ArticleLiterature Review

NRF2 targeting: A promising therapeutic strategy in chronic obstructive pulmonary disease

April 2011
Trends in Molecular Medicine 17(7):363-71

DOI:10.1016/j.molmed.2011.02.006

Source
PubMed

Authors:

Elise Artaud-Macari

Académie de Rouen

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Several convergent destructive mechanisms such as oxidative stress, alveolar cell apoptosis, extracellular matrix proteolysis and chronic inflammation contribute to chronic obstructive pulmonary disease (COPD) development. Evidence suggests that oxidative stress contributes to the pathophysiology of COPD, particularly during exacerbations. Nuclear factor erythroid-2-related factor 2 (NRF2), a transcription factor expressed predominantly in epithelium and alveolar macrophages, has an essential protective role in the lungs through the activation of antioxidant response element-regulated antioxidant and cytoprotective genes. Animal models and human studies have identified NRF2 and several NRF2 target genes as a protective system against inflammation and oxidative stress from cigarette smoke, a major causative factor in COPD development. Hence, NRF2 targeting might provide clinical benefit by reducing both oxidative stress and inflammation in COPD.

Nrf2 activation through the inhibition of Keap1–Nrf2 protein–protein interaction

Article

Full-text available

May 2020
MED CHEM RES

Activation of the transcription factor Nrf2 via the Keap1–Nrf2–ARE signaling system regulates the transcription and subsequent expression of cellular cytoprotective proteins and plays a crucial role in preventing pathological conditions exacerbated by the overproduction of oxidative stress. In addition to electrophilic modulators, direct noncovalent inhibitors that interrupt the Keap1–Nrf2 protein–protein interaction (PPI) leading to Nrf2 activation have attracted a great deal of attention as potential preventive and therapeutic agents for oxidative stress-related diseases. Structural studies of Keap1-binding ligands, development of biochemical and cellular assays, and new structure-based design approaches have facilitated the discovery of small molecule PPI inhibitors. This perspective reviews the Keap1–Nrf2–ARE system, its physiological functions, and the recent progress in the discovery and the potential applications of direct inhibitors of Keap1–Nrf2 PPI.

Caspase-independent apoptosis in infected macrophages triggered by sulforaphane via Nrf2/p38 signaling pathways

Article

Aug 2015

Mycobacterium abscessus (Mabs), a non-tuberculous mycobacterium, is an emerging and rapidly growing opportunistic pathogen that is frequently found in patients with cystic fibrosis and in immunosuppressed patients. Its high tolerance to antibiotics is of great concern for public health. In this study, our results showed that human THP-1-derived macrophages infected with M. abscessus presented an increase in ROS production and cell necrosis. In addition, M. abscessus infection triggered activation of the Nuclear factor E2-related factor 2 (Nrf2) signaling pathway, and the induction of HO-1 and NQO1 expression levels. Interestingly, pretreatment of macrophages with sulforaphane (SFN), an activator of the antioxidant key regulator Nrf2, followed by M. abscessus infection significantly decreased mycobacterial burden. We demonstrated that this reduction in mycobacterial growth was due to an activation in cell apoptosis in SFN-pretreated and M. abscessus-infected macrophages. Pretreatment with specific MAPK inhibitors, PD98059, SP600125, and SB203580 to ERK, JNK, and p38 respectively, failed to inhibit induction of Nrf2 expression, suggesting that Nrf2 signaling pathway was upstream of MAPK signaling. Activation of cell apoptosis was caspase 3/7 independent but p38 MAPK dependent. Moreover, p38 MAPK induction was abolished in macrophages transfected with Nrf2 siRNA. In addition, p38 inhibitor abolished Nrf2-dependent apoptosis in infected macrophages. Taken together, our results indicate that modulation of the Nrf2 signaling using Nrf2 activators may help potentiate the actual drug therapies used to treat mycobacterial infection.

Miconazole Contributes to NRF2 Activation by Noncanonical P62-KEAP1 Pathway in Bladder Cancer Cells

Article

Full-text available

Mar 2020

Purpose: Nuclear factor (erythroid-derived 2)-like 2, also known as NFE2L2 or NRF2, a transcription factor capable of upregulating antioxidant response element (ARE)-mediated expression and cytoprotective proteins, plays critical roles in chemoprevention, inflammation and aging. NRF2 has recently been proposed as a novel target for cancer chemoprevention. The fungicide miconazole has shown promising antiproliferative effects in cancer cells. Materials and methods: After miconazole treatment, the p62-KEAP1-NRF2 activation was analyzed by qPCR and Western blot. The nuclear translocation indicating NRF2 activation was further confirmed by immunofluorescence. Finally, the ROS production was detected by CM-H2DCFDA staining. Results: We demonstrate in this study that miconazole dramatically increases NRF2 activation in bladder cancer cells, in a dose- and time-dependent manner. Interestingly, levels of expression of p62, a noncanonical pathway that mediates NRF2 activation, appeared to increase in accordance with NRF2. We also investigated levels of the negative regulator kelch-like ECH-associated protein 1 (KEAP1), which is involved in NRF2 activation. As expected, a decrease in KEAP1 expression was found after miconazole exposure. Confirmation of NRF2 nuclear translocation was monitored by immunofluorescence. Miconazole-induced generation of reactive oxygen species (ROS) promoted NRF2 activation. Pretreatment of bladder cancer cells with ROS scavengers abolished NRF2 expression and nuclear translocation, indicating that miconazole activates the noncanonical p62-KEAP1-NRF2 pathway, which is regulated by ROS production. Conclusion: Our study elucidates the mechanisms through which miconazole stimulates NRF2 which may contribute to cancer chemopreventive effects.

Therapeutic role of nuclear factor erythroid-2 related factor 2 (Nrf2) in liver disorders

Article

Aug 2020

The progression of liver disease involves the most crucial pathogenic events; oxidative stress and inflammation. Nuclear erythroid 2-related factor 2 (Nrf2) has been regarded as the master regulator of gene expression that are involved in cellular protection via exerting antioxidant, cytoprotective and anti-inflammatory effects. Numerous compounds have been tested as Nrf2 activators including natural (resveratrol, epigallocatechin-3 and curcumin) as well as synthetic compounds (bardoxolone methyl, oltipraz and anethole dithiol ethione) that prevent interaction of Nrf2 with Kelch-like ECH-associated protein 1 (Keap1) due to modification in cysteine sulfhydryl groups of Keap1. The current review demonstrates the function of Nrf2 in various liver diseases; acute liver failure, alcoholic liver disease, non-alcoholic fatty liver disease (NAFLD) and also holds recent studies for the identification of novel drug upregulating Nrf2. There is no approved treatment for liver diseases. Currently oltipraz which is an activator of Nrf2 is in Phase III clinical evaluation for NAFLD; more preclinical and clinical evaluation of drugs as Nrf2 activators is still needed to develop a new treatment strategy for liver disorders.

Article

Full-text available

Mar 2022

Mitochondria are well known as the centre of energy metabolism in eukaryotic cells. However, they can not only generate ATP through the tricarboxylic acid cycle and oxidative phosphorylation but also control the mode of cell death through various mechanisms, especially regulated cell death (RCD), such as apoptosis, mitophagy, NETosis, pyroptosis, necroptosis, entosis, parthanatos, ferroptosis, alkaliptosis, autosis, clockophagy and oxeiptosis. These mitochondria-associated modes of cell death can lead to a variety of diseases. During cell growth, these modes of cell death are programmed, meaning that they can be induced or predicted. Mitochondria-based treatments have been shown to be effective in many trials. Therefore, mitochondria have great potential for the treatment of many diseases. In this review, we discuss how mitochondria are involved in modes of cell death, as well as basic research and the latest clinical progress in related fields. We also detail a variety of organ system diseases related to mitochondria, including nervous system diseases, cardiovascular diseases, digestive system diseases, respiratory diseases, endocrine diseases, urinary system diseases and cancer. We highlight the role that mitochondria play in these diseases and suggest possible therapeutic directions as well as pressing issues that need to be addressed today. Because of the key role of mitochondria in cell death, a comprehensive understanding of mitochondria can help provide more effective strategies for clinical treatment.

