),21592-600.
69.Katoh,Y.;Itoh,K.;Yoshida,E.;Miyagishi,M.;Fukamizu,A.;Yamamoto,M.,Two domains of Nrf2 cooperatively bind CBP,a CREB binding protein,and synergistically activate transcription.Genes Cells 2001,6,(10),857-68.
70.Banning,A.;Deubel,S.;Kluth,D.;Zhou,Z.;Brigelius-Flohe,R.,The GI-GPx gene is a target for Nrf2.Mol Cell Biol 2005,25,(12),4914-23.
71.Ishii,T.;Itoh,K.;Takahashi,S.;Sato,H.;Yanagawa,T.;Katoh,Y.;Bannai,S.;Yamamoto,M.,Transcription factor Nrf2 coordinately regulates a group of oxidative stress-inducible genes in macrophages.J Biol Chem 2000,275,(21),16023-9.
72.Kim,Y.C.;Masutani,H.;Yamaguchi,Y.;Itoh,K.;Yamamoto,M.;Yodoi,J.,Hemin-induced activation of the thioredoxin gene by Nrf2.A differential regulation of the antioxidant responsive element by a switch of its binding factors.J Biol Chem 2001,276,(21),18399-406.
73.Sakurai,A.;Nishimoto,M.;Himeno,S.;Imura,N.;Tsujimoto,M.;Kunimoto,M.;Hara,S.,Transcriptional regulation of thioredoxin reductase 1 expression by cadmium in vascular endothelial cells:role of NF-E2-related factor-2.J Cell Physiol 2005,203,(3),529-37.
74.Yueh,M.F.;Tukey,R.H.,Nrf2-Keap1 signaling pathway regulates human UGT1A1 expression in vitro and in transgenic UGT1 mice.J Biol Chem 2007,282,(12),8749-58.
75.Ishii,T.;Yanagawa,T.,Stress-induced peroxiredoxins.Subcell Biochem 2007,44,375-84.
76.Li,X.;Zhang,D.;Hannink,M.;Beamer,L.J.,Crystal structure of the Kelch domain of human Keapl.J Biol Chem 2004,279,(52),54750-8.
77.Lo,S.C.;Li,X.;Henzl,M.T.;Beamer,L.J.;Hannink,M.,Structure of the Keap1:Nrf2 interface provides mechanistic insight into Nrf2 signaling.Embo J 2006,25,(15),3605-17.
78.Hong,F.;Sekhar,K.R.;Freeman,M.L.;Liebler,D.C.,Specific patterns of electrophile adduction trigger Keapl ubiquitination and Nrf2 activation.J Biol Chem 2005,280,(36),31768-75.
79.Eggler,A.L.;Luo,Y.;van Breemen,R.B.;Mesecar,A.D.,Identification of the highly reactive cysteine 151 in the chemopreventive agent-sensor Keap1protein is method-dependent.Chem Res Toxicol 2007,20,(12),1878-84.
80.Eggler,A.L.;Liu,G.;Pezzuto,J.M.;van Breemen,R.B.;Mesecar,A.D.,Modifying specific cysteines of the electrophile-sensing human Keap1 protein is insufficient to disrupt binding to the Nrf2 domain Neh2.Proc Natl Acad Sci USA 2005,102,(29),10070-5.
81.Zhang,D.D.;Hannink,M.,Distinct cysteine residues in Keap1 are required for Keap1-dependent ubiquitination of Nrf2 and for stabilization of Nrf2 by chemopreventive agents and oxidative stress.Mol Cell Biol 2003,23,(22),8137-51.
82.Dinkova-Kostova,A.T.;Holtzclaw,W.D.;Cole,R.N.;Itoh,K.;Wakabayashi,N.;Katoh,Y.;Yamamoto,M.;Talalay,P.,Direct evidence that sulfhydryl groups of Keapl are the sensors regulating induction of phase 2 enzymes that protect against carcinogens and oxidants.Proc Natl Acad Sci U S A 2002,99,(18),11908-13.
83.Levonen,A.L.;Landar,A.;Ramachandran,A.;Ceaser,E.K.;Dickinson,D.A.;Zanoni,G.;Morrow,J.D.;Darley-Usmar,V.M.,Cellular mechanisms of redox cell signalling:role of cysteine modification in controlling antioxidant defences in response to electrophilic lipid oxidation products.Biochem J 2004,378,(Pt 2),373-82.
84.Stewart,D.;Killeen,E.;Naquin,R.;Alam,S.;Alam,J.,Degradation of transcription factor Nrf2 via the ubiquitin-proteasome pathway and stabilization by cadmium.J Biol Chem 2003,278,(4),2396-402.
85.Cope,G.A.;Deshaies,R.J.,COP9 signalosome:a multifunctional regulator of SCF and other cullin-based ubiquitin ligases.Cell 2003,114,(6),663-71.
86.Hochstrasser,M.,Ubiquitin-dependent protein degradation.Annu Rev Genet 1996,30,405-39.
87.Pickart,C.M.,Mechanisms underlying ubiquitination.Annu Rev Biochem 2001,70,503-33.
88.Cullinan,S.B.;Gordan,J.D.;Jin,J.;Harper,J.W.;Diehl,J.A.,The Keapl-BTB protein is an adaptor that bridges Nrf2 to a Cul3-based E3 ligase:oxidative stress sensing by a Cul3-Keap1 ligase.Mol Cell Biol 2004,24,(19),8477-86.
89.Furukawa,M.;Xiong,Y.,BTB protein Keap1 targets antioxidant transcription factor Nrf2 for ubiquitination by the Cullin 3-Roc1 ligase.Mol Cell Biol 2005,25,(1),162-71.
90.Sun,Z.;Zhang,S.;Chan,J.Y.;Zhang,D.D.,Keap1 controls postinduction repression of the Nrf2-mediated antioxidant response by escorting nuclear export of Nrf2.Mol Cell Biol 2007,27,(18),6334-49.
91.Wang,X.J.;Sun,Z.;Chen,W.;Eblin,K.E.;Gandolfi,J.A.;Zhang,D.D.,Nrf2 protects human bladder urothelial cells from arsenite and monomethylarsonous acid toxicity.Toxicol Appl Pharmacol 2007,225,(2),206-13.
92.Rossman,T.G.,Mechanism of arsenic carcinogenesis:an integrated approach.Mutat Res 2003,533,(1-2),37-65.
93.Cohen,S.M.;Shirai,T.;Steineck,G.,Epidemiology and etiology of premalignant and malignant urothelial changes.Scand J Urol Nephrol Suppl 2000,(205),105-15.
94.Smith,K.R.;Klei,L.R.;Barchowsky,A.,Arsenite stimulates plasma membrane NADPH oxidase in vascular endothelial cells.Am J Physiol Lung Cell Mol Physiol 2001,280,(3),L442-9.
95.Scott,N.;Hatlelid,K.M.;MacKenzie,N.E.;Carter,D.E.,Reactions of arsenic(Ⅲ) and arsenic(Ⅴ) species with glutathione.Chem Res Toxicol 1993,6, (1),102-6.
