Nrf2-ARE信号通路参与肝脏疾病病理机制研究进展

核因子NF-E2相关因子2(Nrf2)是细胞抵御氧化应激的一个重要转录因子,它能够在活性氧或亲电试剂的刺激下,转位进入细胞核,并与抗氧化反应元件(ARE)相互作用,从而诱导下游保护性Ⅱ相解毒酶和抗氧化酶的表达,达到细胞保护的作用。氧化应激是诸多肝脏疾病共同的发病机制,而Nrf2-ARE是体内一条极为重要的抗氧化应激信号通路,该通路在肝脏疾病的发生、发展及预防过程中起着非常重要的作用,Nrf2或将成为肝脏疾病治疗的新靶点。该文综述了Nrf2-ARE信号通路参与肝脏疾病病理机制的最新研究进展,以期为日后相关研究提供参考。     

Nrf2在神经退行性疾病中的作用及激活剂的研究进展

氧化应激被认为是多种神经退行性疾病的发病机制之一,在疾病的发生发展过程中起重要作用。核转录因子E2相关因子2(nuclear factor erythroid 2 related factor 2,Nrf2)是内源性抗氧化防御系统的关键调节蛋白,在氧化应激条件下核转录因子Nrf2发生核转位,与抗氧反应元件(antioxidant response element,ARE)结合,启动下游大量的抗氧化酶基因的转录,发挥抗氧化的保护作用。Nrf2诱导剂在多种神经退行性疾病模型中能减缓氧化应激,表现出良好的神经保护作用。如何有效地激活Nrf2-ARE通路已经越来越受到研究者的重视。本文概述了Nrf2-ARE通路的作用机制并具体阐述了激活Nrf2在不同的神经退行性疾病中所发挥的保护作用,同时统计了目前研究中的Nrf2激活剂。     

姜黄素对D-半乳糖致衰老大鼠氧化应激及Nrf2/ARE通路的影响

目的 研究姜黄素对D-半乳糖致衰老模型大鼠氧化应激及Nrf2/ARE通路的影响.方法 将大鼠随机均分为空白组、模型组和姜黄素组于模型组和姜黄素组大鼠每日sc 125 mg· kg-1D-半乳糖造模,姜黄素组大鼠同时ip 10 mg·kg-1姜黄素,空白组大鼠给予生理盐水,连续7周.测定各组大鼠血清中丙二醛(MDA)、肝组织中蛋白质糖基、谷胱甘肽过氧化物(GSH)的含量和血清超氧化物歧化酶(SOD)、全血谷胱甘肽过氧化物酶(GSH-Px)的活性;用Western blot法分析肝组织中Nrf2蛋白和HO-1蛋白的表达水平.结果 与模型组比较,姜黄素组大鼠血清中SOD的活性和MDA、肝中GSH、蛋白质羰基的含量均降低,全血中GSH-Px的活性显著升高,Nfr2和HO-1蛋白的表达水平也明显升高.结论 姜黄素能够缓解D-半乳糖导致的大鼠氧化应激,Nrf2/ARE通路参与了姜黄素的抗氧化活性.     

18α-甘草酸二铵对大鼠肝脏细胞色素P450和II相酶的影响

目的研究18α-甘草酸二铵(18α-GL)对肝脏药物代谢酶的影响。方法♂Wistar大鼠ig18α-GL12.5和50mg·kg^-1,分别给药3,6和12d,对照组给等容量的溶媒。酶学测定肝微粒体细胞色素P450(CYP),尿苷二磷酸葡萄糖醛酸转移酶(GT)和谷胱甘肽巯基转移酶(GST)活性。结果18α-GL抑制苯胺羟化酶,乙氧异唑脱乙基酶和红霉素脱甲基酶活性,抑制率分别可达53.2%,47.3%和34.3%;增加GT₁(底物为7-甲基-4-羟基-香豆素),GT₂(底物为4-羟基联苯)和GST活性,分别可达29.9%,70.3%和48.3%。结论18α-GL对大鼠肝微粒体I相酶(CYP2E1,CYP1A1和CYP3A)主要是抑制作用,对II相酶(GT₁,GT₂和GST)是诱导。     

