转录因子Nrf2在肝脏疾病中的作用

NF-E2相关因子2(nuclear erythroid 2-related factor 2,Nrf2)是一种能调节肝脏中大量解毒和抗氧化防御基因表达的重要转录因子.氧化应激与各种形式的肝损伤有密切的关系.Nrf2由亲电体压力或氧化应激激活,并通过结合抗氧化反应元件(antioxidant response element,ARE)诱导其靶基因,从而对细胞产生保护作用.因此,Nrf2通路在肝脏疾病中的作用已被深入研究.多种动物模型研究结果表明,Nrf2通路通过靶基因表达,在对抗病毒性肝炎、药物性肝损伤、酒精性肝病、非酒精性脂肪肝及肝癌方面表现出了不同的生物功能.根据Nrf2及其信号通路在对抗肝损伤中产生保护作用的相关文献,本文综述并讨论了其作为治疗肝损伤的药物作用靶点方面可能的应用前景.     

Isolation, identification, and biological evaluation of Nrf2-ARE activator from the leaves of green perilla (Perilla frutescens var. crispa f. viridis)

The nuclear factor erythroid 2-related factor 2 (Nrf2)-antioxidant response element (ARE) pathway is a cellular defense system against oxidative stress. Activation of this pathway increases expression of antioxidant enzymes. Epidemiological studies have demonstrated that the consumption of fruits and vegetables is associated with reduced risk of contracting a variety of human diseases. The aim of this study is to find Nrf2-ARE activators in dietary fruits and vegetables. We first attempted to compare the potency of ARE activation in six fruit and six vegetables extracts. Green perilla (Perilla frutescens var. crispa f. viridis) extract exhibited high ARE activity. We isolated the active fraction from green perilla extract through bioactivity-guided fractionation. Based on nuclear magnetic resonance and mass spectrometric analysis, the active ingredient responsible for the ARE activity was identified as 2',3'-dihydroxy-4',6'-dimethoxychalcone (DDC). DDC induced the expression of antioxidant enzymes, such as γ-glutamylcysteine synthetase (γ-GCS), NAD(P)H: quinone oxidoreductase-1 (NQO1), and heme oxygenase-1. DDC inhibited the formation of intracellular reactive oxygen species and the cytotoxicity induced by 6-hydroxydopamine. Inhibition of the p38 mitogen-activated protein kinase pathway abolished ARE activation, the induction of γ-GCS and NQO1, and the cytoprotective effect brought about by DDC. Thus, this study demonstrated that DDC contained in green perilla enhanced cellular resistance to oxidative damage through activation of the Nrf2-ARE pathway.     

Nuclear factor Nrf2 and antioxidant response element regulate NRH:quinone oxidoreductase 2 (NQO2) gene expression and antioxidant induction

Human NRH:quinone oxidoreductase 2 (NQO2) is a cytosolic protein that catalyzes the metabolic reduction of quinones and provides protection against myelogenous hyperplasia and chemical carcinogenesis. NQO2 gene expression is induced in response to antioxidant tert-butylhydroquinone (tBHQ). Sequence analysis revealed six putative antioxidant response elements (ARE1 through 6) in the human NQO2 gene promoter. Deletion mutagenesis and transfection studies suggested that the ARE region between nucleotides -1433 and -1424 is essential for basal expression and antioxidant induction of NQO2 gene expression. Mutation of this ARE from 3.8 kb NQO2 gene promoter significantly repressed expression and abrogated the induction in response to antioxidant in transfected cells. Band shift, supershift, and chromatin immunoprecipitation (ChIP) assays demonstrated binding of nuclear factors Nrf2 and JunD with human NQO2 gene ARE. Coimmunoprecipitation experiments revealed an association between Nrf2 and JunD. Overexpression of Nrf2 upregulated and overexpression of Nrf2 dominant-negative mutant downregulated ARE-mediated NQO2 gene expression. The treatment of Hep-G2 cells with Nrf2-specific RNAi significantly reduced Nrf2 and NQO2 gene expression and tBHQ induction. The results combined demonstrated that Nrf2 associates with JunD, binds to ARE at nucleotide -1433, and regulates human NQO2 gene expression and induction in response to antioxidants.     