96.Delnomdedieu,M.;Basti,M.M.;Otvos,J.D.;Thomas,D.J.,Reduction and binding of arsenate and dimethylarsinate by glutathione:a magnetic resonance study.Chem Biol Interact 1994,90,(2),139-55.
97.Kala,S.V.;Neely,M.W.;Kala,G.;Prater,C.I.;Atwood,D.W.;Rice,J.S.;Lieberman,M.W.,The MRP2/cMOAT transporter and arsenic-glutathione complex formation are required for biliary excretion of arsenic.J Biol Chem 2000,275,(43),33404-8.
98.Vernhet,L.;Seite,M.P.;Allain,N.;Guillouzo,A.;Fardel,O.,Arsenic induces expression of the multidrug resistance-associated protein 2(MRP2) gene in primary rat and human hepatocytes.J Pharmacol Exp Ther 2001,298,(1),234-9.
99.Leslie,E.M.;Haimeur,A.;Waalkes,M.P.,Arsenic transport by the human multidrug resistance protein 1(MRP1/ABCC1).Evidence that a tri-glutathione conjugate is required.J Biol Chem 2004,279,(31),32700-8.
100.Aposhian,H.V.;Gurzau,E.S.;Le,X.C.;Gurzau,A.;Healy,S.M.;Lu,X.;Ma,M.;Yip,L.;Zakharyan,R.A.;Maiorino,R.M.;Dart,R.C.;Tircus,M.G.;Gonzalez-Ramirez,D.;Morgan,D.L.;Avram,D.;Aposhian,M.M.,Occurrence of monomethylarsonous acid in urine of humans exposed to inorganic arsenic.Chem Res Toxicol 2000,13,(8),693-7.
101.Mandal,B.K.;Ogra,Y.;Suzuki,K.T.,Identification of dimethylarsinous and monomethylarsonous acids in human urine of the arsenic-affected areas in West Bengal,India.Chem Res Toxicol 2001,14,(4),371-8.
102.Petrick,J.S.;Ayala-Fierro,F.;Cullen,W.R.;Carter,D.E.;Vasken Aposhian,H.,Monomethylarsonous acid(MMA(Ⅲ)) is more toxic than arsenite in Chang human hepatocytes.Toxicol Appl Pharmacol 2000,163,(2),203-7.
103.Venugopal,R.;Jaiswal,A.K.,Nrf1 and Nrf2 positively and c-Fos and Fral negatively regulate the human antioxidant response element-mediated expression of NAD(P)H:quinone oxidoreductasel gene.Proc Natl Acad Sci U S A 1996,93,(25),14960-5.
104.Itoh,K.;Ishii,T.;Wakabayashi,N.;Yamamoto,M.,Regulatory mechanisms of cellular response to oxidative stress.Free Radic Res 1999,31,(4),319-24.
105.Wild,A.C.;Moinova,H.R.;Mulcahy,R.T.,Regulation of gamma-glutamylcysteine synthetase subunit gene expression by the transcription factor Nrf2.J Biol Chem 1999,274,(47),33627-36.
106.McMahon,M.;Itoh,K.;Yamamoto,M.;Chanas,S.A.;Henderson,C.J.;McLellan,L.I.;Wolf,C.R.;Cavin,C.;Hayes,J.D.,The Cap'n'Collar basic leucine zipper transcription factor Nrf2(NF-E2 p45-related factor 2) controls both constitutive and inducible expression of intestinal detoxification and glutathione biosynthetic enzymes.Cancer Res 2001,61,(8),3299-307.
107.Jaiswal,A.K.,Nrf2 signaling in coordinated activation of antioxidant gene expression.Free Radic Biol Med 2004,36,(10),1199-207.
108.Kobayashi,A.;Ohta,T.;Yamamoto,M.,Unique function of the Nrf2-Keapl pathway in the inducible expression of antioxidant and detoxifying enzymes.Methods Enzymol 2004,378,273-86.
109.Motohashi,H.;Yamamoto,M.,Nrf2-Keapl defines a physiologically important stress response mechanism.Trends Mol Med 2004,10,(11),549-57.
110.Cho,H.Y.;Reddy,S.P.;Kleeberger,S.R.,Nrf2 defends the lung from oxidative stress.Antioxid Redox Signal 2006,8,(1-2),76-87.
111.Zhang,D.D.;Lo,S.C.;Cross,J.V.;Templeton,D.J.;Hannink,M.,Keapl is a redox-regulated substrate adaptor protein for a Cul3-dependent ubiquitin ligase complex.Mol Cell Biol 2004,24,(24),10941-53.
112.Wang,X.J.;Sun,Z.;Chen,W.;Li,Y.;Villeneuve,N.F.;Zhang,D.D.,Activation of Nrf2 by arsenite and monomethylarsonous acid is independent of Keap1-C151:enhanced Keap1-Cu13 interaction.Toxicol Appl Pharmacol 2008,230,(3),383-9.
113.Kwak,M.K.;Itoh,K.;Yamamoto,M.;Sutter,T.R.;Kensler,T.W.,Role of transcription factor Nrf2 in the induction of hepatic phase 2 and antioxidative enzymes in vivo by the cancer chemoprotective agent,3H-1,2-dimethiole-3-thione.Mol Med 2001,7,(2),135-45.
114.Ramos-Gomez,M.;Kwak,M.K.;Dolan,P.M.;Itoh,K.;Yamamoto,M.;Talalay,P.;Kensler,T.W.,Sensitivity to carcinogenesis is increased and chemoprotective efficacy of enzyme inducers is lost in nrf2 transcription factor-deficient mice.Proc Natl Acad Sci USA 2001,98,(6),3410-5.
115.Iida,K.;Itoh,K.;Kumagai,Y.;Oyasu,R.;Hattori,K.;Kawai,K.;Shimazui,T.;Akaza,H.;Yamamoto,M.,Nrf2 is essential for the chemopreventive efficacy of oltipraz against urinary bladder carcinogenesis.Cancer Res 2004,64,(18),6424-31.
116.Umemura,T.;Kuroiwa,Y.;Kitamura,Y.;Ishii,Y.;Kanki,K.;Kodama,Y.;Itoh,K.;Yamamoto,M.;Nishikawa,A.;Hirose,M.,A crucial role of Nrf2 in in vivo defense against oxidative damage by an environmental pollutant,pentachlorophenol.Toxicol Sci 2006,90,(1),111-9.
117.Rangasamy,T.;Guo,J.;Mitzner,W.A.;Roman,J.;Singh,A.;Fryer,A.D.;Yamamoto,M.;Kensler,T.W.;Tuder,R.M.;Georas,S.N.;Biswal,S.,Disruption of Nrf2 enhances susceptibility to severe airway inflammation and asthma in mice.J Exp Med 2005,202,(1),47-59.
118.Iizuka,T.;Ishii,Y.;Itoh,K.;Kiwamoto,T.;Kimura,T.;Matsuno,Y.;Morishima,Y.;Hegab,A.E.;Homma,S.;Nomura,A.;Sakamoto,T.;Shimura,M.;Yoshida,A.;Yamamoto,M.;Sekizawa,K.,Nrf2-deficient mice are highly susceptible to cigarette smoke-induced emphysema.Genes Cells 2005,10,(12),1113-25.