萝卜硫素对肾小管上皮细胞氧化应激及Nrf2/HO 1信号通路的影响

摘 要 目的：探讨萝卜硫素（SFN）对肾小管上皮细胞（HK-2）的氧化应激及核转录因子E2相关因子2（Nrf2）/血红素加氧酶（HO）-1信号通路的影响。方法: 体外培养HK-2细胞,将细胞分为空白对照组、H2O2处理组、H2O2+10 μmol·K^-1 SFN组、H2O2+20 μmol·K^-1 SFN组和H2O2+40 μmol·K^-1 SFN组;测定HK-2细胞中丙二醛（MDA）、一氧化氮（NO）、谷胱甘肽（GSH）的含量及超氧化物歧化酶（SOD）的活性;RT-PCR和Western Blot法分别检测Nrf2和HO-1 mRNA及蛋白表达水平。结果: 与空白对照组相比,H2O2处理组中MDA、NO含量明显升高,GSH水平和SOD酶活性显著降低（P<0.05）;经高、中、低3种浓度的萝卜硫素处理后MDA、NO含量显著降低,GSH水平显著升高,SOD酶活性也显著升高,Nrf2和HO 1mRNA及蛋白表达明显上调（P<0.05）。结论:萝卜硫素有抗HK 2细胞氧化应激损伤的作用,其作用机制可能与激活Nrf2/HO-1信号通路有关。     

Nrf2-ARE信号通路在左乙拉西坦抗癫痫中的作用

目的探讨Nrf2-ARE信号通路在左乙拉西坦抗癫痫中的作用,以及应用左乙拉西坦对认知功能的影响。方法成年♂SD大鼠36只,250~300 g,随机分为生理盐水对照(control)组、戊四唑(1,5-pentamethylene-1H-tetrazole,PTZ)癫痫模型组、左乙拉西坦(levetiracetam,LEV)对照组以及左乙拉西坦治疗组,每组9只。以Morris水迷宫测试大鼠的空间学习记忆能力;用免疫印迹法测定海马组织中Nrf2、HO-1和NQO1蛋白表达量。结果与癫痫模型组相比,给予左乙拉西坦治疗的癫痫大鼠在Morris水迷宫测试中潜伏期明显缩短(P<0.05),海马Nrf2、HO-1、NQO1蛋白表达均明显增高(P<0.01)。结论左乙拉西坦能够改善癫痫大鼠的认知功能,其机制可能通过Nrf2-ARE通路,使HO-1、NQO1蛋白表达增多,起到抗癫痫的作用。     

18α-甘草酸二铵对大鼠肝脏细胞色素P450和II相酶的影响

目的　研究 18α 甘草酸二铵 (18α GL)对肝脏药物代谢酶的影响。方法　♂Wistar大鼠ig 18α GL 12 5和 5 0mg·kg-1,分别给药 3 ,6和 12d ,对照组给等容量的溶媒。酶学测定肝微粒体细胞色素P45 0 (CYP) ,尿苷二磷酸葡萄糖醛酸转移酶 (GT)和谷胱甘肽巯基转移酶 (GST)活性。结果　 18α GL抑制苯胺羟化酶 ,乙氧异唑脱乙基酶和红霉素脱甲基酶活性 ,抑制率分别可达 5 3 2 % ,47 3%和 34 3 % ;增加GT1(底物为 7 甲基 4 羟基 香豆素 ) ,GT2 (底物为 4 羟基联苯 )和GST活性 ,分别可达 2 9 9% ,70 3%和 48 3 %。结论　 18α GL对大鼠肝微粒体I相酶(CYP2E1,CYP1A1和CYP3A)主要是抑制作用 ,对II相酶 (GT1,GT2 和GST)是诱导     

丝氨酸/苏氨酸蛋白磷酸酶1/2A在缺氧预处理诱导人脐静脉内皮细胞耐受中的作用(英文)

