Nrf2信号通路在防治肝、肾损伤中的研究进展

核因子E2相关因子2(nuclear factor E2 related factor 2, Nrf2)是诸多细胞信号通路中最重要的一种,是一种基础转录调节因子,主要编码表达多种重要的细胞保护因子,包括解毒酶、抗氧化蛋白、外排型转运体、抗凋亡蛋白、抗炎因子以及其他的应激反应因子。在肝脏或肾脏因外界条件造成损伤时,Nrf2通过调节细胞对毒物的代谢排泄、细胞凋亡以及在应激状态下细胞的抗炎抗氧化进程而发挥作用。对Nrf2在肝脏、肾脏损伤中的保护作用及其分子机制做一综述,以期为新药研发提供理论依据。     

Nrf2/ARE通路调节机制的研究进展

核因子 E2 相关因子 Nrf2 是一个参与多种蛋白表达的核转录因子, 是机体氧化应激反应的调节中枢, 它与抗氧化反应元件 （ARE） 结合后可启动下游多个抗氧化、 抗炎蛋白及解毒酶等的表达, 其介导的这一信号通路参与了炎症、 肿瘤等多种病理过程的发生发展。本综述在阐述其基本结构、 生物学效应以及介导的信号通路的基础上, 针对各种参与该信号通路正负向调控的因素及调控机制的最新研究进展进行了概述, 为抗炎症、 抗氧化以及抗肿瘤等生物化学治疗提供了新的靶点, 在此基础上, 本文也展望了生物信息学技术的应用将会为这一靶点的干预提供更好     

Nrf2在糖尿病心肌病中的作用及中药干预研究进展

糖尿病心肌病(diabetic cardiomyopathy, DCM)作为糖尿病的心血管并发症,严重影响糖尿病患者的预后。近年来,糖尿病心肌病的发病率和死亡率持续增高,成为人们重点关注的研究问题。糖尿病心肌病的发病机制复杂,多种信号通路参与其进展过程。核因子E2相关因子2(nuclear factor NF-E2-related factor 2, Nrf2)作为一种强效抗氧化基因,通过与其他信号因子相互关联增强心肌抵抗氧化应激的能力,并发挥抗炎症反应、抗心肌纤维化以及抗细胞凋亡等作用。大量研究文献显示,中药有效成分可以通过增强Nrf2的表达,影响Nrf2与其他信号因子的相互关联来改善糖尿病心肌病的心肌损伤。故本文就Nrf2在糖尿病心肌病中的作用及中药干预研究进行文献综述,为糖尿病心肌病的防治提供参考。     

Nrf2-ARE信号通路功能与临床疾病及相关药物的研究新进展

Nrf2（NF-E2-related factor 2）核因子E2相关因子是一种机体抵抗内界和外界氧化或化学等刺激的中枢调节者。Nrf2-ARE则是近年来新发现的细胞氧化应激反应的关键传导通路,当其在体内被有毒有害物质激活后转位进入细胞核能与抗氧化反应元件（antioxidant response element,ARE）结合形成Nrf2-ARE信号通路,从而调控下游抗氧化蛋白、氧化酶和Ⅱ相解毒酶等。研究发现该通路在抗衰老、抗肿瘤、抗炎症、神经损伤、眼科等多方面均有重要作用。以Nrf2为靶点的药物有望用于肿瘤、糖尿病、神经退行性疾病等。本文综述了Nrf2-ARE信号通路功能及以其为靶点的药物研究的进展。     

溴氰菊酯对大鼠大脑组织CuZn-SOD、GR、GCS亚单位和Nrf2基因表达的影响

目的 研究溴氰菊酯（DM）染毒对大鼠大脑皮层和海马组织铜、锌超氧化物歧化酶（Cu、Zn-SOD）、谷胱苷肽还原酶（GR）和γ-谷氨酰半胱氨酸合成酶（GCS）轻亚单位（GCS1）mRNA表达的影响，以及DM对GCS重亚单位（GCSh）基因和转录因子NF-E2相关因子2（Nrf2）基因mRNA和蛋白表达的影响，并探讨DM对这些酶活力影响的机制。     