119.Aoki,Y.;Sato,H.;Nishimura,N.;Takahashi,S.;Itoh,K.;Yamamoto,M.,Accelerated DNA adduct formation in the lung of the Nrf2 knockout mouse exposed to diesel exhaust.Toxicol Appl Pharmacol 2001,173,(3),154-60.
120.Enomoto,A.;Itoh,K.;Nagayoshi,E.;Haruta,J.;Kimura,T.;O'Connor,T.;Harada,T.;Yamamoto,M.,High sensitivity of Nrf2 knockout mice to acetaminophen hepatotoxicity associated with decreased expression of ARE-regulated drug metabolizing enzymes and antioxidant genes.Toxicol Sci 2001,59,(1),169-77.
121.Surh,Y.J.,Cancer chemoprevention with dietary phytochemicals.Nat Rev Cancer 2003,3,(10),768-80.
122.Lee,J.S.;Surh,Y.J.,Nrf2 as a novel molecular target for chemoprevention.Cancer Lett 2005,224,(2),171-84.
123.Zhang,Y.;Kensler,T.W.;Cho,C.G.;Posner,G.H.;Talalay,P.,Anticarcinogenic activities of sulforaphane and structurally related synthetic norbomyl isothiocyanates.Proc Natl Acad Sci USA 1994,91,(8),3147-50.
124.Suganuma,M.;Okabe,S.;Sueoka,N.;Sueoka,E.;Matsuyama,S.;Imai,K.;Nakachi,K.;Fujiki,H.,Green tea and cancer chemoprevention.Mutat Res 1999,428,(1-2),339-44.
125.Orsolic,N.;Sver,L.;Terzic,S.;Tadic,Z.;Basic,I.,Inhibitory effect of water-soluble derivative of propolis and its polyphenolic compounds on tumor growth and metastasizing ability:a possible mode of antitumor action.Nutr Cancer 2003,47,(2),156-63.
126.Shen,G.;Xu,C.;Hu,R.;Jain,M.R.;Nair,S.;Lin,W.;Yang,C.S.;Chan,J.Y.;Kong,A.N.,Comparison of(-)-epigallocatechin-3-gallate elicited liver and small intestine gene expression profiles between C57BL/6J mice and C57BL/6J/Nrf2(-/-) mice.Pharm Res 2005,22,(11),1805-20.
127.Kensler,T.W.;Curphey,T.J.;Maxiutenko,Y.;Roebuck,B.D.,Chemoprotection by organosulfur inducers of phase 2 enzymes:dithiolethiones and dithiins.Drug Metabol Drug Interact 2000,17,(1-4),3-22.
128.Pae,H.O.;Choi,B.M.;Oh,G.S.;Lee,M.S.;Ryu,D.G.;Rhew,H.Y.;Kim,Y.M.;Chung,H.T.,Roles of heme oxygenase-1 in the antiproliferative and antiapoptotic effects of nitric oxide on Jurkat T cells.Mol Pharmacol 2004,66,(1),122-8.
129.Garg,R.;Gupta,S.;Maru,G.B.,Dietary curcumin modulates transcriptional regulators of phase Ⅰ and phase Ⅱ enzymes in benzo[a]pyrene-treated mice:mechanism of its anti-initiating action.Carcinogenesis 2008,29,(5),1022-32.
130.Na,H.K.;Surh,Y.J.,Modulation of Nrf2-mediated antioxidant and detoxifying enzyme induction by the green tea polyphenol EGCG.Food Chem Toxicol 2008,46,(4),1271-8.
131.Kode,A.;Rajendrasozhan,S.;Caito,S.;Yang,S.R.;Megson,I.L.;Rahman,I.,Resveratrol induces glutathione synthesis by activation of Nrf2 and protects against cigarette smoke-mediated oxidative stress in human lung epithelial cells.Am J Physiol Lung Cell Mol Physiol 2008,294,(3),L478-88.
132.Chen,C.Y.;Jang,J.H.;Li,M.H.;Surh,Y.J.,Resveratrol upregulates heme oxygenase-1 expression via activation of NF-E2-related factor 2 in PC12 cells.Biochem Biophys Res Commun 2005,331,(4),993-1000.
133.Higgins,L.G.;Cavin,C.;Itoh,K.;Yamamoto,M.;Hayes,J.D.,Induction of cancer chemopreventive enzymes by coffee is mediated by transcription factor Nrf2.Evidence that the coffee-specific diterpenes cafestol and kahweol confer protection against acrolein.Toxicol Appl Pharmacol 2008,226,(3),328-37.
134.Cavin,C.;Holzhaeuser,D.;Scharf,G.;Constable,A.;Huber,W.W.;Schilter,B.,Cafestol and kahweol,two coffee specific diterpenes with anticarcinogenic activity.Food Chem Toxicol 2002,40,(8),1155-63.
135.Ben-Dor,A.;Steiner,M.;Gheber,L.;Danilenko,M.;Dubi,N.;Linnewiel,K.;Zick,A.;Sharoni,Y.;Levy,J.,Carotenoids activate the antioxidant response element transcription system.Mol Cancer Ther 2005,4,(1),177-86.
136.Shinkai,Y.;Sumi,D.;Fukami,I.;Ishii,T.;Kumagai,Y.,Sulforaphane,an activator of Nrf2,suppresses cellular accumulation of arsenic and its cytotoxicity in primary mouse hepatocytes.FEBS Lett 2006,580,(7),1771-4.
137.Hayashi,A.;Suzuki,H.;Itoh,K.;Yamamoto,M.;Sugiyama,Y.,Transcription factor Nrf2 is required for the constitutive and inducible expression of multidrug resistance-associated protein 1 in mouse embryo fibroblasts.Biochem Biophys Res Commun 2003,310,(3),824-9.
138.Ohtsubo,T.;Kamada,S.;Mikami,T.;Murakami,H.;Tsujimoto,Y.,Identification of NRF2,a member of the NF-E2 family of transcription factors,as a substrate for caspase-3(-like) proteases.Cell Death Differ 1999,6,(9),865-72.
2.Bagchi,D.;Bagchi,M.;Stohs,S.J.;Das,D.K.;Ray,S.D.;Kuszynski,C.A.;Joshi,S.S.;Pruess,H.G.,Free radicals and grape seed proanthocyanidin extract:importance in human health and disease prevention.Toxicology 2000,148,(2-3),187-97.
3.Vinson,J.A.;Mandarano,M.A.;Shuta,D.L.;Bagchi,M.;Bagchi,D.,Beneficial effects of a novel IH636 grape seed proanthocyanidin extract and a niacin-bound chromium in a hamster atherosclerosis model.Mol Cell Biochem 2002,240,(1-2),99-103.
4.马亚兵;高海青;伊永亮.,葡萄籽原花青素降低动脉粥样硬化兔血清C反应蛋白水平.中国动脉硬化杂志 2004,12,(5),549-552.
5.Pataki,T.;Bak,I.;Kovacs,P.;Bagchi,D.;Das,D.K.;Tosaki,A.,Grape seed proanthocyanidins improved cardiac recovery during reperfusion after ischemia in isolated rat hearts.Am J Clin Nutr 2002,75,(5),894-9.