Alternative approaches for the treatment of Asthma and COPD: Focus on Cell-based therapies, Epigenetics, and Gene silencing approaches

Article

Full-text available

May 2020

espite many organized health initiatives and critically acclaimed guidelines for proper management of asthma therapy, there is still a large population of severe asthmatics having an uncontrolled disease. Severe persistent asthma, characterized by chronic airway inflammation, increased eosinophils and serum IgE is currently managed by using inhaled corticosteroids. It is quite challenging to get the best treatment guidelines for bronchial asthma in severe asthmatics, particularly in the presence of steroid resistance and the non-responsiveness to β-agonists. For that purpose, other methodologies are required to reverse the uncontrolled airway remodeling in steroid-resistant severe asthma. These advanced alternative approaches should be able to treat asthma symptoms and to improve the inflammatory conditions underlying characteristic pathological features of asthma. The current review focuses and summarizes the alternative approaches used in severe asthma patients. Agents targeting inflammatory cytokines, phosphodiesterase inhibitors, antibodies, oligonucleotides, stem cells, and target drug delivery using gene silencing, offer promise in treating severe asthma. D Abstract www.als-journal.com/ ISSN 2310-5380/ May 2020

Hepatoprotective Effect of Pericarpium zanthoxyli Extract Is Mediated via Antagonism of Oxidative Stress

Article

Full-text available

Jul 2020
EVID-BASED COMPL ALT

Pericarpium zanthoxyli has been extensively used in traditional Oriental medicine to treat gastric disorders and has anti-inflammatory and antioxidative activities. Therefore, the present study examined a possible hepatoprotective effect of a P. zanthoxyli extract (PZE) and investigated the underlying molecular mechanisms. We employed an in vitro model of arachidonic acid (AA) + iron-induced hepatocyte damage and an in vivo model of CCl4-induced liver injury to assess the effects of PZE and evaluated the relevant molecular targets using biochemical assays, flow cytometry analysis, Western blot, and histopathological analysis. The PZE inhibited AA + iron-induced hepatotoxicity in HepG2 cells, improved mitochondrial dysfunction, and reversed an increase in the cellular H2O2 production and a decrease in the reduced GSH levels induced by AA + iron. Treatment with either 30 or 100 μg/ml PZE significantly increased the expression of nuclear factor erythroid 2-related factor 2 (Nrf2) protein, and the latter dose also increased the antioxidant response element- (ARE-) driven luciferase activity and enhanced the protein expressions of glutamate-cysteine ligase catalytic subunit and NAD(P)H:quinone oxidoreductase 1. In addition, treatment with 100 μg/ml PZE for 3 or 6 h increased the phosphorylation rates of Nrf2 and the extracellular signal-regulated kinase. In the in vivo experiment, oral treatment with both 100 and 300 mg/kg PZE inhibited the plasma aspartate aminotransferase activity, and the latter also inhibited the plasma alanine aminotransferase activity. In addition, both doses of PZE ameliorated the parenchymal degeneration and necrosis in the liver induced by CCl4 administration, which was associated with reduced expressions of cleaved caspase-3, cleaved poly (ADP-ribose) polymerase, nitrotyrosine, and 4-hydroxynonenal by PZE. These findings suggest that PZE has protective effects against hepatotoxicity both in vitro and in vivo, which are mainly mediated via its antioxidant activity. 1. Introduction Nowadays, liver disease is still one of the biggest health problems worldwide [1]. Not only is acute liver injury associated with high mortality rates, but it also initiates severe hepatic damage such as liver fibrosis, hepatic cirrhosis, and liver cancer [2]. Excessive oxidative stress caused by stressful events has been shown to be the mechanism underpinning the pathophysiological process in various acute liver injuries [3], highlighting the possibility that that counteracting hepatic oxidative stress can improve both acute liver damage and severe chronic hepatic diseases. Consequently, medical agents with hepatoprotective potential are usually determined for their effects on hepatic oxidative stress [4]. Fortunately, there are several in vitro and in vivo experimental models available to evaluate drug’s effects on hepatic oxidative stress. For example, in vitro, simultaneous application of arachidonic acid (AA) and iron to the cultured hepatic origin cells (i.e., HepG2 cells) is widely used to induce hepatic oxidative stress. AA is a polyunsaturated fatty acid that is released from membranes and promotes oxidative stress, apoptosis, necrosis, and inflammatory response [5–7]. In addition, surplus iron catalyzes the release of AA by altering membrane phospholipids [8, 9]; moreover, it synergizes with AA to induce mitochondrial damage and oxidative stress; thereby, AA + iron is typically toxic to hepatocytes [6]. Carbon tetrachloride (CCl4) is a hepatotoxicant used widely in animals to investigate in vivo the effects of hepatoprotective drugs on toxicant-induced liver injury. CCl4 is mainly metabolized in the liver by cytochrome P450 2E1, producing directly or indirectly a variety of free radical metabolites such as trichloromethyl, trichloromethyl peroxyl, and peroxynitrite, which further generate reactive oxygen species (ROS), constituting the molecular basis for the CCl4-induced hepatotoxicity [10]. Excessive ROS launch lipid peroxidation of the cellular membrane and endoplasmic reticulum and create disturbance of membrane permeability, reduction of protein synthesis, and impairment of DNA, eventually leading to hepatic degeneration and necrosis [11]. An excessive oxidative stress is fundamentally the result of the imbalance of prooxidant and antioxidant functioning in the tissue; that is to say, antagonization of oxidative stress can be implemented by boosting the antioxidant capacity in the tissue. A transcription factor named nuclear factor erythroid 2-related factor 2 (Nrf2) appears to be an important antioxidant molecule in cells [12, 13]. Nrf2 is a basic leucine zipper protein that initiates the expression of antioxidant proteins which protect against the oxidative damage triggered by endogenous and exogenous toxicants [14]. Nrf2 can be detected in a wide range of tissues, including in the liver. Accordingly, the role of Nrf2 in the liver disorders has been frequently evaluated to identify therapeutic candidates [4]. Pericarpium zanthoxyli is the dried pericarp of the ripe fruit from Zanthoxylum bungeanum Maxim. or Zanthoxylum schinifolium Siebold and Zucc. (Rutaceae), which are distributed in China, Japan, and Korea. Extracts of P. zanthoxyli (PZE) have been empirically used in traditional Oriental medicine for treating cold perspiration of the stomach and spleen, stomach pain, indigestion, diarrhea, gastritis, and toothache [15–17]. P. zanthoxyli contains many biologically active constituents, such as (−)-aromadendrene, (−)-isopulegol, (+)-gamma-cadinene, (+)-beta-pinene, (−)-N-acetylanonaine (R-type), hydroxyl-γ-sanshool, hydroxy-α-sanshool, hydroxy-β-sanshool, linalool, nerol, zanthoxylin, zanthobungeanine, α-pinene, piperitone, skimmianine, β-sitosterol, γ-sanshool, terpinen-4-ol, (+, −)α-sanshool, α-terpineol, α-thujene, β-sanshool, and trans-ocimene [18]. Modern scientific experiments have revealed that P. zanthoxyli has antiparasitic, antibacterial, anti-inflammatory, antioxidative, and antidiabetic effects [19]. For example, PZE lowered the plasma levels of IL-1β, cyclooxygenase-2 (COX-2), and TNF-α in rats with cervical spondylotic radiculopathy [20]; flavonoids from P. zanthoxyli effectively scavenged hydroxyl free radicals in an in vitro experiment [21]; PZE inhibited lipid peroxidation induced by lipopolysaccharide in macrophage RAW 264.7 cells and suppressed the expressions of inducible nitric oxide synthase and COX-2 [22]. These preclinical facts along with the empirical use of P. zanthoxyli in treating human digestive diseases prompt us to hypothesize that P. zanthoxyli has hepatoprotective effects which may be mediated via its antioxidant properties. To test this hypothesis, in the present study, we examined whether PZE protects hepatocytes against AA plus iron-induced oxidative stress by manipulating mitochondrial dysfunction, modulating glutathione (GSH) levels and H2O2 production, and interfering with the apoptotic process; in addition, we examined whether this cytoprotective effect is linked to the induction of antioxidant genes through ERK-mediated Nrf2 signaling. Moreover, in in vivo experiments, the possible hepatoprotective effect of PZE was also determined in CCl4-treated mice by measuring the plasma activities of the marker enzymes for hepatic functioning and by analyzing histomorphometrically the histopathological profiles of the hepatic damage. 2. Materials and Methods 2.1. Reagents and Antibodies AA was obtained from Calbiochem (San Diego, CA, USA). Antibodies against procaspase-3, cleaved caspase-3, poly (ADP-ribose) polymerase (PARP), Bcl-2, lamin A/C, ERK1/2, phospho-ERK1/2, and NAD(P)H:quinone oxidoreductase 1 (NQO1) along with horseradish peroxidase-conjugated goat anti-mouse antibodies were provided by Cell Signaling Technology (Beverly, MA, USA). Anti-Nrf2 and anti-cleaved PARP antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The anti-phospho-Nrf2, anti-glutamate-cysteine ligase catalytic subunit (GCLC), and anti-4-hydroxynonenal (4-HNE) polyclonal antibodies were purchased from Abcam (Cambridge, MA, USA). The Fugene® HD and luciferase assay kit were obtained from Promega (Madison, WI, USA). Anti-nitrotyrosine (NT) polyclonal antibody was purchased from Millipore Corporation (Bedford, MA, USA). The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT), rhodamine 123 (Rh123), 2′,7′-dichlorofluorescein diacetate (DCFH-DA), silymarin (SIL), anti-β-actin antibody, and other reagents were supplied by Sigma-Aldrich (St. Louis, MO, USA). 2.2. Preparation of PZE Pericarpium zanthoxyli was obtained from Daewon Pharmacy (located in Daegu, Republic of Korea), and a voucher specimen (number: DHU-GHFM78) was stored in Daegu Haany University (Republic of Korea). PZE was prepared by extracting 100 g of P. zanthoxyli in 1.2 liter of boiling water for 3 h. The PZE was filtered using a 0.22 μm filter (Nalgene, Rochester, NY, USA), lyophilized by a vacuum evaporator, and deposited at −20°C until use. The yield of lyophilized PZE was 14.5%. The ultra-performance liquid chromatography (UPLC) profile of PZE was analyzed using a Waters ACQUITY UPLC system (Waters Corp., Milford, MA, USA) with a Waters ACQUITY photodiode array detector and Waters ACQUITY BEH C18 column (1.7 μm, 2.1 mm × 100 mm) assisted by the Empower software (Figure 1). The PZE includes hyperoside (21.047 ± 0.53 μg/g), 7-methoxycoumarin (19.395 ± 0.63 μg/g), bergapten (2.816 ± 0.096 μg/g), and xanthoxylin (1.756 ± 0.043 μg/g). (a)