Mechanisms of Nrf2 Protection in Astrocytes as Identified by Quantitative Proteomics and siRNA Screening

The Nrf2 (NF-E2 related factor 2)-ARE (antioxidant response element) pathway controls a powerful array of endogenous cellular antioxidant systems and is an important pathway in the detoxification of reactive oxygen species (ROS) in the brain. Using a combination of quantitative proteomics and siRNA screening, we have identified novel protective mechanisms of the Nrf2-ARE pathway against oxidative stress in astrocytes. Studies from our lab and others have shown Nrf2 overexpression protects astrocytes from oxidative stress. However, the exact mechanisms by which Nrf2 elicits these effects are unknown. In this study, we show that induction of Nrf2 reduces levels of reactive oxygen species (ROS) produced by various oxidative stressors and results in robust cytoprotection. To identify the enzymes responsible for these effects, we used stable isotope labeling by amino acids in cell culture (SILAC) and quantitative shotgun proteomics to identify 72 Nrf2-regulated proteins in astrocytes. We hypothesized a subset of these proteins might play a critical role in Nrf2 protection. In order to identify these critical proteins, we used bioinformatics to narrow our target list of proteins and then systematically screened each candidate with siRNA to assess the role of each in Nrf2 protection. We screened each target against H₂O₂, tert-butyl hydroperoxide, and 4-hydroxynonenal and subsequently identified three enzymes–catalase, prostaglandin reductase-1, and peroxiredoxin-6–that are critical for Nrf2-mediated protection in astrocytes.     

Effects of different exercise durations on Keap1-Nrf2-ARE pathway activation in mouse skeletal muscle

Abstract

The purpose of this study was to investigate the effects of acute exercise stress on the nuclear factor-erythroid2 p45-related factor 2 (Nrf2)/antioxidant response element (ARE) transactivation, Kelch-like ECH-associated protein 1 (Keap1) cytosolic protein and Nrf2 nucleoprotein expressions, Nrf2 target genes mRNA expressions, and glutathione redox (GSH/GSSG) ratio level; with a particular focus on the changes in Keap1-Nrf2-ARE pathway activation following different durations of exercise. Wild-type mice (C57BL/6J, two months old) were separated into one-hour and six-hour treadmill running groups, as well as a non-exercise control group (n = 10 in each group). Measurements of Nrf2/ARE transactivation, Nrf2 nucleoprotein expressions, Keap1 cytosolic protein expression, Nrf2 target genes’ mRNA expressions (superoxide dismutase-1 [SOD1], superoxide dismutase-2 [SOD2], γ-glutamyl cysteine ligase-modulatory [GCLm], γ-glutamyl cysteine ligase-catalytic [GCLc], glutathione reductase [GR], glutathione peroxidase-1 [Gpx1], catalase [CAT], and hemoxygenase-1 [Ho-1]), and GSH/GSSG ratio were carried out immediately after exercise. The results showed significant increases in Keap1-Nrf2-ARE pathway activation and the mRNA expressions of six measured enzymes in skeletal muscle after six hours of exercise; while in the one-hour exercise group, there was no change in Keap1-Nrf2-ARE pathway activation and only two enzymes’ mRNA expressions were increased. It is suggested that the changes in Keap1-Nrf2-ARE pathway activation and its target genes’ mRNA expressions were dependent on the exercise duration, with longer duration associated with higher responses.     

Selenium deficiency activates mouse liver Nrf2-ARE but vitamin E deficiency does not

Selenium (Se) and vitamin E are antioxidant micronutrients. Se functions through selenoproteins and vitamin E reacts with oxidizing molecules in membranes. The relationship of these micronutrients with the Nrf2-antioxidant response element (ARE) pathway was investigated using ARE-reporter mice and Nrf2-/- mice. Weanling males were fed Se-deficient (0 Se), vitamin E-deficient (0 E), or control diet for 16 or 22 weeks. The ARE reporter was elevated 450-fold in 0 Se liver but was not elevated in 0 E liver. Antioxidant enzymes induced by Nrf2-ARE (glutathione S-transferase (GST), NAD(P)H quinone oxidoreductase (NQOR), and heme oxygenase-1 (HO-1)) were elevated in 0 Se livers but not in 0 E livers. Deletion of Nrf2 had varying effects on the inductions, with GST induction being abolished by it but induction of NQOR and HO-1 still occurring. Thus, Se deficiency, but not vitamin E deficiency, induces a number of enzymes that protect against oxidative stress and modify xenobiotic metabolism through Nrf2-ARE and other stress-response pathways. We conclude that Se deficiency causes cytosolic oxidative stress but that vitamin E deficiency does not. This suggests that the oxidant defense mechanisms in which these antioxidant nutrients function are independent of one another.