6.Peng,N.;Clark,J.T.;Prasain,J.;Kim,H.;White,C.R.;Wyss,J.M.,Antihypertensive and cognitive effects of grape polyphenols in estrogen-depleted,female,spontaneously hypertensive rats.Am J Physiol Regul Integr Comp Physiol 2005,289,(3),R771-5.
7.Shao,Z.H.;Becker,L.B.;Vanden Hoek,T.L.;Schumacker,P.T.;Li,C.Q.;Zhao,D.;Wojcik,K.;Anderson,T.;Qin,Y.;Dey,L.;Yuan,C.S.,Grape seed proanthocyanidin extract attenuates oxidant injury in cardiomyocytes.Pharmacol Res 2003,47,(6),463-9.
8.Kensler,T.W.;Chen,J.G.;Egner,P.A.;Fahey,J.W.;Jacobson,L.P.;Stephenson,K.K.;Ye,L.;Coady,J.L.;Wang,J.B.;Wu,Y.;Sun,Y.;Zhang,Q.N.;Zhang,B.C.;Zhu,Y.R.;Qian,G.S.;Carmella,S.G.;Hecht,S.S.;Benning, L.;Gange,S.J.;Groopman,J.D.;Talalay,P.,Effects of glucosinolate-rich broccoli sprouts on urinary levels of aflatoxin-DNA adducts and phenanthrene tetraols in a randomized clinical trial in He Zuo township,Qidong,People's Republic of China.Cancer Epidemiol Biomarkers Prev 2005,14,(11 Pt 1),2605-13.
9.E1-Alfy,A.T.;Ahmed,A.A.;Fatani,A.J.,Protective effect of red grape seeds proanthocyanidins against induction of diabetes by alloxan in rats.Pharmacol Res 2005,52,(3),264-70.
10.Ray,S.D.;Parikh,H.;Bagchi,D.,Proanthocyanidin exposure to B6C3F1 mice significantly attenuates dimethylnitrosamine-induced liver tumor induction and mortality by differentially modulating programmed and unprogrammed cell deaths.Mutat Res 2005,579,(1-2),81-106.
11.Roy,A.M.;Baliga,M.S.;Elmets,C.A.;Katiyar,S.K.,Grape seed proanthocyanidins induce apoptosis through p53,Bax,and caspase 3 pathways.Neoplasia 2005,7,(1),24-36.
12.Joshi,S.S.;Kuszynski,C.A.;Bagchi,D.,The cellular and molecular basis of health benefits of grape seed proanthocyanidin extract.Curr Pharm Biotechnol 2001,2,(2),187-200.
13.Sangeetha,P.;Balu,M.;Haripriya,D.;Panneerselvam,C.,Age associated changes in erythrocyte membrane surface charge:Modulatory role of grape seed proanthocyanidins.Exp Gerontol 2005,40,(10),820-8.
14.Sharma,S.D.;Katiyar,S.K.,Dietary grape-seed proanthocyanidin inhibition of ultraviolet B-induced immune suppression is associated with induction of IL-12.Carcinogenesis 2006,27,(1),95-102.
15.Yahara,N.;Tofani,I.;Maki,K.;Kojima,K.;Kojima,Y.;Kimura,M.,Mechanical assessment of effects of grape seed proanthocyanidins extract on tibial bone diaphysis in rats.J Musculoskelet Neuronal Interact 2005,5,(2),162-9.
16.Tseng,C.H.,An overview on peripheral vascular disease in blackfoot disease-hyperendemic villages in Taiwan.Angiology 2002,53,(5),529-37.
17.Khanna,S.;Roy,S.;Bagchi,D.;Bagchi,M.;Sen,C.K.,Upregulation of oxidant-induced VEGF expression in cultured keratinocytes by a grape seed proanthocyanidin extract.Free Radic Biol Med 2001,31,(1),38-42.
18.Khanna,S.;Venojarvi,M.;Roy,S.;Sharma,N.;Trikha,P.;Bagchi,D.;Bagchi,M.;Sen,C.K.,Dermal wound healing properties of redox-active grape seed proanthocyanidins.Free Radic Biol Med 2002,33,(8),1089-96.
19.Yamakoshi,J.;Saito,M.;Kataoka,S.;Kikuchi,M.,Safety evaluation of proanthocyanidin-rich extract from grape seeds.Food Chem Toxicol 2002,40,(5),599-607.
20.Bentivegna,S.S.;Whitney,K.M.,Subchronic 3-month oral toxicity study of grape seed and grape skin extracts.Food Chem Toxicol 2002,40,(12),1731-43.
21.Shao,Z.H.;Vanden Hoek,T.L.;Xie,J.;Wojcik,K.;Chan,K.C.;Li,C.Q.;Hamann,K.;Qin,Y.;Schumacker,P.T.;Becker,L.B.;Yuan,C.S.,Grape seed proanthocyanidins induce pro-oxidant toxicity in cardiomyocytes.Cardiovasc Toxicol 2003,3,(4),331-9.
22.Shao,Z.H.;Vanden Hoek,T.L.;Li,C.Q.;Schumacker,P.T.;Becker,L.B.;Chan,K.C.;Qin,Y.;Yin,J.J.;Yuan,c.S.,Synergistic effect of Scutellaria baicalensis and grape seed proanthocyanidins on scavenging reactive oxygen species in vitro.Am J Chin Med 2004,32,(1),89-95.
23.Corbe,C.;Boissin,J.P.;Siou,A.,[Light vision and chorioretinal circulation.Study of the effect of procyanidolic oligomers(Endotelon)].J Fr Ophtalmol 1988,11,(5),453-60.
24.Yamakoshi,J.;Sano,A.;Tokutake,S.;Saito,M.;Kikuchi,M.;Kubota,Y.;Kawachi,Y.;Otsuka,F.,Oral intake of proanthocyanidin-rich extract from grape seeds improves chloasma.Phytother Res 2004,18,(11),895-9.
25.Pieur,C.;Rigaud,J.;Cheynier,V.;Moutounet,M.,Oligomeric and polymeric procyanidins from grape seeds.Phytochemistry 1994,36,781-784.
26.de Vries,J.H.;Hollman,P.C.;Meyboom,S.;Buysman,M.N.;Zock,P.L.;van Staveren,W.A.;Katan,M.B.,Plasma concentrations and urinary excretion of the antioxidant flavonols quercetin and kaempferol as biomarkers for dietary intake..Am.J..Clin.Nutr.1998,68,(60 - 65).
27.Fan,P.;Lou,H.,Effects of polyphenols from grape seeds on oxidative damage to cellular DNA.Moll.Cell Biochem.2004,267,(1-2),67-74.
28.Budihardjo,I.;Oliver,H.;Lutter,M.;Luo,X.;Wang,X.,Biochemical pathways of caspase activation during apoptosis.Annu.Rev.Cell Dev.Biol.1999,15,269-90.
29.Griendling,K.K.;FitzGerald,G.A.,Oxidative stress and cardiovascular injury:Part Ⅱ:animal and human studies.Circulation 2003,108,(17),2034-40.