黄连素激活Nrf2通路缓解新生大鼠哮喘模型氧化应激反应和对心肺组织的保护作用

目的：探讨黄连素（berberine,BB）对新生大鼠哮喘模型氧化应激反应的影响和对心肺组织的保护作用以及潜在的分子机制。方法：将新生SD雄性大鼠随机分成健康对照组、健康加药组、哮喘模型组和模型加药组;卵清蛋白诱导新生大鼠哮喘模型;收集肺泡灌洗液（broncho alveokar lavage fluid,BALF）并分析细胞类型和细胞数目;苏木伊红（hematoxylin eosin,HE）染色观察心肌组织和肺组织病理变化;末端标记法（terminal deoxynucleoitidyl transferase mediated nick end labeling,TUNEL）染色检测心肌组织和肺组织细胞凋亡情况;试剂盒检测血清中超氧化物歧化酶（superoxide dismutase,SOD）的活性和丙二醛（malondialdehyde,MDA）、一氧化氮（nitric oxide,NO）的含量;蛋白质印记检测肺组织中Kelch样ECH相关蛋白1（Kelch-like ECH-associated protein 1,Keap1）、NFE2相关因子2（NF-E2 related factor 2,Nrf2）和血红素氧合酶1（heme oxygenase 1,HMOX-1）的表达水平。结果：哮喘模型组与对照组相比,肺泡灌洗液中炎性细胞的数目显著增加;心肌组织细胞排列不规则,部分细胞结构不清晰;肺组织中有大量的炎性细胞浸润,黏膜下水肿,气道上皮断裂脱落,支气管壁明显增厚;心肌组织和肺组织中凋亡细胞比例显著增加;血清中SOD的活性显著降低,MDA和NO的含量显著增加;肺组织中Keap1、Nrf2和HMOX-1的表达水平显著升高。模型加药组与模型组相比,肺泡灌洗液中炎性细胞的数目明显减少;心肌组织较规则,细胞排列较整齐;肺组织中仍有部分炎性细胞浸润,部分肺泡壁增厚;心肌组织和肺组织中凋亡细胞比例显著降低;SOD的活性显著升高,MDA和NO的含量显著降低;Keap1、Nrf2和HMOX-1的表达水平显著升高。结论：黄连素可激活Nrf2通路,缓解新生大鼠哮喘模型的氧化应激反应,对心肺组织具有保护作用。     

Nrf2信号通路与胰腺β细胞功能障碍

核转录因子E2相关因子2（nuclear factor elytroid-derived factor 2-related factor,Nrf2）是维持细胞氧化还原稳态的中枢调节者,激活Nrf2在细胞水平和动物模型中均能拮抗氧化应激,表现出抗氧化、抗凋亡作用。近年来,大量的实验研究表明,氧化应激与2型糖尿病的形成和发展有着密切关系,而Nrf2信号通路在调节胰腺β细胞功能障碍和胰岛素分泌过程中具有重要作用。本文围绕Nrf2在胰腺β细胞功能障碍中的作用及其机制展开综述,以进一步了解Nrf2在2型糖尿病形成、发展及防治中的作用。     

丹参酮胶囊对咪喹莫特诱导的银屑病模型小鼠的抗炎和抗氧化应激作用

目的　探讨丹参酮胶囊对咪喹莫特诱导的银屑病模型小鼠的抗炎和抗氧化应激作用,寻找银屑病的治疗方法。方法　使用咪喹莫特乳膏构建银屑病样小鼠模型,通过PASI评分和组织病理评估丹参酮胶囊对银屑病样小鼠模型皮损炎性反应的影响;通过酶联免疫吸附实验检测小鼠血清中白细胞介素（IL）-17A和肿瘤坏死因子（TNF）-α的水平;通过免疫组化和实时荧光定量PCR检测小鼠皮损组织中核因子E2相关因子-2（Nrf2）、抗氧化基因［血红素加氧酶1（HO-1）和超氧化物歧化酶2（SOD2）］和二相解毒酶基因［NAD（P）H醌氧还原酶-1（NQO1）］的表达水平。统计学方法采用单因素方差分析。结果　丹参酮胶囊能明显改善银屑病样小鼠炎性反应程度,包括减轻皮肤红斑、鳞屑和皮损厚度。丹参酮胶囊能降低银屑病样小鼠血清中IL-17A和TNF-α水平（均P<0.05）。丹参酮胶囊还能诱导银屑病样小鼠皮肤组织中Nrf2、HO-1和SOD2水平升高（均P<0.05）。结论　丹参酮胶囊通过降低银屑病样小鼠血清中IL-17A和TNF-α水平以及激活Nrf2信号通路,从而发挥抗炎和抗氧化应激作用。     