Mitochondrial alterations triggered by repeated exposure to fine (PM2.5-0.18) and quasi-ultrafine (PM0.18) fractions of ambient particulate matter

Article

Full-text available

Sep 2020
ENVIRON INT

Nowadays ambient particulate matter (PM) levels still regularly exceed the guideline values established by World Health Organization in most urban areas. Numerous experimental studies have already demonstrated the airway toxicity of the fine fraction of PM (FP), mainly triggered by oxidative stress-induced airway inflammation. However, only few studies have actually paid close attention to the ultrafine fraction of PM (UFP), which is likely to be more easily internalized in cells and more biologically reactive. Mitochondria are major endogenous sources of reactive oxygen species (ROS) through oxidative metabolism, and coordinate many critical cellular signaling processes. Mitochondria have been often studied in the context of PM toxicity and generally associated with apoptosis activation. However, little is known about the underlying adaptation mechanisms that could occur following exposure at sub-apoptotic doses of ambient PM. Here, normal human bronchial epithelial BEAS-2B cells were acutely or repeatedly exposed to relatively low doses (5 µg.cm⁻²) of FP (PM2.5-0.18) or quasi-UFP (Q-UFP; PM0.18) to better access the critical changes in mitochondrial morphology, functions, and dynamics. No significant cytotoxicity nor increase of apoptotic events were reported for any exposure. Mitochondrial membrane potential (ΔΨm) and intracellular ATP content were also not significantly impaired. After cell exposure to sub-apoptotic doses of FP and notably Q-UFP, oxidative phosphorylation was increased as well as mitochondrial mass, resulting in increased production of mitochondrial superoxide anion. Given this oxidative boost, the NRF2-ARE signaling pathway was significantly activated. However, mitochondrial dynamic alterations in favor of accentuated fission process were observed, in particular after Q-UFP vs FP, and repeated vs acute exposure. Taken together, these results supported mitochondrial quality control and metabolism dysfunction as an early lung underlying mechanism of toxicity, thereby leading to accumulation of defective mitochondria and enhanced endogenous ROS generation. Therefore, these features might play a key role in maintaining PM-induced oxidative stress and inflammation within lung cells, which could dramatically contribute to the exacerbation of inflammatory chronic lung diseases. The prospective findings of this work could also offer new insights into the physiopathology of lung toxicity, arguably initiate and/or exacerbate by acutely and rather repeated exposure to ambient FP and mostly Q-UFP.

The methanol extract of Guettarda speciosa Linn. Ameliorates acute lung injury in mice

Article

Full-text available

Feb 2020

Background: Guettarda speciosa is mainly found in tropical areas in Asia. Although G. speciosa is traditionally used to treat some of the inflammatory disorders, the experimental evidence supporting the anti-inflammatory effect of G. speciosa is limited. Here, we sought to obtain evidence that G. speciosa has anti-inflammatory activity using an acute lung injury (ALI) mouse model and to explore possible underlying mechanisms for the activity. Methods: The methanol extract of G. speciosa Linn. (MGS) was fingerprinted by HPLC. Cytotoxicity was determined by MTT and flow cytometer. As for an ALI mouse model, C57BL/6 mice received an intratracheal (i.t.) injection of lipopolysaccharide (LPS). The effects of MGS on lung inflammation in the ALI mice were assessed by differential cell counting and FACS of inflammatory cells and hematoxylin and eosin staining of lung tissue. Proteins were analyzed by immunoprecipitation and immunoblotting, and gene expression was by real-time qPCR. Neutrophil elastase activity was measured by ELISA. Results: MGS did not cause metabolic disarray or produce reactive oxygen species that could induce cytotoxicity. Similar to ALI patients, C57BL/6 mice that received an i.t. LPS developed a high level of neutrophils, increased pro-inflammatory cytokines, and inflicted tissue damage in the lung, which was suppressed by i.t. MGS administered at 2 h after LPS. Mechanistically, MGS activated Nrf2, which was related to MGS interrupting the ubiquitin-dependent degradation of Nrf2. MGS suppressed the nuclear localization of NF-κB induced by LPS, suggesting the inhibition of NF-κB activity. Furthermore, MGS inhibited the enzymatic activity of neutrophil elastase. Conclusion: MGS could suppress lung inflammation in an ALI mouse model, the effect of which could be attributed to multiple mechanisms, including the activation of Nrf2 and the suppression of NF-κB and neutrophil elastase enzymatic activity by MGS.

Primary Amine Modified Gold Nanodots Regulate Macrophage Function and Antioxidant Response: Potential Therapeutics Targeting of Nrf2

Article

Full-text available

Oct 2020
INT J NANOMED

Background: Gold nanoparticles with high biocompatibility and immunomodulatory properties have potential applications in the development of new diagnostic and therapeutic strategies for nanomedicine. Nanoparticles targeting macrophages can manipulate or control immunological diseases. This study assessed the activity of dendrimer-encapsulated gold nanodots (AuNDs) with three surface modifications [ie, outfacing groups with primary amine (AuNDs-NH2), hydroxyl (AuNDs-OH), and quaternary ammonium ions (AuNDs-CH3)] regulated macrophage function and antioxidant response through Nrf2-dependent pathway. Methods: AuNDs were prepared and characterized. Intracellular distribution of AuNDs in human macrophages was observed through confocal microscopy. The activity of AuNDs was evaluated using macrophage functions and antioxidant response in the human macrophage cell line THP-1. Results: AuNDs-NH2 and AuNDs-CH3, but not AuNDs-OH, drove the obvious Nrf2-antioxidant response element pathway in THP-1 cells. Of the three, AuNDs-NH2 considerably increased mRNA levels and antioxidant activities of heme oxygenase 1 and NAD(P)H quinone dehydrogenase 1 in THP-1 cells. IL-6 mRNA and protein expression was mediated through Nrf2 activation in AuNDs-NH2-treated macrophages. Furthermore, Nrf2 activation by AuNDs-NH2 increased the phagocytic ability of THP-1 macrophages. Conclusion: AuNDs-NH2 had immunomodulatory activities in macrophages. The findings of the present work suggested that AuNDs have potential effects against chronic inflammatory diseases via the Nrf2 pathway. Keywords: gold nanodots, macrophage, antioxidant, inflammation

CD28 Deficiency Ameliorates Thoracic Blast Exposure-Induced Oxidative Stress and Apoptosis in the Brain through the PI3K/Nrf2/Keap1 Signaling Pathway