30.Bagchi,D.;Sen,C.K.;Ray,S.D.;Das,D.K.;Bagchi,M.;Preuss,H.G.;Vinson,J.A.,Molecular mechanisms of cardioprotection by a novel grape seed proanthocyanidin extract.Mutat.Res.2003,523-524,87-97.
31.Oldenburg,O.;Cohen,M.V.;Yellon,D.M.;Downey,J.M.,Mitochondrial K(ATP) channels:role in cardioprotection.Cardiovasc Res 2002,55,(3),429-37.
32.Tang,X.L.;Takano,H.;Rizvi,A.;Turrens,J.F.;Qiu,Y.;Wu,W.J.;Zhang,Q.;Bolli,R.,Oxidant species trigger late preconditioning against myocardial stunning in conscious rabbits.Am J Physiol Heart Circ Physiol 2002,282,(1),H281-91.
33.Kehrer,J.P.,Free radicals as mediators of tissue injury and disease.Crit Rev Toxicol 1993,23,(1),21-48.
34.Formigli,L.;Ibba-Manneschi,L.;Perna,A.M.;Nediani,C.;Liguori,P.;Tani,A.;Zecchi-Orlandini,S.,Ischemia--reperfusion-induced apoptosis and p53expression in the course of rat heterotopic heart transplantation.Microvasc Res 1998,56,(3),277-81.
35.Greenlund,L.J.;Deckwerth,T.L.;Johnson,E.M.,Jr.,Superoxide dismutase delays neuronal apoptosis:a role for reactive oxygen species in programmed neuronal death.Neuron 1995,14,(2),303-15.
36.Wattanapitayakul,S.K.;Bauer,J.A.,Oxidative pathways in cardiovascular disease:roles,mechanisms,and therapeutic implications.Pharmacol Ther 2001,89,(2),187-206.
37.Vollrath,V.;Wielandt,A.M.;Iruretagoyena,M.;Chianale,J.,Role of Nrf2 in the regulation of the Mrp2(ABCC2) gene.Biochem J 2006,395,(3),599-609.
38.Wakabayashi,N.;Dinkova-Kostova,A.T.;Holtzclaw,W.D.;Kang,M.I.;Kobayashi,A.;Yamamoto,M.;Kensler,T.W.;Talalay,P.,Protection against electrophile and oxidant stress by induction of the phase 2 response:fate of cysteines of the Keap1 sensor modified by inducers.Proc Natl Acad Sci U S A 2004,101,(7),2040-5.
39.Hsueh,Y.M.;Huang,Y.L.;Huang,C.C.;Wu,W.L.;Chen,H.M.;Yang,M.H.;Lue,L.C.;Chen,C.J.,Urinary levels of inorganic and organic arsenic metabolites among residents in an arseniasis-hyperendemic area in Taiwan.J Toxicol Environ Health A 1998,54,(6),431-44.
40.Orhan,B.,Doxorubicin cardiotoxicity:growing importance.J.Clin.Oncol.1999,17,(7),2294-6.
41.Silber,J.H.;Barber,G.,Doxorubicin-induced cardiotoxicity.N.Engl.J.Med.1995,333,(20),1359-60.
42.De Beer,E.L.;Bottone,A.E.;Voest,E.E.,Doxorubicin and mechanical performance of cardiac trabeculae after acute and chronic treatment:a review.Eur.J.Pharmacol.2001,415,(1),1-11.
43.Balogun,E.;Hoque,M.;Gong,P.;Killeen,E.;Green,C.J.;Foresti,R.;Alam,J.;Motterlini,R.,Curcumin activates the haem oxygenase-1 gene via regulation of Nrf2 and the antioxidant-responsive element.Biochem J 2003,371,(Pt 3),887-95.
44.Antonsson,B.;Martinou,J.C.,The Bcl-2 protein family.Exp.Cell Res.2000,256,(1),50-7.
45.Crompton,M.,Bax,Bid and the permeabilization of the mitochondrial outer membrane in apoptosis.Curr.Opin.Cell Biol.2000,12,(4),414-9.
46.Shihab,F.S.;Andoh,T.F.;Tanner,A.M.;Yi,H.;Bennett,W.M.,Expression of apoptosis regulatory genes in chronic cyclosporine nephrotoxicity favors apoptosis.Kidney Int.1999,56,(6),2147-59.
47.Wang,J.S.;Shen,X.;He,X.;Zhu,Y.R.;Zhang,B.C.;Wang,J.B.;Qian,G.S.; Kuang,S.Y.;Zarba,A.;Egner,P.A.;Jacobson,L.P.;Munoz,A.;Helzlsouer,K.J.;Groopman,J.D.;Kensler,T.W.,Protective alterations in phase 1 and 2metabolism of aflatoxin B1 by oltipraz in residents of Qidong,People's Republic of China.J Natl Cancer Inst 1999,91,(4),347-54.
48.Kumar,D.;Kirshenbaum,L.;Li,T.;Danelisen,I.;Singal,P.,Apoptosis in isolated adult cardiomyocytes exposed to adriamycin.Ann.N.Y.Acad.Sci.1999,874,156-68.
49.Kumagai,Y.;Sumi,D.,Arsenic:signal transduction,transcription factor,and biotransformation involved in cellular response and toxicity.Annu Rev Pharmacol Toxicol 2007,47,243-62.
50.Smith,A.H.;Lingas,E.O.;Rahman,M.,Contamination of drinking-water by arsenic in Bangladesh:a public health emergency.Bull World Health Organ 2000,78,(9),1093-103.
51.Tchounwou,P.B.;Patlolla,A.K.;Centeno,J.A.,Carcinogenic and systemic health effects associated with arsenic exposure--a critical review.Toxicol Pathol 2003,31,(6),575-88.
52.Smith,A.H.;Marshall,G.;Yuan,Y.;Ferreccio,C.;Liaw,J.;von Ehrenstein,O.;Steinmaus,C.;Bates,M.N.;Selvin,S.,Increased mortality from lung cancer and bronchiectasis in young adults after exposure to arsenic in utero and in early childhood.Environ Health Perspect 2006,114,(8),1293-6.
53.Steinmaus,C.;Moore,L.;Hopenhayn-Rich,C.;Biggs,M.L.;Smith,A.H.,Arsenic in drinking water and bladder cancer.Cancer Invest 2000,18,(2),174-82.
54.申家英;杨波.,冬凌草甲素对HL-60细胞的生长抑制作用及其对细胞端粒酶活性的调节.白血病·淋巴瘤 2003,12(4),224-226.
55.Ikezoe,T.;Yang,Y.;Bandobashi,K.;Saito,T.;Takemoto,S.;Machida,H.;Togitani,K.;Koeffler,H.P.;Taguchi,H.,Oridonin,a diterpenoid purified from Rabdosia rubescens,inhibits the proliferation of cells from lymphoid malignancies in association with blockade of the NF-kappa B signal pathways.Mol Cancer Ther 2005,4,(4),578-86.
56.吴婕;袁守军;杨德宣.,冬凌草甲素抑制BGC823细胞的生长及MMP22M MP29的表达解放军药学学报 2007,23,(5),3442-3471.
57.刘明月;范魁生;樊青霞.,冬凌草甲素诱导结肠癌细胞凋亡的实验研究.河南肿瘤学杂志 2004,17,(3),167-168.