荷丹三七降脂片对高脂血症大鼠脂代谢及Nrf2、γ-GCS基因mRNA表达的影响

目的：探讨荷丹三七降脂片对高脂血症大鼠血脂代谢的影响。方法：将72只大鼠随机分为正常对照组、高脂血症模型组、荷丹三七降脂片高剂量组、中剂量组、低剂量组、阳性对照组（辛伐他汀）6个小组,每小组12只。采用高脂饲料建立大鼠高脂血症模型,连续给药30 d后,测体质量、肝脏湿重和肝脏系数;取血测定各剂量组高脂血症大鼠血清中总胆固醇（TC）、三酰甘油（TG）、高密度脂蛋白胆固醇（HDL-C）、低密度脂蛋白胆固醇（LDL-C）、谷草转氨酶（AST）、谷丙转氨酶（ALT）水平;测定肝组织的抗氧化指标：超氧化物歧化酶（SOD）、丙二醛（MDA）、谷胱甘肽过氧化物酶（GSH-Px）、过氧化氢酶（CAT）、总抗氧化能力（T-AOC）;采用实时荧光定量RT-PCR法测定高脂血症大鼠肝组织中核因子NF-E₂相关因子（nuclear factor erythroid 2-related factor₂,Nrf₂）、谷氨酰半胱氨酸合酶（γ-glutamylcysteine synthethase,γ-GCS） mRNA表达;显微镜下观察肝组织形态学变化;通过小鼠急性毒性试验记录小鼠状况。结果：与模型组比较,荷丹三七降脂片各剂量组显著降低TC、TG、LDL-C、MDA水平（P<0.01,P<0.05）;显著升高SOD、CAT、GSH-Px、T-AOC （P<0.01,P<0.05）以及肝组织中的Nrf₂、γ-GCS mRNA表达显著升高（P<0.01,P<0.05）并明显减轻肝组织病理损伤;急性毒性试验表明荷丹三七降脂片无毒性。结论：荷丹三七降脂片安全无毒,对高脂血症大鼠具有良好的辅助降血脂及肝脏保护作用,其可能作用机制是通过调节Nrf₂基因表达的作用,促进γ-GCS表达,进而启动机体的抗氧化防御系统,减轻氧化应激对机体的损害作用。     

基于Keap1/Nrf2信号通路研究紫檀芪对对乙酰氨基酚诱导的小鼠急性肝损伤的保护作用

目的 观察紫檀芪（pterostilbene，PTE）对对乙酰氨基酚（acetaminophen，APAP）诱导的小鼠急性肝损伤的保护作用，并探讨相关机制。方法 利用APAP （250 mg·kg^-1）制备小鼠急性肝损伤模型，PTE分别给予15，30，60 mg·kg^-1，连续灌胃15 d预保护。自动生化分析仪检测谷丙转氨酶（alanine aminotransferase，ALT）、谷草转氨酶（aspartate aminotransferase，AST）、总蛋白（total protein，TP）、白蛋白（albumin，Alb）并计算白球比（Alb/Glb，A/G）；苏木精-伊红（HE）染色法进行肝组织病理学检测并进行分级评价；试剂盒检测超氧化物歧化酶（superoxide dismutase，SOD）、还原型谷胱甘肽（glutathione，GSH）、丙二醛（malondialdehyde，MDA）水平；JC-1检测线粒体膜电位；TUNEL染色法检测肝细胞凋亡情况；Western blotting检测Bax、cytochrome C、转录因子NF-E2相关因子2（nuclear factor erythroid 2-related factor-2，Nrf2）、Kelch样环氧氯丙烷相关蛋白1（Keleh-like ECH-associated protein l，Keap1）的蛋白表达情况。结果 与APAP模型组相比，不同浓度PTE组小鼠血清ALT、AST值显著降低（P<0.01），Alb、TP及A/G比值显著升高（P<0.05或P<0.01）；肝组织病理损伤分级降低；肝组织SOD、GSH显著升高（P<0.01），MDA显著降低（P<0.01）；线粒体膜电位增加（P<0.01）；TUNEL染色结果显示PTE组小鼠肝细胞凋亡明显减少；PTE组与APAP模型组相比，细胞质中的Bax表达显著增加（P<0.05或P<0.01），cytochrome C表达显著降低（P<0.01），而对应的线粒体中Bax表达显著降低（P<0.01），cytochrome C表达显著增加（P<0.01），同时伴有肝组织总的Nrf2表达增加（P<0.01），Keap1表达降低（P<0.01），细胞质Nrf2表达增加（P<0.01），细胞核Nrf2表达增加（P<0.01）。结论 PTE可以保护APAP诱导的小鼠急性肝损伤，其机制可能与PTE上调Nrf2水平，抑制氧化应激，保护线粒体功能有关。     