目的研究丝氨酸/苏氨酸蛋白磷酸酶1/2A(PP1/2A)在调节人脐静脉内皮细胞(HUVEC)对缺氧耐受相关信号转导中的作用。方法采用缺氧预处理诱导HUVEC对缺氧损伤的耐受。采用细胞存活率、乳酸脱氢酶(LDH)释放及总抗氧化能力(T-AOC)评价HUVEC的耐受性。免疫细胞化学联合蛋白质印迹法检测核因子E2相关因子2(Nrf2)的亚细胞定位。蛋白质印迹法检测应激蛋白血红素氧合酶1(HO-1)的表达。结果缺氧90 min导致HUVEC存活率及T-AOC降低,LDH释放增加。缺氧预处理(缺氧10 min后4, 8及24 h)可提高HUVEC对随后缺氧90 min的耐受,细胞存活率及T-AOC较缺氧组显著提高,LDH释出显著降低,并诱导Nrf2由胞浆向胞核移位,上调其下游信号分子HO-1的表达。缺氧预处理前用PP1/2A特异性抑制剂冈田酸(40 nmol.L-1)孵育HUVEC 10 min可部分抑制缺氧预处理诱导的Nrf2向核移位、HO-1表达及细胞耐受性。结论 PP1/2A至少部分参与缺氧预处理诱导HUVEC对缺氧损伤的耐受,其机制可能与调节Nrf2向核移位及HO-1表达有关。     

心肌缺血再灌注损伤对大鼠肝细胞色素P450酶代谢的影响

目的探讨心肌缺血再灌注状态下,大鼠肝代谢功能和相关的氧化/抗氧化能力变化。方法雄性SD大鼠随机分为5组,除假手术组外,制备在体心肌缺血再灌注模型,并于缺血40min、再灌注15,60和180min分别处死大鼠,检测血浆丙氨酸转氨酶(ALT)和天冬氨酸转氨酶(AST)活性,肝匀浆丙二醛(MDA)含量、超氧化物歧化酶(SOD)活性;以红霉素N-脱甲基酶、五氧基异噁唑O-脱乙基酶和苯胺羟化酶法为探针测定肝细胞色素P450(CYP)3A,CYP2B1和CYP2E1催化功能;RT-PCR法检测肝Ⅰ相药物代谢酶CYP3A1,CYP2B1/2,CYP2E1,以及Ⅱ相解毒酶NAD(P)H醌氧化还原酶(NQO1)及其上游因子NF-E2相关因子(Nrf2)mRNA水平。结果再灌注60 min,肝匀浆MDA含量升高(P<0.05),SOD活力下降(P<0.01);再灌注180 min时,血浆ALT和AST活性升高(P<0.05)。Nrf2基因于再灌注60 min时显著激活(P<0.05),下游因子NQO1 mRNA于再灌注180 min时明显上调(P<0.05)。CYP3A催化功能和mRNA水平分别于再灌注60和180 min开始明显降低(P<0.05);CYP2B1/2 mRNA和催化功能水平分别于再灌注15和180 min开始明显降低(P<0.05);CYP2E1催化功能无明显改变。结论大鼠心肌缺血再灌注可引起肝组织氧化应激及并导致功能损伤。在再灌注早期,具有抗氧化功能的NQO1在转录水平显著上调,其机制可能与上游因子Nrf2被激活相关;CYP3A和CYP2B催化功能在转录和(或)转录后水平明显下调。     

Acute exposure to 3‐methylcholanthrene induces hepatic oxidative stress via activation of the Nrf2/ARE signaling pathway in mice