Article

Full-text available

Dec 2019
OXID MED CELL LONGEV

Blast exposure is a worldwide public health concern, but most related research has been focused on direct injury. Thoracic blast exposure-induced neurotrauma is a type of indirect injuries where research is lacking. As CD28 stimulates T cell activation and survival and contributes to inflammation initiation, it may play a role in thoracic blast exposure-induced neurotrauma. However, it has not been investigated. To explore the effects of CD28 on thoracic blast exposure-induced brain injury and its potential molecular mechanisms, a mouse model of thoracic blast exposure-induced brain injury was established. Fifty C57BL/6 wild-type (WT) and fifty CD28 knockout (CD28 -/- ) mice were randomly divided into five groups (one control group and four model groups), with ten mice (from each of the two models) for each group. Lung and brain tissue and serum samples were collected at 12 h, 24 h, 48 h, and 1 week after thoracic blast exposure. Histopathological changes were detected by hematoxylin-eosin staining. The expressions of inflammatory-related factors were detected by ELISA. Oxidative stress in the brain tissue was evaluated by determining the generation of reactive oxygen species (ROS) and the expressions of thioredoxin (TRX), malondialdehyde (MDA), SOD-1, and SOD-2. Apoptosis in the brain tissue was evaluated by TUNEL staining and the levels of Bax, Bcl-xL, Bad, Cytochrome C, and caspase-3. In addition, proteins of related pathways were also studied by western blotting and immunofluorescence. We found that CD28 deficiency significantly reduced thoracic blast exposure-induced histopathological changes and decreased the levels of inflammatory-related factors, including IL-1 β , TNF- α , and S100 β . In the brain tissue, CD28 deficiency also significantly attenuated thoracic blast exposure-induced generation of ROS and expressions of MDA, TRX, SOD-1, and SOD-2; lowered the number of apoptotic cells and the expression of Bax, cleaved caspase-3, Cytochrome C, and Bad; and maintained Bcl-xL expression. Additionally, CD28 deficiency significantly ameliorated thoracic blast exposure-induced increases of p-PI3K and Keap1 and the decrease of Nrf2 expression in the brain. Our results indicate that CD28 deficiency has a protective effect on thoracic blast exposure-induced brain injury that might be associated with the PI3K/Nrf2/Keap1 signaling pathway.

Epigenetic Targets for Therapeutic Approaches in COPD and Asthma. Nutrigenomics – Possible or Illusive

Article

Full-text available

Sep 2019

Oxidative stress generated by cigarette smoking, environmental pollution, or other noxious particles leads to epigenetic changes in the cells of the respiratory tract. They reflect cell adaptation in response to chronic exposure to external factors. Although there is no change in the genetic code, epigenetic changes may be heritable and translated from one generation to another, accumulating abnormalities and rendering cells into entirely different phenotype, causing disease. DNA methylation, post-translation histone modification, ubiquitination, sumoylation and miRNA transcriptional regulation are the major processes that are responsible for the epigenetic control of gene expression. All of them are reversible. They can be regulated by targeting specific enzymes/proteins involved in the process in order to mitigate inflammation. Chronic respiratory diseases have epigenetic signatures that affect gene expression in the lung. Targeting them provides the development of novel diagnostic and therapeutic approaches in respiratory medicine. Nutrigenomics reveals the beneficial effect of natural phytochemicals, affecting key steps in the signaling pathways of chronic respiratory diseases.

Emerging Screening Approaches in the Development of Nrf2-Keap1 Protein-Protein Interaction Inhibitors

Article

Full-text available

Sep 2019
INT J MOL SCI

Due to role of the Keap1-Nrf2 protein-protein interaction (PPI) in protecting cells from oxidative stress, the development of small molecule inhibitors that inhibit this interaction has arisen as a viable approach to combat maladies caused by oxidative stress, such as cancers, neurodegenerative disease and diabetes. To obtain specific and genuine Keap1-Nrf2 inhibitors, many efforts have been made towards developing new screening approaches. However, there is no inhibitor for this target entering the clinic for the treatment of human diseases. New strategies to identify novel bioactive compounds from large molecular databases and accelerate the developmental process of the clinical application of Keap1-Nrf2 protein-protein interaction inhibitors are greatly needed. In this review, we have summarized virtual screening and other methods for discovering new lead compounds against the Keap1-Nrf2 protein-protein interaction. We also discuss the advantages and limitations of different strategies, and the potential of this PPI as a drug target in disease therapy.

Alveolar macrophages generate a noncanonical NRF2-driven transcriptional response to Mycobacterium tuberculosis in vivo

Article

Jul 2019

Alveolar macrophages (AMs) are the first cells to be infected during Mycobacterium tuberculosis (M.tb.) infection. Thus, the AM response to infection is the first of many steps leading to initiation of the adaptive immune response required for efficient control of infection. A hallmark of M.tb. infection is the slow initiation of the adaptive response, yet the mechanisms responsible for this are largely unknown. To study the initial AM response to infection, we developed a system to identify, sort, and analyze M.tb.-infected AMs from the lung within the first 10 days of infection. In contrast to what has been previously described using in vitro systems, M.tb.-infected AMs up-regulate a cell-protective antioxidant transcriptional signature that is dependent on the lung environment but not bacterial virulence. Computational approaches including pathway analysis and transcription factor motif enrichment analysis identify NRF2 as a master regulator of the response. Using knockout mouse models, we demonstrate that NRF2 drives expression of the cell-protective signature in AMs and impairs the control of early bacterial growth. AMs up-regulate a substantial pro-inflammatory response to M.tb. infection only 10 days after infection, yet comparisons with bystander AMs from the same infected animals demonstrate that M.tb.-infected AMs generate a less robust inflammatory response than the uninfected cells around them. Our findings demonstrate that the initial macrophage response to M.tb. in the lung is far less inflammatory than has previously been described by in vitro systems and may impede the overall host response to infection.

Mir-29b Mediates the Regulation of Nrf2 on Airway Epithelial Remodeling and Th1/Th2 Differentiation in COPD Rats

Article

Full-text available

Jul 2019

COPD, or Chronic obstructive pulmonary disease, is an inflammation-related disease and lead to cachexia and muscle wasting. Altered nuclear factor erythroid 2-related factor 2 (Nrf2) expression is found in patients of COPD because it is involved in pulmonary protective effects. MiR-29b could be activated by Nrf2. We hypothesized that miR-29b might mediate the regulation of Nrf2 on Th1/Th2 differentiation and airway epithelial remodeling in COPD rats. SD rats were exposed to smoke for COPD induction. Expression of Nrf2 mRNA and miR-29b in lung tissues was quantified. Expression of Nrf2 and matrix metalloproteinase 2 (MMP2) were also detected by immunohistochemistry and western blot. Th1 markers and Th2 markers were measured by ELISA in peripheral blood. Flow cytometry was used to detect the Th1/Th2 ratio. miR-29b and Nrf2 was manipulated at mRNA level in A549 cells using transfection. Cellular growth and migration were measured in transfectants. In lung tissues of COPD rats, expression of Nrf2 and miR-29b decreased. MMP2, a target of miR-29b, had an opposite expression to miR-29b in peripheral blood. Levels of inflammatory factors and Th1/Th2 ratio increased. MiR-29b mediated the regulation of Nrf2 on remodeling of lung epithelial cells. Blocking Nrf2 expression in A549 cells led to the opposite expression of miR-29b and further decreased MMP2 production; meanwhile, cell growth and motility were improved. Different miR-29b levels affected MMP2 expression and cellular characteristics. The findings suggested that miR-29b was a regulator the pathological progress of COPD. It mediates the effect of Nrf2 on Th1/Th2 differentiation and on remodeling process of airway epithelial cells. Keywords: T helper cell differentiation, Chronic obstructive pulmonary disease, Nuclear factor erythroid 2-related factor, MiR-29b, Matrix metalloproteinase

Pharmacological Applications of Nrf2 Inhibitors as Potential Antineoplastic Drugs

Article

Full-text available

Apr 2019
INT J MOL SCI

Oxidative stress (OS) is associated with many diseases ranging from cancer to neurodegenerative disorders. Nuclear factor-erythroid 2 p45-related factor 2 (Nrf2) is one of the most effective cytoprotective controller against OS. Modulation of Nrf2 pathway constitutes a remarkable strategy in the antineoplastic treatments. A big number of Nrf2-antioxidant response element activators have been screened for use as chemo-preventive drugs in OS associated diseases like cancer even though activation of Nrf2 happens in a variety of cancers. Research proved that hyperactivation of the Nrf2 pathway produces a situation that helps the survival of normal as well as malignant cells, protecting them against OS, anticancer drugs, and radiotherapy. In this review, the modulation of the Nrf2 pathway, anticancer activity and challenges associated with the development of an Nrf2-based anti-cancer treatment approaches are discussed.