Nrf2/ARE信号通路及其调控的抗氧化蛋白

机体在应对活性氧(reactive oxygen species,ROS)损害时形成了一套复杂的氧化应激应答系统,当暴露于ROS时,机体自身能诱导出一系列保护性蛋白,以缓解细胞所受的损害。这一协调反应是由这些保护性基因上游调节区的抗氧化反应元件(antioxidant responsive element,ARE)来调控的。而近年来的研究发现,核因子NF-E2相关因子(nuclear fac-tor erythroid 2-related factor 2,Nrf2)是ARE的激活因子。Nrf2是外源性有毒物质和氧化应激的感受器,在参与细胞抗氧化应激和外源性有毒物质诱导的主要防御机制中发挥重要的作用。Nrf2-ARE通路是迄今为止发现的最为重要的内源性抗氧化应激通路。     

Chotosan ameliorates cognitive impairment and hippocampus neuronal loss in experimental vascular dementia via activating the Nrf2-mediated antioxidant pathway

Recent studies suggested that Chotosan has ameliorative effects on vascular dementia through antioxidative pathways. Nevertheless, no systematic pharmacological research was conducted to evaluate the contribution of nuclear factor-E2-related factor 2 (Nrf2), a crucial regulator of antioxidative system, on Chotosan-induced neuroprotection invascular dementia. The present study aimed to investigate the neuroprotective effect of Chotosan on vascular dementia and reveal the possible molecular mechanism involving Nrf2. We found that Chotosan treatment could ameliorate memory impairment and reduce neuron cell loss induced by common carotid artery occlusion surgery. Furthermore, Chotosan could significantly reverse reactive oxygen species production, neuronal apoptosis and microglia over-activation in hippocampus. In addition, Chotosan enhanced Nrf2 expression and its nuclear translocation as well as its downstream antioxidant protein expression, NAD(P)H/quinone oxidoreductase 1 and heme oxygenase-1. These findings suggest that Chotosan exert neuroprotection in an animal model of vascular dementia via activating Nrf2-mediated antioxidant pathway. Chotosan may serve as a potential candidate and promising Nrf2 activator for treating vascular dementia.     

Role of Nrf2 in preventing ethanol-induced oxidative stress and lipid accumulation