Polycyclic aromatic hydrocarbons (PAHs) are the most common contaminants in the environment. The primary focus on the toxicity of PAHs is their ability to activate the aryl hydrocarbon receptor (AhR)‐mediated pathway and lead to carcinogenesis in different organisms. However, the influence of PAHs on the antioxidant system in mammalian systems has received only limited attention. In the present study, we observed that the intraperitoneal injection of 100 mg/kg 3‐methylcholanthrene (3MC) into mice significantly increased reactive oxygen species (ROS) levels and malondialdehyde (MDA) contents and decreased glutathione (GSH) contents and the activity of total antioxidant capacity (T‐AOC), indicating that serious oxidative stress had been induced in the liver of mice. Then, the oxidative stress signal activated the nuclear factor erythroid 2‐related factor 2/antioxidant response element (Nrf2/ARE) pathway by enhancing the mRNA levels of Nrf2, p38, and Erk2. Moreover, the mRNA levels of Nrf2/ARE target genes, including glutathione peroxidase (Gpx), glutathione reductase (GR), glutathione synthetase (GS), NAD(P)H: quinone oxidoreductase 1 (Nqo1), superoxide dismutase 1 (Sod1), and Sod2, increased significantly after treatment with 3MC for 24 hours. The hepatic levels of NQO1 and the activities of GR and GS were also significantly enhanced at 24 hours after 3MC treatment. Because the expression of NQO1 is co‐regulated by Nrf2/ARE and AhR/XRE in mammalian tissues, NQO1 may play an important role in protecting against the oxidative stress induced by 3MC. Taken together, our findings suggested that acute exposure to 3MC altered the cellular redox balance in hepatocytes to trigger Nrf2‐regulated antioxidant responses, which may represent an adaptive cell defense mechanism against the oxidative stress induced by PAHs. © 2013 Wiley Periodicals, Inc. Environ Toxicol 29: 1399–1408, 2014.     

Study of inducibility and mechanism of phase 2 enzymes by quercetin and rutin

Objective The increasing recognition of the role for oxidative stress in hepatic disorders has led to extensive investigation on the protection by exogenous antioxidants against hepatic injury.In this study,we choose two typical polyphenol,quercetin and rutin,to investigate the mechanism of induction of cellular antioxidants and phase 2 enzymes in human HepG2 cells.Methods The HepG2 cells were treated with various concentrations of quercetin and rutin for 6 h and 24 h.The activities of NAD(P)H:quinone oxidoreductase(NQO1)in HepG2 cells were measured by 2,6-dichloroindophenol reduction method.The content of superoxide dismutase(SOD)was determined with the method of chemical colorimetry.The protein expressions of NQO1 and NF-E2-related factor 2(Nrf2)in HepG2 cells were detected by Western blotting.Results Incubation of HepG2 cells with quercetin and rutin resulted in a marked concentration-and time-dependent induction of a number of cellular antioxidants and phase 2 enzymes,including NQO1,SOD.Quercetin and rutin treatment of HepG2 cells also caused increase in protein expressions of NQO1 and Nrf2.Conclusions This study demonstrates that a series of phase 2 enzymes in HepG2 cells can be induced by quercetin and rutin in a concentration-and time-dependent fashion by upregulation the protein expression of nrf2.     

A polyacetylene from Gymnaster koraiensis exerts hepatoprotective effects in vivo and in vitro

In the present study, we isolated a polyacetylene, gymnasterkoreayne B (GKB), from Gymnaster koraiensis and investigated the effect of GKB on the protection from oxidative stress-induced cytotoxicity through induction of the expression of cellular defense enzymes. GKB induced mRNA expression and enzyme activity of NAD(P)H:quinone oxidoreductase (NQO1) in vitro and in vivo, and potently increased expression of many cellular defense genes including glutathione-S-transferases, UDP-glucuronosyltransferase, and glutathione reductase (GSR) in normal rat liver. The nuclear factor erythroid 2-related factor 2 (Nrf2) which is known to induce various antioxidant and cytoprotective genes, and the genes containing the antioxidant response element (ARE), including NQO1, hemeoxygenease-1, GSR were induced by GKB in HepG2 human hepatocarcinoma cells. Pre-treatment of the cells with GKB accelerated the production of glutathione and mitigated menadione-induced cytotoxicity in HepG2 cells. Taken together, we found that GKB was a novel inducer of phase II detoxification enzymes and cellular defense enzymes, resulting in protection of the cells from oxidative stress and hepatotoxicity through regulation of detoxifying and antioxidant systems.     