Potential Applications of NRF2 Inhibitors in Cancer Therapy

Article

Full-text available

Apr 2019
OXID MED CELL LONGEV

The NRF2/KEAP1 pathway represents one of the most important cell defense mechanisms against exogenous or endogenous stressors. Indeed, by increasing the expression of several cytoprotective genes, the transcription factor NRF2 can shelter cells and tissues from multiple sources of damage including xenobiotic, electrophilic, metabolic, and oxidative stress. Importantly, the aberrant activation or accumulation of NRF2, a common event in many tumors, confers a selective advantage to cancer cells and is associated to malignant progression, therapy resistance, and poor prognosis. Hence, in the last years, NRF2 has emerged as a promising target in cancer treatment and many efforts have been made to identify therapeutic strategies aimed at disrupting its prooncogenic role. By summarizing the results from past and recent studies, in this review, we provide an overview concerning the NRF2/KEAP1 pathway, its biological impact in solid and hematologic malignancies, and the molecular mechanisms causing NRF2 hyperactivation in cancer cells. Finally, we also describe some of the most promising therapeutic approaches that have been successfully employed to counteract NRF2 activity in tumors, with a particular emphasis on the development of natural compounds and the adoption of drug repurposing strategies.

Promising effects of exercise on the cardiovascular, metabolic and immune system during COVID-19 period

Article

Full-text available

Sep 2020

With 4 billion people in lockdown in the world, COVID-19 outbreak may result in excessive sedentary time, especially in the population of vulnerable and disabled subjects. In many chronic disorders and diseases including type 2 diabetes mellitus and hypertension, cardiovascular and immune beneficial effects of exercise interventions should be reminded.

The Effects of Naringin on Cigarette Smoke-Induced Dynamic Changes in Oxidation/Antioxidant System in Lung of Mice

Article

Full-text available

Aug 2020

Naringin possesses strong antioxidative activity and can protect against some respiratory diseases. Oxidative stress is thought to be a major factor in the development of many tobacco-caused diseases. The nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway plays a critical role in the regulation of oxidative stress. The dynamic changes in the antioxidant system in the lung that are induced by cigarette smoke (CS) are not well investigated, and how naringin affects these changes remains unknown. This study aimed to investigate the dynamic changes between the oxidation and antioxidant systems resulting from CS exposure and the effects of naringin on these changes in mice. Mice were chronically exposed to CS for 30 days. The levels of malondialdehyde (MDA), glutathione (GSH), interleukin (IL)-6, and tumor necrosis factor-alpha (TNF-α); the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px); and the expressions of Nrf2, heme oxygenase-1 (HO-1), and nicotinamide adenine dinucleotide phosphate quinone dehydrogenase 1 (NQO1) in lung tissue were measured on days 2, 7, and 30. The levels of MDA, GSH, IL-6, and TNF-α in the lung were found to increase throughout the exposure. SOD and GSH-Px activities showed an increase on day 2 and a decrease on days 7 and 30. The messenger ribonucleic acid expressions of Nrf2, HO-1, and NQO1 were elevated on day 2 and decreased on day 7; Nrf2 and HO-1 expressions were continually decreased, but NQO1 expression was increased again, on day 30. Naringin restored the levels of these biochemical indices to normal throughout the experiment, suggesting that naringin protected against the CS-induced oxidative damage by suppressing the increase of antioxidants resulting from the early stage of CS exposure, as well as inhibiting the depletion of antioxidants due to long-term oxidative stress. Naringin also suppressed lung inflammation by inhibiting IL-6 and TNF-α. These results indicate that naringin possesses a powerful ability to maintain the balance of the oxidation/antioxidant system in the lung when subjected to CS exposure, probably by regulating the Nrf2 signaling pathway.

Beneficial Effects of Naringenin in Cigarette Smoke-Induced Damage to the Lung Based on Bioinformatic Prediction and In Vitro Analysis

Article

Full-text available

Oct 2020
MOLECULES

Naringenin is found mainly in citrus fruits, and is thought to be beneficial in the prevention and control of lung diseases. This study aims to investigate the mechanisms of naringenin against the damage in the lung caused by cigarette smoke. A system bioinformatic approach was proposed to predict the mechanisms of naringenin for protecting lung health. Then, we validated this prediction in BEAS-2B cells treated with cigarette smoke extract (CSE). System bioinformatic analysis indicated that naringenin exhibits protective effects on lung through the inhibition of inflammation and suppression of oxidative stress based on a multi-pathways network, mainly including oxidative stress pathway, Nrf2 pathway, Lung fibrosis pathway, IL-3 signaling pathway, and Aryl hydrocarbon receptor pathway. The in vitro results showed that naringenin significantly attenuated CSE-induced up-regulation of IL-8 and TNF-α. CSE stimulation increased the mRNA expressions of Nrf2, HO-1, and NQO1; the levels of total protein and nuclear protein of Nrf2; and the activity of SOD on days 2 and 4; but decreased these indexes on day 6. Naringenin can balance the antioxidant system by regulating Nrf2 and its downstream genes, preliminarily validating that Nrf2 pathway is involved in the protection offered by naringenin against cigarette smoke-induced damage to the lung. It suggests that dietary naringenin shows possible potential use in the management of lung health.

Allyl isothiocyanate increases MRP1 expression in cigarette smoke extract‑stimulated human bronchial epithelial cells via the JNK/Nrf2 pathway

Article

Full-text available

Feb 2021

Multidrug resistance-related protein 1 (MRP1) is involved in the biological transport of several molecules with diverse structural characteristics outside of the cell. In addition to its transport activity, MRP1 exhibits multiple defense mechanisms in vivo. MRP1 is highly expressed in normal lung tissues and plays a protective role in the process of chronic obstructive pulmonary disease. In the present study, human bronchial epithelial cells (16HBE14o-cells) were stimulated by cigarette smoke extract (CSE) in vitro to simulate a smoking environment. On this basis, the mechanism of Allyl isothiocyanate (AITC) administration on the expression of MRP1 in CSE-stimulated 16HBE14o-cells was investigated. The effects of CSE on the viability of 16 HBE14o-cells were investigated by an MTT assay. The changes in the mRNA expression levels of nuclear erythroid factor 2 (Nrf2) and MRP1 were investigated in CSE-stimulated 16HBE14o-cells using western blotting and reverse transcription quantitative PCR (RT-qPCR). Immunofluorescence analysis was used to detect Nrf2 nuclear translocation. Incubation of the cells with 5% CSE for 24 h had minor effects on cell viability and resulted in the activation of the JNK and p38MAPK signaling pathways. AITC activated the JNK pathway, inhibited the activation of the p38MAPK pathway in 16HBE14o-cells stimulated by 5% CSE and upregulated the expression levels of Nrf2 and MRP1 in a time-dependent manner. The upregulation of Nrf2, MRP1 and of Nrf2, and MRP1 mRNA expression levels in CSE-stimulated cells was inhibited by pretreatment with SP600125 (a JNK pathway inhibitor). Furthermore, the fluorescence intensity in the nucleus was significantly enhanced following AITC pretreatment and the analysis indicated nuclear translocation of Nrf2 in the cells. These results indicated that Nrf2 and MRP1 expression levels in CSE-stimulated cells were altered following AITC pretreatment. Thus demonstrating that the primary mechanism may be associated with activation of the JNK pathway, while the p38MAPK pathway may not be involved.

Large‐scale plasma proteomics can reveal distinct endotypes in chronic obstructive pulmonary disease and severe asthma

Article

Full-text available

Dec 2021

Background Chronic airway diseases including chronic obstructive pulmonary disease (COPD) and asthma are heterogenous in nature and endotypes within are underpinned by complex biology. This study aimed to investigate the utility of proteomic profiling of plasma combined with bioinformatic mining, and to define molecular endotypes and expand our knowledge of the underlying biology in chronic respiratory diseases. Methods The plasma proteome was evaluated using an aptamer-based affinity proteomics platform (SOMAscan®), representing 1238 proteins in 34 subjects with stable COPD and 51 subjects with stable but severe asthma. For each disease, we evaluated a range of clinical/demographic characteristics including bronchodilator reversibility, blood eosinophilia levels, and smoking history. We applied modified bioinformatic approaches used in the evaluation of RNA transcriptomics. Results Subjects with COPD and severe asthma were distinguished from each other by 365 different protein abundancies, with differential pathway networks and upstream modulators. Furthermore, molecular endotypes within each disease could be defined. The protein groups that defined these endotypes had both known and novel biology including groups significantly enriched in exosomal markers derived from immune/inflammatory cells. Finally, we observed associations to clinical characteristics that previously have been under-explored. Conclusion This investigational study evaluating the plasma proteome in clinically-phenotyped subjects with chronic airway diseases provides support that such a method can be used to define molecular endotypes and pathobiological mechanisms that underpins these endotypes. It provided new concepts about the complexity of molecular pathways that define these diseases. In the longer term, such information will help to refine treatment options for defined groups.