58.Maher,J.M.;Cheng,X.;Slitt,A.L.;Dieter,M.Z.;Klaassen,C.D.,Induction of the multidrug resistance-associated protein family of transporters by chemical activators of receptor-mediated pathways in mouse liver.Drug Metab Dispos 2005,33,(7),956-62.
59.张守伟;刘加军,复方冬凌草甲素对肺癌SPCA21细胞的端粒酶活性的影响 临床肺科杂志 2005,8,(3),2042-2061.
60.朱国臣;肖大江;张亚男,冬凌草甲素对人鼻咽癌细胞CNE-2的抑制作用.时珍国医国药 2007,18,(4),804-805 of Nrf-2 to an electrophile responsive element.Biochem Biophys Res Commun 1999,261,(3),661-8.
68.McMahon,M.;Itoh,K.;Yamamoto,M.;Hayes,J.D.,Keap1-dependent proteasomal degradation of transcription factor Nrf2 contributes to the negative regulation of antioxidant response element-driven gene expression.J Biol Chem 2003,278,(24),21592-600.
69.Katoh,Y.;Itoh,K.;Yoshida,E.;Miyagishi,M.;Fukamizu,A.;Yamamoto,M.,Two domains of Nrf2 cooperatively bind CBP,a CREB binding protein,and synergistically activate transcription.Genes Cells 2001,6,(10),857-68.
70.Banning,A.;Deubel,S.;Kluth,D.;Zhou,Z.;Brigelius-Flohe,R.,The GI-GPx gene is a target for Nrf2.Mol Cell Biol 2005,25,(12),4914-23.
71.Ishii,T.;Itoh,K.;Takahashi,S.;Sato,H.;Yanagawa,T.;Katoh,Y.;Bannai,S.;Yamamoto,M.,Transcription factor Nrf2 coordinately regulates a group of oxidative stress-inducible genes in macrophages.J Biol Chem 2000,275,(21),16023-9.
72.Kim,Y.C.;Masutani,H.;Yamaguchi,Y.;Itoh,K.;Yamamoto,M.;Yodoi,J.,Hemin-induced activation of the thioredoxin gene by Nrf2.A differential regulation of the antioxidant responsive element by a switch of its binding factors.J Biol Chem 2001,276,(21),18399-406.
73.Sakurai,A.;Nishimoto,M.;Himeno,S.;Imura,N.;Tsujimoto,M.;Kunimoto,M.;Hara,S.,Transcriptional regulation of thioredoxin reductase 1 expression by cadmium in vascular endothelial cells:role of NF-E2-related factor-2.J Cell Physiol 2005,203,(3),529-37.
74.Yueh,M.F.;Tukey,R.H.,Nrf2-Keap1 signaling pathway regulates human UGT1A1 expression in vitro and in transgenic UGT1 mice.J Biol Chem 2007,282,(12),8749-58.
75.Ishii,T.;Yanagawa,T.,Stress-induced peroxiredoxins.Subcell Biochem 2007,44,375-84.
76.Li,X.;Zhang,D.;Hannink,M.;Beamer,L.J.,Crystal structure of the Kelch domain of human Keapl.J Biol Chem 2004,279,(52),54750-8.
77.Lo,S.C.;Li,X.;Henzl,M.T.;Beamer,L.J.;Hannink,M.,Structure of the Keap1:Nrf2 interface provides mechanistic insight into Nrf2 signaling.Embo J 2006,25,(15),3605-17.
78.Hong,F.;Sekhar,K.R.;Freeman,M.L.;Liebler,D.C.,Specific patterns of electrophile adduction trigger Keapl ubiquitination and Nrf2 activation.J Biol Chem 2005,280,(36),31768-75.
79.Eggler,A.L.;Luo,Y.;van Breemen,R.B.;Mesecar,A.D.,Identification of the highly reactive cysteine 151 in the chemopreventive agent-sensor Keap1protein is method-dependent.Chem Res Toxicol 2007,20,(12),1878-84.
80.Eggler,A.L.;Liu,G.;Pezzuto,J.M.;van Breemen,R.B.;Mesecar,A.D.,Modifying specific cysteines of the electrophile-sensing human Keap1 protein is insufficient to disrupt binding to the Nrf2 domain Neh2.Proc Natl Acad Sci USA 2005,102,(29),10070-5.
81.Zhang,D.D.;Hannink,M.,Distinct cysteine residues in Keap1 are required for Keap1-dependent ubiquitination of Nrf2 and for stabilization of Nrf2 by chemopreventive agents and oxidative stress.Mol Cell Biol 2003,23,(22),8137-51.
82.Dinkova-Kostova,A.T.;Holtzclaw,W.D.;Cole,R.N.;Itoh,K.;Wakabayashi,N.;Katoh,Y.;Yamamoto,M.;Talalay,P.,Direct evidence that sulfhydryl groups of Keapl are the sensors regulating induction of phase 2 enzymes that protect against carcinogens and oxidants.Proc Natl Acad Sci U S A 2002,99,(18),11908-13.
83.Levonen,A.L.;Landar,A.;Ramachandran,A.;Ceaser,E.K.;Dickinson,D.A.;Zanoni,G.;Morrow,J.D.;Darley-Usmar,V.M.,Cellular mechanisms of redox cell signalling:role of cysteine modification in controlling antioxidant defences in response to electrophilic lipid oxidation products.Biochem J 2004,378,(Pt 2),373-82.
84.Stewart,D.;Killeen,E.;Naquin,R.;Alam,S.;Alam,J.,Degradation of transcription factor Nrf2 via the ubiquitin-proteasome pathway and stabilization by cadmium.J Biol Chem 2003,278,(4),2396-402.
85.Cope,G.A.;Deshaies,R.J.,COP9 signalosome:a multifunctional regulator of SCF and other cullin-based ubiquitin ligases.Cell 2003,114,(6),663-71.
86.Hochstrasser,M.,Ubiquitin-dependent protein degradation.Annu Rev Genet 1996,30,405-39.
87.Pickart,C.M.,Mechanisms underlying ubiquitination.Annu Rev Biochem 2001,70,503-33.
88.Cullinan,S.B.;Gordan,J.D.;Jin,J.;Harper,J.W.;Diehl,J.A.,The Keapl-BTB protein is an adaptor that bridges Nrf2 to a Cul3-based E3 ligase:oxidative stress sensing by a Cul3-Keap1 ligase.Mol Cell Biol 2004,24,(19),8477-86.
89.Furukawa,M.;Xiong,Y.,BTB protein Keap1 targets antioxidant transcription factor Nrf2 for ubiquitination by the Cullin 3-Roc1 ligase.Mol Cell Biol 2005,25,(1),162-71.
90.Sun,Z.;Zhang,S.;Chan,J.Y.;Zhang,D.D.,Keap1 controls postinduction repression of the Nrf2-mediated antioxidant response by escorting nuclear export of Nrf2.Mol Cell Biol 2007,27,(18),6334-49.
