An isopentenyl-substituted flavonoid norartocarpin activates Nrf2 signalling pathway and prevents oxidative insults in human lung epithelial cells

The nuclear factor erythroid 2-related factor 2 (Nrf2) plays a crucial role in regulating the intracellular oxidative stress, and thus activation of Nrf2 by nature-derived molecules effectively alleviates the pathological process of oxidative stress-induced chronic diseases. The isopentenyl-substituted flavonoid norartocarpin (NOR) induced the activity of NAD(P)H: quinone reductase (QR), implying that it might be a potential Nrf2 activator. Further studies indicated that NOR upregulated the protein levels of Nrf2 and its downstream genes, NAD(P)H quinone oxidoreductase 1 (NQO1), and γ-glutamyl cysteine synthetase (GCLM) through facilitating the nuclear translocation of Nrf2 and enhancing Nrf2 protein stability. NOR-induced activation of Nrf2 pathway was associated with multiple upstream kinases, including mitogen-activated protein kinase (MAPK), phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K), protein kinase C (PKC), and protein kinase R-like endoplasmic reticulum kinase (PERK). Moreover, NOR protected human lung epithelial Beas-2B cells against sodium arsenite [As(III)]-induced cytotoxicity in an Nrf2-dependent manner. Collectively, NOR was firstly identified to be an Nrf2 activator, which demonstrated the capability of preventing oxidative insults in human lung epithelial cells.     

NAD(P)H:quinone oxidoreductase 1 activity reduces hypertrophy in 3T3-L1 adipocytes

The nuclear factor E2-related factor 2 (Nrf2)/Kelch-like ECH-associated protein 1 (Keap1) pathway responds to oxidative stress via control of several antioxidant defense gene expressions. Recent efforts demonstrate that Nrf2 modulates development of adiposity and adipogenesis. One of the major Nrf2-regulated proteins, NAD(P)H:quinone oxidoreductase 1 (NQO1), is implicated in the development of adipose tissue and obesity. However, little is known about in situ disposition of Nrf2, Keap1, and NQO1 during adipogenesis in isolated adipocytes. Based on literature data, we hypothesized that adipocyte differentiation would increase expression of the Nrf2/Keap1 pathway and NQO1. Using murine 3T3-L1 preadipocytes, we mapped an increase in NQO1 protein at limited clonal expansion and postmitotic growth arrest (Days 1-3) stages and a decrease in terminally differentiated (Day 8) adipocytes that lasted for several days afterward. Conversely, NQO1, Nrf2, and Keap1 mRNA expressions were all increased in differentiated adipocytes (Days 11-14), indicating a discrepancy between steady-state mRNA levels and resulting protein. Treatment of differentiated 3T3-L1 adipocytes with glycogen synthase kinase-3β (GSK-3β) inhibitor, LiCl, led to 1.9-fold increase in NQO1 protein. Sulforaphane enhanced NQO1 protein (10.5-fold) and blunted triglyceride and FABP4 accumulation. The decrement in triglyceride content was partially reversed when NQO1 activity was pharmacologically inhibited. These data demonstrate a biphasic response of Nrf2 and NQO1 during adipocyte differentiation that is regulated by Keap1- and GSK-3β-dependent mechanisms, and that hypertrophy is negatively regulated by NQO1 activity.     

虫草发酵菌丝体对胰岛素抵抗大鼠氧化应激的缓解作用

探究虫草发酵菌丝体对胰岛素抵抗(IR)大鼠氧化应激的影响。采用低中高剂量虫草发酵菌丝体(剂量分别为1.65g/kg饲料,3.30g/kg饲料和6.60g/kg饲料)分别干预IR大鼠。口服葡萄糖耐量实验观察不同剂量虫草发酵菌丝体的降糖效果,同时观察血脂、氧化应激水平的变化情况。RT-PCR法测肌肉、肝脏组织氧化应激相关基因Nrf2、HO-1和NQO1mRNA相对表达量,Westernblot法测其蛋白相对表达量。结果发现CM干预能够显著改善IR大鼠空腹血糖、血脂和机体氧化应激水平,肌肉、肝脏组织Nrf2、HO-1和NQO1mRNA相对表达量显著上升,肝脏Nrf2和NQO1蛋白相对表达量显著上调,并且具有剂量依赖性。说明CM具有缓解IR大鼠氧化应激的作用。     