Bitter Gourd Honey Ameliorates Hepatic and Renal Diabetic Complications on Type 2 Diabetes Rat Models by Antioxidant, Anti-Inflammatory, and Anti-Apoptotic Mechanisms

Article

Full-text available

Nov 2021

Honey has several pharmacological effects, including anti-diabetic activity. However, the effectiveness of bitter gourd honey (BGH) in the treatment of diabetes mellitus (DM) is unknown. The aim of this study was to determine the antioxidant, anti-inflammatory, and anti-apoptotic properties of BGH on the kidney and liver of a streptozotocin-induced diabetes rat model. Methods: A single dose (nicotinamide 110 mg/kg, streptozotocin (STZ) 55 mg/kg, intraperitoneal (i.p.)) was used to induce DM in male rats. For 28 days, normal or diabetic rats were administered 1 g/kg/day and 2 g/kg/day of BGH orally. After the treatment, blood, liver, and kidney samples were collected and analysed for biochemical, histological, and molecular parameters. In addition, liquid chromatography-mass spectrometry (LC-MS) was used to identify the major bioactive components in BGH. Results: The administration of BGH to diabetic rats resulted in significant reductions in alanine transaminase (ALT),aspartate aminotransferase (AST), creatinine, and urea levels. Diabetic rats treated with BGH showed lesser pathophysiological alterations in the liver and kidney as compared to non-treated control rats. BGH-treated diabetic rats exhibited reduced levels of oxidative stress (MDA levels), inflammatory (MYD88, NFKB, p-NFKB, IKKβ), and apoptotic (caspase-3) markers, as well as higher levels of antioxidant enzymes (SOD, CAT, and GPx) in the liver and kidney. BGH contains many bioactive compounds that may have antioxidative stress, anti-inflammatory, and anti-apoptotic effects. Conclusion: BGH protected the liver and kidney in diabetic rats by reducing oxidative stress, inflammation, and apoptosis-induced damage. As a result, BGH can be used as a potential therapy to ameliorate diabetic complications.

An Update on the Role of Nrf2 in Respiratory Disease: Molecular Mechanisms and Therapeutic Approaches

Article

Full-text available

Aug 2021
INT J MOL SCI

Nuclear factor erythroid 2-related factor (Nrf2) is a transcriptional activator of the cell protection gene that binds to the antioxidant response element (ARE). Therefore, Nrf2 protects cells and tissues from oxidative stress. Normally, Kelch-like ECH-associated protein 1 (Keap1) inhibits the activation of Nrf2 by binding to Nrf2 and contributes to Nrf2 break down by ubiquitin proteasomes. In moderate oxidative stress, Keap1 is inhibited, allowing Nrf2 to be translocated to the nucleus, which acts as an antioxidant. However, under unusually severe oxidative stress, the Keap1-Nrf2 mechanism becomes disrupted and results in cell and tissue damage. Oxide-containing atmospheric environment generally contributes to the development of respiratory diseases, possibly leading to the failure of the Keap1-Nrf2 pathway. Until now, several studies have identified changes in Keap1-Nrf2 signaling in models of respiratory diseases, such as acute respiratory distress syndrome (ARDS)/acute lung injury (ALI), chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis (IPF), and asthma. These studies have confirmed that several Nrf2 activators can alleviate symptoms of respiratory diseases. Thus, this review describes how the expression of Keap1-Nrf2 functions in different respiratory diseases and explains the protective effects of reversing this expression.

Rhodiola rosea L. Attenuates Cigarette Smoke and Lipopolysaccharide-Induced COPD in Rats via Inflammation Inhibition and Antioxidant and Antifibrosis Pathways

Article

Full-text available

Mar 2021
EVID-BASED COMPL ALT

The root cause behind the development of chronic obstructive pulmonary disease (COPD) is cigarette smoke that induces the inflammation of the lung tissue and alveolar destruction. Long-term cigarette smoking can lead to deterioration in lung parenchymal function and cause structural changes in the lung, further resulting in pulmonary fibrosis. Rhodiola rosea L., a traditional medicinal perennial herb, is well known for its numerous pharmacological benefits, including anti-inflammation, antioxidant, antifatigue, antidepressive, and antifibrotic properties. Here, we evaluated the pharmacological effects and mechanisms of the Rhodiola rosea L. (RRL) macroporous resin extract on COPD caused by lipopolysaccharide (LPS) and cigarette smoke (CS) in rats. The RRL significantly improved the pathological structure of the lung tissue. Additionally, RRL decreased the infiltration of inflammatory cells and, subsequently, oxidative stress. Furthermore, the RNAseq assay indicated that RRL attenuated the CS and LPS-induced COPD via anti-inflammatory, antifibrotic, and antiapoptotic activities. Western blot analysis substantiated that the RRL resulted in upregulated levels of Nrf2 and HO-1 as well as downregulated levels of IκBα, NF-κB p65, α-SMA, and TGF-β1. Interestingly, the RRL could protect rats from CS and LPS-induced COPD by inhibiting the ERK1/2 and Smad3 signaling pathways and apoptosis. Thus, the RRL could attenuate CS and LPS-induced COPD through inflammation inhibition and antioxidant and antifibrosis pathways.

Interactions with the macrophages: An emerging targeted approach using novel drug delivery systems in respiratory diseases

Article

Mar 2019
CHEM-BIOL INTERACT

Macrophages are considered as the most flexible cells of the hematopoietic system that are distributed in the tissues to act against pathogens and foreign particles. Macrophages are essential in maintaining homeostatic tissue processes, repair and immunity. Also, play important role in cytokine secretion and signal transduction of the infection so as to develop acquired immunity. Accounting to their involvement in pathogenesis, macrophages present a therapeutic target for the treatment of inflammatory respiratory diseases. This review focuses on novel drug delivery systems (NDDS) including nanoparticles, liposomes, dendrimers, microspheres etc that can target alveolar macrophage associated with inflammation, intracellular infection and lung cancer. The physiochemical properties and functional moieties of the NDDS attributes to enhanced macrophage targeting and uptake. The NDDS are promising for sustained drug delivery, reduced therapeutic dose, improved patient compliance and reduce drug toxicity. Further, the review also discuss about modified NDDS for specificity to the target and molecular targeting via anti-microbial peptides, kinases, NRF-2 and phosphodiesterase.

Potential Role of Antioxidant and Anti-Inflammatory Therapies to Prevent Severe SARS-Cov-2 Complications

Article

Full-text available

Feb 2021

The coronavirus disease 2019 (COVID-19) pandemic is caused by a novel severe acute respiratory syndrome (SARS)-like coronavirus (SARS-CoV-2). Here, we review the molecular pathogenesis of SARS-CoV-2 and its relationship with oxidative stress (OS) and inflammation. Furthermore, we analyze the potential role of antioxidant and anti-inflammatory therapies to prevent severe complications. OS has a potential key role in the COVID-19 pathogenesis by triggering the NOD-like receptor family pyrin domain containing 3 inflammasome and nuclear factor-kB (NF-kB). While exposure to many pro-oxidants usually induces nuclear factor erythroid 2 p45-related factor2 (NRF2) activation and upregulation of antioxidant related elements expression, respiratory viral infections often inhibit NRF2 and/or activate NF-kB pathways, resulting in inflammation and oxidative injury. Hence, the use of radical scavengers like N-acetylcysteine and vitamin C, as well as of steroids and inflammasome inhibitors, has been proposed. The NRF2 pathway has been shown to be suppressed in severe SARS-CoV-2 patients. Pharmacological NRF2 inducers have been reported to inhibit SARS-CoV-2 replication, the inflammatory response, and transmembrane protease serine 2 activation, which for the entry of SARS-CoV-2 into the host cells through the angiotensin converting enzyme 2 receptor. Thus, NRF2 activation may represent a potential path out of the woods in COVID-19 pandemic.