Nrf2 and c-Jun regulation of antioxidant response element (ARE)-mediated expression and induction of gamma-glutamylcysteine synthetase heavy subunit gene

gamma-Glutamylcysteine synthetase (gamma-GCS) is a rate-limiting enzyme in the de novo synthesis of glutathione, a known scavenger of electrophiles and reactive oxygen species (ROS). The gamma-GCS gene is expressed ubiquitously and induced coordinately with NAD(P)H:quinone oxidoreductase(1) (NQO1) and glutathione S-transferase Ya (GST Ya) in response to xenobiotics and antioxidants. The antioxidant response element (ARE) is required for expression and induction of these genes. In the current report, we demonstrated that ARE-mediated gamma-GCS gene expression and induction is regulated by similar Nrf and Jun factors as reported earlier for the NQO1 and GST Ya genes. The gamma-GCS gene ARE competed with the binding of nuclear proteins (Nrf + Jun) to the NQO1 gene ARE (hARE). In addition, the overexpression of Nrf2 and Nrf1 with c-Jun significantly up-regulated gamma-GCS ARE-mediated basal expression and beta-naphthoflavone induction of the chloramphenicol acetyltransferase gene in transfected HepG2 cells. Interestingly, Nrf2 + c-Jun was more effective than Nrf1 + c-Jun in the regulation of ARE-mediated gamma-GCS gene expression. Further experiments demonstrated that the c-Jun level within the cells is an important determinant of the level of ARE-mediated gamma-GCS gene expression. Therefore, at higher concentrations of c-Jun, gamma-GCS gene expression is repressed, presumably due to generation of a sufficient amount of c-Jun + c-Fos complex that interferes with the binding of Nrf2 + c-Jun complex to the ARE.     

Detoxifying effects of optimal hyperoxia (40% oxygenation) exposure on benzo[a]pyrene-induced toxicity in human keratinocytes

ABSTRACT

Detoxifying effects of hyperoxia, which is widely used in clinical practice, were investigated using HaCat cells (human keratinocytes) treated with benzo[a]pyrene (B[a]P) as a model agent to induce adverse effects in the skin. It is well-established that B[a]P may produce toxicities including cancer, endocrine disruption, and phototoxicity involving DNA damage, free radical generation, and down regulation of nuclear factor erythroid 2-related factor 2 (Nrf2). It is well-known that Nrf2 is associated increase of antioxidant enzyme catalase (CAT) or detoxification enzyme glutathione S-transferase (GST) in HaCat cells treated with B[a]P under optimal condition of hyperoxia (40% oxygenation) conditions. To further examine the underlying basis of this phenomenon, factors affecting the expression of Nrf2 were determined. Nrf2 was upregulated accompanied by a rise in p38 MAPK, sequestosome-1 (also known as p62) and NF-κB. In contrast, Nrf2 was downregulated associated with an elevation in glycogen synthase kinase 3 beta (GSK-3β) and peroxisome proliferator-activated receptor alpha (PPARα). Hyperoxia was also found to diminish DNA damage and generation of free radicals initiated in B[a]P-treated cells which was attributed to an significant rise of Nrf2, leading to elevated antioxidant activities or detoxification proteins including heme oxygenase 1 (HO-1), superoxide dismutase (SOD), glutathione peroxidase-1/2 (GPX-1/2), CAT, GST and glutathione (GSH). In addition, factors related to skin aging were also altered by hyperoxia. Data suggest that optimal hyperoxia exposure of 40% oxygenation may reduce cellular toxicity induced by B[a]P in HaCat cells as evidenced by inhibition of DNA damage, free radical generation, and down-regulation of Nrf2.     