Interplay between vascular endothelial growth factor (VEGF) and nuclear factor erythroid 2-related factor-2 (Nrf2): implications for preeclampsia

Several recently published studies have suggested that decreasing VEGF levels result in placental oxidative stress in preeclampsia, although the question as to how decreased VEGF concentrations increase oxidative stress still remains unanswered. Here, we show that VEGF activated Nrf2, the main regulating factor of the intracellular redox balance, in the cytotrophic cell line BeWo. In turn, this activated the production of antioxidative enzymes thioredoxin, thioredoxin reductase, and heme oxygenase-1, which showed a decrease in their expression in the placentas of preeclamptic women. Nevertheless, this activation occurred without oxidative stress stimulus. As a consequence, the activation of Nrf2 protected BeWo cells against H(2)O(2)/Fe(2+)-induced oxidative damage. We further show that VEGF up-regulated the expression of itself. A positive feedback loop was described in which VEGF activated Nrf2 in an ERK1/2-dependent manner; the up-regulation of HO-1 expression by Nrf2 augmented the production of carbon monoxide, which in turn up-regulated VEGF expression. In conclusion, VEGF induces the Nrf2 pathway to protect against oxidative stress and, via a positive feedback loop, to elevate VEGF expression. Therefore, decreased VEGF bioavailability during preeclampsia may result in higher vulnerability to placental oxidative cell damage and a further reduction of VEGF bioavailability, a vicious circle that may end up in preeclampsia.     

Induction of NAD(P)H:quinone oxidoreductase 1 expression by cysteine via Nrf2 activation in human intestinal epithelial LS180 cells

In this study, we examined the effects of 20 amino acids on the expression level of NAD(P)H:quinone oxidoreductase 1 (NQO1) in human intestinal LS180 cells. Five amino acids were associated with significant increases in NQO1 mRNA expression; the most substantial increase was induced by cysteine, which markedly increased the NQO1 mRNA level in a time- and dose-dependent manner. Cysteine also increased the protein level of NQO1 and its enzymatic activity in LS180 cells. Furthermore, cysteine significantly up-regulated NQO1 promoter activity, and this induction was completely abolished by mutation of the antioxidant response element, a binding site of the nuclear factor erythroid 2-related factor 2 (Nrf2). Knockdown experiment using siRNA against Nrf2 showed the involvement of Nrf2 on cysteine-induced increase in NQO1 mRNA expression. Further, cysteine treatment increased the amount of Nrf2 protein in the nucleus and decreased the amount of Kelch-like ECH-associated protein 1 (a suppressor protein of Nrf2) in the cytosol, suggesting that Nrf2 was activated by cysteine. Oral administration of cysteine to mice significantly increased NQO1 mRNA levels in the mouse intestinal mucosa. These findings show that cysteine induces NQO1 expression in both in vitro and in vivo systems and also suggest that Nrf2 activation is involved in this induction.     

NAD(P)H:quinone oxidoreductase 1 protects lungs from oxidant-induced emphysema in mice