AITC induces MRP1 expression by protecting against CS/CSE-mediated DJ-1 protein degradation via activation of the DJ-1/Nrf2 axis

Article

Full-text available

Nov 2020

The present study aimed to examine the effect of allyl isothiocyanate (AITC) on chronic obstructive pulmonary disease and to investigate whether upregulation of multidrug resistance-associated protein 1 (MRP1) associated with the activation of the PARK7 (DJ-1)/nuclear factor erythroid 2-related factor 2 (Nrf2) axis. Lung function indexes and histopathological changes in mice were assessed by lung function detection and H&E staining. The expression levels of Nrf2, MRP1, heme oxygenase-1 (HO-1), and DJ-1 were determined by immunohistochemistry, Western blotting and reverse transcription-quantitative polymerase chain reaction. Next, the expression of DJ-1 in human bronchial epithelial (16HBE) cells was silenced by siRNA, and the effect of DJ-1 expression level on cigarette smoke extract (CSE)-stimulated protein degradation and AITC-induced protein expression was examined. The expression of DJ-1, Nrf2, HO-1, and MRP1 was significantly decreased in the wild type model group, while the expression of each protein was significantly increased after administration of AITC. Silencing the expression of DJ-1 in 16HBE cells accelerated CSE-induced protein degradation, and significantly attenuated the AITC-induced mRNA and protein expression of Nrf2 and MRP1. The present study describes a novel mechanism by which AITC induces MRP1 expression by protecting against CS/CSEmediated DJ-1 protein degradation via activation of the DJ-1/Nrf2 axis.

Understanding the antioxidant and carbonyl sequestering activity of carnosine: direct and indirect mechanisms

Article

Dec 2020

Carnosine is an endogenous dipeptide whose oral administration has been found to prevent several oxidative based diseases including lung disease, type 2 diabetes and its micro and macrovascular complications, cardiovascular disorders, neurodegenerative and kidney disease. While it is generally accepted that the beneficial effects of carnosine are due to its antioxidant, anti-advanced glycation end product (AGE) and -advanced lipoxidation end product (ALE) and anti-inflammatory properties, the molecular mechanisms explaining such effects have not yet been clearly defined. Studies indicate that carnosine acts by a direct antioxidant mechanism and by sequestering reactive carbonyls (RCS), the byproducts of lipid and glucose oxidation, thus inhibiting AGE and ALE which are the reaction products of RCS with proteins. Moreover, carnosine has also been found to act indirectly by activating the Nrf2 transcription factor, a mechanism that would explain many of the effects evoked by this peptide such as anti-inflammatory, antioxidant, antiglycation and anti-carbonyl effects and taken together would explain its therapeutic effect. The present review reports and discusses the most recent studies on the molecular mechanisms of carnosine which need to be fully clarified before promoting carnosine and derivatives as therapeutic agents.

Trans-4,4′-dihydroxy stilbene ameliorates cigarette smoke-induced progression of chronic obstructive pulmonary disease via inhibiting oxidative stress and inflammatory response

Article

Nov 2019
FREE RADICAL BIO MED

Chronic obstructive pulmonary disease (COPD) is a chronic inflammatory disease resulted from airflow obstructions, and there is a driving requirement for novel and effective preventive and therapeutic agents of COPD. Nuclear factor-erythroid 2-related factor 2 (Nrf2) has been regarded to be a promising therapeutic target for COPD. Resveratrol is a natural Nrf2 activator with antioxidant and anti-inflammatory properties, however, its application is limited by its relative low efficiency and poor bioavailability. Herein, based on the skeleton of resveratrol, trans-4,4'-dihydroxystilbene (DHS) has been firstly identified to be an Nrf2 activator, which is more potent than the well-known sulforaphane (SF) and resveratrol. Our results indicate that DHS blocks Nrf2 ubiquitylation through specifically reacting with Cys151 cysteine in Keap1 protein to activate Nrf2-regulated defensive response, and thus enhances intracellular antioxidant capability. Furthermore, DHS relieves lipopolysaccharide (LPS)-stimulated inflammatory response via inhibition of NF-κB. Importantly, DHS significantly ameliorates pathological alterations (e.g. infiltration of leukocytes and fibrosis), downregulates the levels of oxidant biomarkers malondialdehyde (MDA) and 8-oxo-7,8-dihydro-2'-deoxyguanosin (8-oxo-dG), and inhibits the overproductions of inflammatory mediators [e.g. tumor necrosis factor α (TNF-α), cyclooxygenase-2 (COX-2), and matrix metalloproteinase-9 (MMP-9)] in a cigarette smoke (CS)-induced pulmonary impairment mice model. Taken together, this study demonstrates that DHS attenuates the CS-induced pulmonary impairments through inhibitions of oxidative stress and inflammatory response targeting Nrf2 and NF-κB in vitro and in vivo, and could be developed into a preventive agent against pulmonary impairments induction by CS.

Flavonoid intakes inversely associate with chronic obstructive pulmonary disease in smokers

Article

Jan 2022

Introduction Higher flavonoid intakes are beneficially associated with pulmonary function parameters, however, their association with chronic obstructive pulmonary disease (COPD) is unknown. This study aimed to examine associations between intakes of 1) total flavonoids, 2) flavonoid subclasses, and 3) major flavonoid compounds and incident COPD in participants from the Danish Diet, Cancer, and Health study. Methods This prospective cohort included 55,413 males and females without COPD, aged 50–65 years at recruitment. Habitual flavonoid intakes at baseline were estimated from a food frequency questionnaire using Phenol-Explorer. Danish nationwide registers were used to identify incident cases of COPD. Associations were modelled using restricted cubic splines within Cox proportional hazards models. Results During 23 years follow-up, 5557 participants were diagnosed with COPD. Of these, 4013 were current-, 1062 were former-, and 482 were never-smokers. After multivariable adjustments, participants with the highest, compared to the lowest, total flavonoid intakes had a 20% lower risk of COPD [Quintile 5 versus Quintile 1 HR (95% CI): 0.80 (0.74, 0.87)]; a 6–22% lower risk was observed for each flavonoid subclass. The inverse association between total flavonoid intake and COPD was present in both males and females but was only present in current [HR: 0.77 (0.70, 0.84)] and former [HR: 0.82 (0.69, 0.97)], but not never, smokers. Furthermore, higher flavonoid intakes appeared to lessen, but not negate, the higher risk of COPD associated with smoking intensity. Conclusion Dietary flavonoids may be important for partially mitigating the risk of smoking-related COPD. However, smoking cessation should remain highest priority.

Structure-Activity and Structure-Conformation Relationships of Aryl Propionic Acid Inhibitors of the Kelch-like ECH-Associated Protein 1/Nuclear Factor Erythroid 2-Related Factor 2 (KEAP1/NRF2) Protein-Protein Interaction

Article

Apr 2019

The KEAP1/NRF2-mediated cytoprotective response plays a key role in cellular homoeostasis. Insufficient NRF2 signalling during chronic oxidative stress may be associated with the pathophysiology of several diseases with an inflammatory component, and pathway activation through direct modulation of the KEAP1-NRF2 protein-protein interaction is being increasingly explored as a potential therapeutic strategy. Nevertheless, the physicochemical nature of the KEAP1-NRF2 interface suggests that achieving high affinity for a cell-penetrant drug-like inhibitor might be challenging. We recently reported the discovery of a highly potent tool compound which was used to probe the biology associated with directly disrupting the interaction of NRF2 with the KEAP1 Kelch domain. We now present a detailed account of the medicinal chemistry campaign leading to this molecule, which included exploration and optimization of protein-ligand interactions in three energetic “hot-spots” identified by fragment screening. In particular, we also discuss how consideration of ligand conformational stabilization was important to its development, and present evidence for preorganization of the lead compound which may contribute to its high affinity and cellular activity.

Clinically-approved CFTR modulators rescue Nrf2 dysfunction in cystic fibrosis airway epithelia

Article

Full-text available

May 2019
J CLIN INVEST

Cystic Fibrosis (CF) is a multi-organ progressive genetic disease caused by loss of functional cystic fibrosis transmembrane conductance regulator (CFTR) channel. Previously, we identified a significant dysfunction in CF cells and model mice of the transcription factor nuclear-factor-E2-related factor-2 (Nrf2), a major regulator of redox balance and inflammatory signaling. Here we report that approved F508del CFTR correctors VX809/VX661 recover diminished Nrf2 function and colocalization with CFTR in CF human primary bronchial epithelia by proximity ligation assay, immunoprecipitation, and immunofluorescence, concordant with CFTR correction. F508del CFTR correctors induced Nrf2 nuclear translocation, Nrf2-dependent luciferase activity, and transcriptional activation of target genes. Rescue of Nrf2 function by VX809/VX661 was dependent on significant correction of F508del and was blocked by inhibition of corrected channel function, or high-level shRNA knockdown of CFTR or F508del-CFTR. Mechanistically, F508del-CFTR modulation restored Nrf2 phosphorylation and its interaction with the coactivator CBP. Our findings demonstrate that sufficient modulation of F508del CFTR function corrects Nrf2 dysfunction in CF.