91.Wang,X.J.;Sun,Z.;Chen,W.;Eblin,K.E.;Gandolfi,J.A.;Zhang,D.D.,Nrf2 protects human bladder urothelial cells from arsenite and monomethylarsonous acid toxicity.Toxicol Appl Pharmacol 2007,225,(2),206-13.
92.Rossman,T.G.,Mechanism of arsenic carcinogenesis:an integrated approach.Mutat Res 2003,533,(1-2),37-65.
93.Cohen,S.M.;Shirai,T.;Steineck,G.,Epidemiology and etiology of premalignant and malignant urothelial changes.Scand J Urol Nephrol Suppl 2000,(205),105-15.
94.Smith,K.R.;Klei,L.R.;Barchowsky,A.,Arsenite stimulates plasma membrane NADPH oxidase in vascular endothelial cells.Am J Physiol Lung Cell Mol Physiol 2001,280,(3),L442-9.
95.Scott,N.;Hatlelid,K.M.;MacKenzie,N.E.;Carter,D.E.,Reactions of arsenic(Ⅲ) and arsenic(Ⅴ) species with glutathione.Chem Res Toxicol 1993,6, (1),102-6.
96.Delnomdedieu,M.;Basti,M.M.;Otvos,J.D.;Thomas,D.J.,Reduction and binding of arsenate and dimethylarsinate by glutathione:a magnetic resonance study.Chem Biol Interact 1994,90,(2),139-55.
97.Kala,S.V.;Neely,M.W.;Kala,G.;Prater,C.I.;Atwood,D.W.;Rice,J.S.;Lieberman,M.W.,The MRP2/cMOAT transporter and arsenic-glutathione complex formation are required for biliary excretion of arsenic.J Biol Chem 2000,275,(43),33404-8.
98.Vernhet,L.;Seite,M.P.;Allain,N.;Guillouzo,A.;Fardel,O.,Arsenic induces expression of the multidrug resistance-associated protein 2(MRP2) gene in primary rat and human hepatocytes.J Pharmacol Exp Ther 2001,298,(1),234-9.
99.Leslie,E.M.;Haimeur,A.;Waalkes,M.P.,Arsenic transport by the human multidrug resistance protein 1(MRP1/ABCC1).Evidence that a tri-glutathione conjugate is required.J Biol Chem 2004,279,(31),32700-8.
100.Aposhian,H.V.;Gurzau,E.S.;Le,X.C.;Gurzau,A.;Healy,S.M.;Lu,X.;Ma,M.;Yip,L.;Zakharyan,R.A.;Maiorino,R.M.;Dart,R.C.;Tircus,M.G.;Gonzalez-Ramirez,D.;Morgan,D.L.;Avram,D.;Aposhian,M.M.,Occurrence of monomethylarsonous acid in urine of humans exposed to inorganic arsenic.Chem Res Toxicol 2000,13,(8),693-7.
101.Mandal,B.K.;Ogra,Y.;Suzuki,K.T.,Identification of dimethylarsinous and monomethylarsonous acids in human urine of the arsenic-affected areas in West Bengal,India.Chem Res Toxicol 2001,14,(4),371-8.
102.Petrick,J.S.;Ayala-Fierro,F.;Cullen,W.R.;Carter,D.E.;Vasken Aposhian,H.,Monomethylarsonous acid(MMA(Ⅲ)) is more toxic than arsenite in Chang human hepatocytes.Toxicol Appl Pharmacol 2000,163,(2),203-7.
103.Venugopal,R.;Jaiswal,A.K.,Nrf1 and Nrf2 positively and c-Fos and Fral negatively regulate the human antioxidant response element-mediated expression of NAD(P)H:quinone oxidoreductasel gene.Proc Natl Acad Sci U S A 1996,93,(25),14960-5.
104.Itoh,K.;Ishii,T.;Wakabayashi,N.;Yamamoto,M.,Regulatory mechanisms of cellular response to oxidative stress.Free Radic Res 1999,31,(4),319-24.
105.Wild,A.C.;Moinova,H.R.;Mulcahy,R.T.,Regulation of gamma-glutamylcysteine synthetase subunit gene expression by the transcription factor Nrf2.J Biol Chem 1999,274,(47),33627-36.
106.McMahon,M.;Itoh,K.;Yamamoto,M.;Chanas,S.A.;Henderson,C.J.;McLellan,L.I.;Wolf,C.R.;Cavin,C.;Hayes,J.D.,The Cap'n'Collar basic leucine zipper transcription factor Nrf2(NF-E2 p45-related factor 2) controls both constitutive and inducible expression of intestinal detoxification and glutathione biosynthetic enzymes.Cancer Res 2001,61,(8),3299-307.
107.Jaiswal,A.K.,Nrf2 signaling in coordinated activation of antioxidant gene expression.Free Radic Biol Med 2004,36,(10),1199-207.
108.Kobayashi,A.;Ohta,T.;Yamamoto,M.,Unique function of the Nrf2-Keapl pathway in the inducible expression of antioxidant and detoxifying enzymes.Methods Enzymol 2004,378,273-86.
109.Motohashi,H.;Yamamoto,M.,Nrf2-Keapl defines a physiologically important stress response mechanism.Trends Mol Med 2004,10,(11),549-57.
110.Cho,H.Y.;Reddy,S.P.;Kleeberger,S.R.,Nrf2 defends the lung from oxidative stress.Antioxid Redox Signal 2006,8,(1-2),76-87.
111.Zhang,D.D.;Lo,S.C.;Cross,J.V.;Templeton,D.J.;Hannink,M.,Keapl is a redox-regulated substrate adaptor protein for a Cul3-dependent ubiquitin ligase complex.Mol Cell Biol 2004,24,(24),10941-53.
112.Wang,X.J.;Sun,Z.;Chen,W.;Li,Y.;Villeneuve,N.F.;Zhang,D.D.,Activation of Nrf2 by arsenite and monomethylarsonous acid is independent of Keap1-C151:enhanced Keap1-Cu13 interaction.Toxicol Appl Pharmacol 2008,230,(3),383-9.
113.Kwak,M.K.;Itoh,K.;Yamamoto,M.;Sutter,T.R.;Kensler,T.W.,Role of transcription factor Nrf2 in the induction of hepatic phase 2 and antioxidative enzymes in vivo by the cancer chemoprotective agent,3H-1,2-dimethiole-3-thione.Mol Med 2001,7,(2),135-45.
114.Ramos-Gomez,M.;Kwak,M.K.;Dolan,P.M.;Itoh,K.;Yamamoto,M.;Talalay,P.;Kensler,T.W.,Sensitivity to carcinogenesis is increased and chemoprotective efficacy of enzyme inducers is lost in nrf2 transcription factor-deficient mice.Proc Natl Acad Sci USA 2001,98,(6),3410-5.
115.Iida,K.;Itoh,K.;Kumagai,Y.;Oyasu,R.;Hattori,K.;Kawai,K.;Shimazui,T.;Akaza,H.;Yamamoto,M.,Nrf2 is essential for the chemopreventive efficacy of oltipraz against urinary bladder carcinogenesis.Cancer Res 2004,64,(18),6424-31.