Emphysema is currently a leading cause of mortality with no known effective therapy to attenuate progressive loss of lung function. Previous work supports that activation of nuclear factor erythroid 2-related factor 2 (Nrf2) is protective to the lung through induction of hundreds of antioxidant genes. In models of lung injury, the expression of NAD(P)H:quinone oxidoreductase 1 (NQO1) is upregulated in a manner dependent on Nrf2 and human emphysema is associated with reduced levels of NQO1. However, the functional role of NQO1 in emphysema remains unknown. In this study, we demonstrate the protective role of NQO1 in the development of emphysema using mouse models. NQO1-deficient animals demonstrated premature age-related emphysema and were more susceptible to both elastase and inhaled lipopolysaccharide models of emphysema. The absence of NQO1 was associated with enhanced markers of oxidant stress. Treatment of NQO1-deficient animals with the antioxidant N-acetylcysteine reversed the NQO1-dependent emphysematous changes. In vitro studies utilizing either inhibition or induction of NQO1 demonstrated a potent antioxidant role of NQO1 in macrophages, suggesting a role for macrophage-derived oxidants in the pathogenesis of emphysema. These novel findings support a functional role for NQO1 in protecting the lung from development of emphysema.     

BRG1 interacts with Nrf2 to selectively mediate HO-1 induction in response to oxidative stress

NF-E2-related factor 2 (Nrf2) regulates antioxidant-responsive element-mediated induction of cytoprotective genes in response to oxidative stress. The purpose of this study was to determine the role of BRG1, a catalytic subunit of SWI2/SNF2-like chromatin-remodeling complexes, in Nrf2-mediated gene expression. Small interfering RNA knockdown of BRG1 in SW480 cells selectively decreased inducible expression of the heme oxygenase 1 (HO-1) gene after diethylmaleate treatment but did not affect other Nrf2 target genes, such as the gene encoding NADPH:quinone oxidoreductase 1 (NQO1). Chromatin immunoprecipitation analysis revealed that Nrf2 recruits BRG1 to both HO-1 and NQO1 regulatory regions. However, BRG1 knockdown selectively decreased the recruitment of RNA polymerase II to the HO-1 promoter but not to the NQO1 promoter. HO-1, but not other Nrf2-regulated genes, harbors a sequence of TG repeats capable of forming Z-DNA with BRG1 assistance. Similarly, replacement of the TG repeats with an alternative Z-DNA-forming sequence led to BRG1-mediated activation of HO-1. These results thus demonstrate that BRG1, through the facilitation of Z-DNA formation and subsequent recruitment of RNA polymerase II, is critical in Nrf2-mediated inducible expression of HO-1.     

Resveratrol protects against polychlorinated biphenyl-mediated impairment of glucose homeostasis in adipocytes

Resveratrol (RSV) is a plant polyphenol that exhibits several favorable effects on glucose homeostasis in adipocytes. Recent studies from our laboratory demonstrated that coplanar polychlorinated biphenyls (PCBs) that are ligands of the aryl hydrocarbon receptor impair glucose homeostasis in mice. PCB-induced impairment of glucose homeostasis was associated with augmented expression of inflammatory cytokines in adipose tissue, a site for accumulation of lipophilic PCBs. This study determined if RSV protects against PCB-77 induced impairment of glucose disposal in vitro and in vivo and if these beneficial effects are associated with enhanced nuclear factor erythoid 2-related factor 2 (Nrf2) signaling in adipose tissue. PCB-77 increased oxidative stress and abolished insulin stimulated 2-deoxy-d-glucose uptake in 3 T3-L1 adipocytes. These effects were restored by RSV, which resulted in a concentration-dependent increase in NAD(P)H:quinone oxidoreductase 1 (NQO1), the downstream target of Nrf2 signaling. We quantified glucose and insulin tolerance and components of Nrf2 and insulin signaling cascades in adipose tissue of male C57BL/6 mice administered vehicle or PCB-77 (50 mg/kg) and fed a diet with or without resVida (0.1%, or 160 mg/kg per day). PCB-77 impaired glucose and insulin tolerance, and these effects were reversed by RSV. PCB-77 induced reductions in insulin signaling in adipose tissue were also abolished by RSV, which increased NQO1 expression. These results demonstrate that coplanar PCB-induced impairment of glucose homeostasis in mice can be prevented by RSV, potentially through stimulation of Nrf2 signaling and enhanced insulin stimulated glucose disposal in adipose tissue.