Oxidative stress and lipid accumulation play important roles in alcohol-induced liver injury. Previous reports showed that, in livers of nuclear factor erythroid 2-related factor 2 (Nrf2)-activated mice, genes involved in antioxidant defense are induced, whereas genes involved in lipid biosynthesis are suppressed. To investigate the role of Nrf2 in ethanol-induced hepatic alterations, Nrf2-null mice, wild-type mice, kelch-like ECH-associated protein 1-knockdown (Keap1-KD) mice with enhanced Nrf2, and Keap1-hepatocyte knockout (Keap1-HKO) mice with maximum Nrf2 activation, were treated with ethanol (5 g/kg, po). Blood and liver samples were collected 6 h thereafter. Ethanol increased alanine aminotransferase and lactate dehydrogenase activities as well as thiobarbituric acid reactive substances in serum of Nrf2-null and wild-type mice, but not in Nrf2-enhanced mice. After ethanol administration, mitochondrial glutathione concentrations decreased markedly in Nrf2-null mice but not in Nrf2-enhanced mice. H₂DCFDA staining of primary hepatocytes isolated from the four genotypes of mice indicates that oxidative stress was higher in Nrf2-null cells, and lower in Nrf2-enhanced cells than in wild-type cells. Ethanol increased serum triglycerides and hepatic free fatty acids in Nrf2-null mice, and these increases were blunted in Nrf2-enhanced mice. In addition, the basal mRNA and nuclear protein levels of sterol regulatory element-binding protein 1(Srebp-1) were decreased with graded Nrf2 activation. Ethanol further induced Srebp-1 mRNA in Nrf2-null mice but not in Nrf2-enhanced mice. In conclusion, Nrf2 activation prevented alcohol-induced oxidative stress and accumulation of free fatty acids in liver by increasing genes involved in antioxidant defense and decreasing genes involved in lipogenesis.     

Nrf2/ARE signaling protects against oxaliplatin-induced hepatotoxicity in mice

Nuclear factor erythroid 2-related factor 2 (Nrf2) controls the expression of a wide array of antioxidant response element (ARE)-driven genes, which are involved in stress response and metabolism regulation. The role of Nrf2/ARE signaling in resistances of cancer cells to radiotherapy and chemotherapy has been widely accepted. However, much less is known about the relevance of Nrf2 to chemotherapy-associated toxicities, such as hepatotoxicity. In the present study, nine chemotherapeutic agents were firstly tested in embryonic fibroblasts (MEFs) and hepatocytes isolated from Nrf2 deficient or wild-type mice. The results indicate that the cytotoxicity of oxaliplatin in hepatocytes was significantly higher than that in MEFs and enhanced by Nrf2 deficiency. Furthermore, oxaliplatin treatment caused more pronounced steatosis and severer liver injury in Nrf2^–/–mice compared with wild-type counterparts, as evidenced by dramatically elevated serum transaminase and bilirubin, increased accumulation of fat, inflammatory infiltration and blood congestion. The increased hepatotoxicity in Nrf2 deficient mice was possibly caused by decreased expression of antioxidant genes and glutathione depletion. Our results demonstrated that oxaliplatin-inducedhepatotoxicity was significantly impacted by Nrf2 status, therefore Nrf2 could potentially serve as a biomarker to predict or a target to prevent hepatotoxicity of oxaliplatin.     

Resveratrol protects human keratinocytes HaCaT cells from UVA-induced oxidative stress damage by downregulating Keap1 expression

Ultraviolet radiation A (UVA)-induced oxidative stress is recognized as an important factor in the development of skin carcinogenesis. Resveratrol is demonstrated to possess remarkable antioxidant activity in the organism. The aim of this study was to investigate the protective role of resveratrol in human keratinocytes (HaCaT) against UVA-induced oxidative damage and the possible mechanism of the translocation of NF-E2-related factor-2 (Nrf2) into the nucleus. The HaCaT cells were UVA-irradiated and the effects of resveratrol on cell viability, reactive oxygen species generation and membrane-lipid peroxidation were measured. The proteins and mRNA of Nrf2 and Kelch-like-ECH-associated protein 1 (Keap1) were determined by immunofluorescence staining, Western blot and quantitative PCR, respectively. UVA exposure led to a decrease in viability and an increase in reactive oxygen species generation in HaCaT cells. Resveratrol could effectively increase the viability of HaCaT cells after UVA exposure and protect them from UVA-induced oxidative stress. Moreover, resveratrol increased the level of Nrf2 protein and facilitated Nrf2 accumulation in the nucleus; as a result, the activity of antioxidant enzymes was also upregulated. The main finding was that Keap1 protein, a repressor of Nrf2 in the cytoplasm, was clearly decreased by resveratrol treatment 12 h and beyond though the level of Keap1 mRNA still increased. Our results suggest that resveratrol can degrade Keap1 protein and facilitate Nrf2 accumulation in the nucleus, thereby protecting HaCaT cells from UVA-induced oxidative stress. Resveratrol could be a more useful natural medicine for the protection of epidermal cells from UVA-induced damage.