116.Umemura,T.;Kuroiwa,Y.;Kitamura,Y.;Ishii,Y.;Kanki,K.;Kodama,Y.;Itoh,K.;Yamamoto,M.;Nishikawa,A.;Hirose,M.,A crucial role of Nrf2 in in vivo defense against oxidative damage by an environmental pollutant,pentachlorophenol.Toxicol Sci 2006,90,(1),111-9.
117.Rangasamy,T.;Guo,J.;Mitzner,W.A.;Roman,J.;Singh,A.;Fryer,A.D.;Yamamoto,M.;Kensler,T.W.;Tuder,R.M.;Georas,S.N.;Biswal,S.,Disruption of Nrf2 enhances susceptibility to severe airway inflammation and asthma in mice.J Exp Med 2005,202,(1),47-59.
118.Iizuka,T.;Ishii,Y.;Itoh,K.;Kiwamoto,T.;Kimura,T.;Matsuno,Y.;Morishima,Y.;Hegab,A.E.;Homma,S.;Nomura,A.;Sakamoto,T.;Shimura,M.;Yoshida,A.;Yamamoto,M.;Sekizawa,K.,Nrf2-deficient mice are highly susceptible to cigarette smoke-induced emphysema.Genes Cells 2005,10,(12),1113-25.
119.Aoki,Y.;Sato,H.;Nishimura,N.;Takahashi,S.;Itoh,K.;Yamamoto,M.,Accelerated DNA adduct formation in the lung of the Nrf2 knockout mouse exposed to diesel exhaust.Toxicol Appl Pharmacol 2001,173,(3),154-60.
120.Enomoto,A.;Itoh,K.;Nagayoshi,E.;Haruta,J.;Kimura,T.;O'Connor,T.;Harada,T.;Yamamoto,M.,High sensitivity of Nrf2 knockout mice to acetaminophen hepatotoxicity associated with decreased expression of ARE-regulated drug metabolizing enzymes and antioxidant genes.Toxicol Sci 2001,59,(1),169-77.
121.Surh,Y.J.,Cancer chemoprevention with dietary phytochemicals.Nat Rev Cancer 2003,3,(10),768-80.
122.Lee,J.S.;Surh,Y.J.,Nrf2 as a novel molecular target for chemoprevention.Cancer Lett 2005,224,(2),171-84.
123.Zhang,Y.;Kensler,T.W.;Cho,C.G.;Posner,G.H.;Talalay,P.,Anticarcinogenic activities of sulforaphane and structurally related synthetic norbomyl isothiocyanates.Proc Natl Acad Sci USA 1994,91,(8),3147-50.
124.Suganuma,M.;Okabe,S.;Sueoka,N.;Sueoka,E.;Matsuyama,S.;Imai,K.;Nakachi,K.;Fujiki,H.,Green tea and cancer chemoprevention.Mutat Res 1999,428,(1-2),339-44.
125.Orsolic,N.;Sver,L.;Terzic,S.;Tadic,Z.;Basic,I.,Inhibitory effect of water-soluble derivative of propolis and its polyphenolic compounds on tumor growth and metastasizing ability:a possible mode of antitumor action.Nutr Cancer 2003,47,(2),156-63.
126.Shen,G.;Xu,C.;Hu,R.;Jain,M.R.;Nair,S.;Lin,W.;Yang,C.S.;Chan,J.Y.;Kong,A.N.,Comparison of(-)-epigallocatechin-3-gallate elicited liver and small intestine gene expression profiles between C57BL/6J mice and C57BL/6J/Nrf2(-/-) mice.Pharm Res 2005,22,(11),1805-20.
127.Kensler,T.W.;Curphey,T.J.;Maxiutenko,Y.;Roebuck,B.D.,Chemoprotection by organosulfur inducers of phase 2 enzymes:dithiolethiones and dithiins.Drug Metabol Drug Interact 2000,17,(1-4),3-22.
128.Pae,H.O.;Choi,B.M.;Oh,G.S.;Lee,M.S.;Ryu,D.G.;Rhew,H.Y.;Kim,Y.M.;Chung,H.T.,Roles of heme oxygenase-1 in the antiproliferative and antiapoptotic effects of nitric oxide on Jurkat T cells.Mol Pharmacol 2004,66,(1),122-8.
129.Garg,R.;Gupta,S.;Maru,G.B.,Dietary curcumin modulates transcriptional regulators of phase Ⅰ and phase Ⅱ enzymes in benzo[a]pyrene-treated mice:mechanism of its anti-initiating action.Carcinogenesis 2008,29,(5),1022-32.
130.Na,H.K.;Surh,Y.J.,Modulation of Nrf2-mediated antioxidant and detoxifying enzyme induction by the green tea polyphenol EGCG.Food Chem Toxicol 2008,46,(4),1271-8.
131.Kode,A.;Rajendrasozhan,S.;Caito,S.;Yang,S.R.;Megson,I.L.;Rahman,I.,Resveratrol induces glutathione synthesis by activation of Nrf2 and protects against cigarette smoke-mediated oxidative stress in human lung epithelial cells.Am J Physiol Lung Cell Mol Physiol 2008,294,(3),L478-88.
132.Chen,C.Y.;Jang,J.H.;Li,M.H.;Surh,Y.J.,Resveratrol upregulates heme oxygenase-1 expression via activation of NF-E2-related factor 2 in PC12 cells.Biochem Biophys Res Commun 2005,331,(4),993-1000.
133.Higgins,L.G.;Cavin,C.;Itoh,K.;Yamamoto,M.;Hayes,J.D.,Induction of cancer chemopreventive enzymes by coffee is mediated by transcription factor Nrf2.Evidence that the coffee-specific diterpenes cafestol and kahweol confer protection against acrolein.Toxicol Appl Pharmacol 2008,226,(3),328-37.
134.Cavin,C.;Holzhaeuser,D.;Scharf,G.;Constable,A.;Huber,W.W.;Schilter,B.,Cafestol and kahweol,two coffee specific diterpenes with anticarcinogenic activity.Food Chem Toxicol 2002,40,(8),1155-63.
135.Ben-Dor,A.;Steiner,M.;Gheber,L.;Danilenko,M.;Dubi,N.;Linnewiel,K.;Zick,A.;Sharoni,Y.;Levy,J.,Carotenoids activate the antioxidant response element transcription system.Mol Cancer Ther 2005,4,(1),177-86.
136.Shinkai,Y.;Sumi,D.;Fukami,I.;Ishii,T.;Kumagai,Y.,Sulforaphane,an activator of Nrf2,suppresses cellular accumulation of arsenic and its cytotoxicity in primary mouse hepatocytes.FEBS Lett 2006,580,(7),1771-4.
137.Hayashi,A.;Suzuki,H.;Itoh,K.;Yamamoto,M.;Sugiyama,Y.,Transcription factor Nrf2 is required for the constitutive and inducible expression of multidrug resistance-associated protein 1 in mouse embryo fibroblasts.Biochem Biophys Res Commun 2003,310,(3),824-9.
138.Ohtsubo,T.;Kamada,S.;Mikami,T.;Murakami,H.;Tsujimoto,Y.,Identification of NRF2,a member of the NF-E2 family of transcription factors,as a substrate for caspase-3(-like) proteases.Cell Death Differ 1999,6,(9),865-72.