Ghrelin attenuates secondary brain injury following intracerebral hemorrhage by inhibiting NLRP3 inflammasome activation and promoting Nrf2/ARE signaling pathway in mice

Ghrelin, a brain-gut peptide, has been proven to exert neuroprotection in different kinds of neurological diseases; however, its role and the potential molecular mechanisms in secondary brain injury (SBI) after intracerebral hemorrhage (ICH) are still unknown. In this study, we investigate whether treatment with ghrelin may attenuate SBI in a murine ICH model, and if so, whether the neuroprotective effects are due to the inhibition of nucleotide-binding oligomerization domain-like receptor pyrin domain-containing 3 (NLRP3) inflammasome activation and promotion of nuclear factor-E2-related factor 2 (Nrf2)/antioxidative response element (ARE) signaling pathway. Stereotactically intrastriatal infusion of autologous blood was performed to mimic ICH. Ghrelin was given intraperitoneally immediately following ICH and again 1 h later. Results showed that ghrelin attenuated neurobehavioral deficits, brain edema, hematoma volume, and perihematomal cell death post-ICH. Ghrelin inhibited the NLRP3 inflammasome activation and subsequently suppressed the neuroinflammatory response as evidenced by reduced microglia activation, neutrophil infiltration, and pro-inflammatory mediators release after ICH. Additionally, ghrelin alleviated ICH-induced oxidative stress according to the chemiluminescence of luminol and lucigenin, malondialdehyde (MDA) content, and total superoxide dismutase (SOD) activity assays. These changes were accompanied by upregulation of Nrf2 expression, Nrf2 nuclear accumulation, and enhanced Nrf2 DNA binding activity, as well as by increased expressions of Nrf2 downstream target antioxidative genes, including NAD(P)H quinine oxidoreductase-1 (NQO1), glutathione cysteine ligase regulatory subunit (GCLC), and glutathione cysteine ligase modulatory subunit (GCLM). Together, our data suggested that ghrelin protected against ICH-induced SBI by inhibiting NLRP3 inflammasome activation and promoting Nrf2/ARE signaling pathway.     

Nrf2-mediated liver protection by sauchinone, an antioxidant lignan, from acetaminophen toxicity through the PKCδ-GSK3β pathway

BACKGROUND AND PURPOSE

Sauchinone, an antioxidant lignan, protects hepatocytes from iron-induced toxicity. This study investigated the protective effects of sauchinone against acetaminophen (APAP)-induced toxicity in the liver and the role of nuclear factor erythroid-2-related factor-2 (Nrf2) in this effect.

EXPERIMENTAL APPROACH

Blood biochemistry and histopathology were assessed in mice treated with APAP or APAP + sauchinone. The levels of mRNA and protein were measured using real-time PCR assays and immunoblottings.

KEY RESULTS

Sauchinone ameliorated liver injury caused by a high dose of APAP. This effect was prevented by a deficiency of Nrf2. Sauchinone treatment induced modifier subunit of glutamate-cysteine ligase, NAD(P)H:quinone oxidoreductase-1 (NQO1) and heat shock protein 32 in the liver, which was abolished by Nrf2 deficiency. In a hepatocyte model, sauchinone activated Nrf2, as evidenced by the increased nuclear accumulation of Nrf2, the induction of NQO1-antioxidant response element reporter gene, and glutamate-cysteine ligase and NQO1 protein induction, which contributed to the restoration of hepatic glutathione content. Consistently, treatment of sauchinone enhanced Nrf2 phosphorylation with a reciprocal decrease in its interaction with Kelch-like ECH-associated protein-1. Intriguingly, sauchinone activated protein kinase C-δ (PKCδ), which led to Nrf2 phosphorylation. In addition, it increased the inhibitory phosphorylation of glycogen synthase kinase-3β (GSK3β), derepressing Nrf2 activity, which was supported by the reversal of sauchinone''s activation of Nrf2 by an activated mutant of GSK3β. Moreover, phosphorylation of GSK3β by sauchinone depended on PKCδ activation.

CONCLUSION AND IMPLICATIONS

Our results demonstrate that sauchinone protects the liver from APAP-induced toxicity by activating Nrf2, and this effect is mediated by PKCδ activation, which induces inhibitory phosphorylation of GSK3β.