Ultraviolet B irradiation induced Nrf2 degradation occurs via activation of TRPV1 channels in human dermal fibroblasts

Article

Jun 2019
FREE RADICAL BIO MED

Ultraviolet (UV) irradiation causes cellular oxidative stress. Under redox imbalance, Keap1-dependent Nrf2 degradation is minimal. In this study, we examined the role of Ca2+ in Nrf2 homeostasis after UVB irradiation using human dermal fibroblasts. UVB irradiation stimulates 12-lipoxygenase and the product 12-hydroxyeicosatetraenoic acid then activates TRPV1 increasing the cell's cytosolic Ca2+ concentration. UVB irradiation induced reactive oxygen species generation and apoptosis are inhibited in the absence of Ca2+ or in the presence of either a 12-lipoxygenase inhibitor or a TRPV1 inhibitor during and after UVB irradiation. Thus, the Ca2+ increase via TRPV1 is a critical factor in UVB irradiation induced oxidative stress. UVB irradiation induces a Ca2+ dependent Nrf2 degradation and thus activation of TRPV1 with 12-hydroxyeicosatetraenoic acid also decreasing Nrf2 levels. UVB irradiation induced Nrf2 degradation is inhibited by co-treatment of cells with W-7, cyclosporin A, SB-216763 or MG-132, which are inhibitors of calmodulin, calcineurin, GSK3β and the proteasome, respectively. Furthermore, UVB irradiation in parallel induces GSK3β dephosphorylation in a Ca2+ dependent manner. Co-immunoprecipitation showed that UVB irradiation induces an increase in Nrf2 phosphorylation, an increase in the binding of β-TrCP and Nrf2, and an increase in Nrf2 ubiquitination; these effects are all Ca2+ dependent. These findings suggest that UVB irradiation induced GSK3β activation in a Ca2+ dependent manner, which then stimulates the phosphorylation and ubiquitination of Nrf2 via β-TrCP. Indeed, silencing of β-TrCP was found to inhibit UVB irradiation-induced oxidative stress, Nrf2 degradation and apoptosis, while it had no effect on the Ca2+ increase. Taken together, our results suggest that a Ca2+ influx via TRPV1 is responsible for UVB irradiation-induced Nrf2 degradation and that modulation of the Ca2+-calmodulin-calcineurin-GSK3β-Nrf2-β-TrCP-Cullin-1 pathway may explain Ca2+ dependent Nrf2 degradation.

Alveolar macrophages: Novel therapeutic targets for respiratory diseases

Article

Nov 2021
Expet Rev Mol Med

Alveolar macrophages (AMs) are lung-resident myeloid cells that sit at the interface of the airway and lung tissue. Under homeostatic conditions, their primary function is to clear debris, dead cells and excess surfactant from the airways. They also serve as innate pulmonary sentinels for respiratory pathogens and environmental airborne particles and as regulators of pulmonary inflammation. However, they have not typically been viewed as primary therapeutic targets for respiratory diseases. Here, we discuss the role of AMs in various lung diseases, explore the potential therapeutic strategies to target these innate cells and weigh the potential risks and challenges of such therapies. Additionally, in the context of the COVID-19 pandemic, we examine the role AMs play in severe disease and the therapeutic strategies that have been harnessed to modulate their function and protect against severe lung damage. There are many novel approaches in development to target AMs, such as inhaled antibiotics, liposomal and microparticle delivery systems, and host-directed therapies, which have the potential to provide critical treatment to patients suffering from severe respiratory diseases, yet there is still much work to be done to fully understand the possible benefits and risks of such approaches.

Fragment-Guided Discovery of Pyrazole Carboxylic Acid Inhibitors of the Kelch-like ECH-Associated Protein 1: Nuclear Factor Erythroid 2 Related Factor 2 (KEAP1:NRF2) Protein−Protein Interaction

Article

Oct 2021

The NRF2-mediated cytoprotective response is central to cellular homoeostasis, and there is increasing interest in developing small-molecule activators of this pathway as therapeutics for diseases involving chronic oxidative stress. The protein KEAP1, which regulates NRF2, is a key point for pharmacological intervention, and we recently described the use of fragment-based drug discovery to develop a tool compound that directly disrupts the protein-protein interaction between NRF2 and KEAP1. We now present the identification of a second, chemically distinct series of KEAP1 inhibitors, which provided an alternative chemotype for lead optimization. Pharmacophoric information from our original fragment screen was used to identify new hit matter through database searching and to evolve this into a new lead with high target affinity and cell-based activity. We highlight how knowledge obtained from fragment-based approaches can be used to focus additional screening campaigns in order to de-risk projects through the rapid identification of novel chemical series.

Nrf2 modulated the restriction of lung function via impairment of intrinsic autophagy upon real-ambient PM2.5 exposure

Article

Dec 2020
J HAZARD MATER

Compelling studies approve that fine particle matter (PM2.5) exposure was associated with high risk of respiratory disorders. However, the available data assessing the detailed influence of PM2.5 on lung was limited. To overcome the difficulty of inhalational PM2.5 exposure, the real-ambient PM2.5 exposure system was constructed. The mice were exposed to filtered air (FA) or real-ambient PM2.5 (PM2.5), and the adverse effect on lung was determined. Nuclear factor E2-related factor 2 (Nrf2) as a transcription factor, was reported to affect autophagy. Autophagy was proposed as a two-edge sword in respiratory disorders. Here, our data presented that PM2.5 exposure dramatically reduced the lung function of WT mice rather than Nrf2-/- mice. Consistently, thickened alveolar walls was observed in WT mice in PM2.5 exposure group, whereas the histological phenotype of Nrf2-/- mice exhibited no obvious alteration. Furthermore, PM2.5 exposure triggered low-grade production of inflammatory profile in WT and Nrf2-/- mice. Moreover, the protein levels of p62, Beclin1 and LC3B of WT mice rather than Nrf2-/- mice were also altered in PM2.5 exposure group. Taken together, the present study applied the real-ambient exposure system, revealed the adverse effect of air pollution on lung, and proposed the underlying mechanism.

Regulation of Antioxidant Nrf2 Signaling: An Important Pathway in COPD

Chapter

Jan 2020

Oxidative stress plays a role in multiple disorders that include lung diseases like chronic obstructive pulmonary disease (COPD) and asthma. The Nrf2 signaling pathway is the principal regulator of the oxidative stress response and protects against various oxidative stress-related diseases. Nrf2, a bZIP transcription factor, regulates the expression of a battery of antioxidant and detoxification genes in response to oxidative stress. Moreover, Nrf2 signaling responds to multiple environmental and physiological inputs such as endoplasmic reticulum (ER) stress and insulin signaling. The effects of these inputs are often mediated through the molecular regulators of Nrf2 such as kinases, acetylases, and other Nrf2-interacting proteins. Additionally, they can also serve as drug targets for therapeutic modulation of Nrf2 signaling. Therefore, a comprehensive understanding of the molecular mechanism of Nrf2 regulation is important to analyze its role in different physiological and pathological conditions and to develop new drugs that modulate Nrf2 activity. Development of new Nrf2-inducing drugs that can complement current therapeutics is of particular importance for improving the treatment of diseases like COPD where Nrf2 activity is suppressed.

Can activation of NRF2 be a strategy against COVID-19?

Article

Full-text available

Jul 2020
TRENDS PHARMACOL SCI

Acute respiratory distress syndrome (ARDS) caused by SARS-CoV-2 is largely the result of a dysregulated host response, followed by damage to alveolar cells and lung fibrosis. Exacerbated pro-inflammatory cytokines release (cytokine storm) and loss of T-lymphocytes (leucopenia) characterize the most aggressive presentation. Here we propose that a multi-faceted anti-inflammatory strategy based on pharmacological activation of nuclear factor erythroid 2 p45-related factor 2 (NRF2), can be deployed against the virus. The strategy provides robust cytoprotection by restoring the redox and protein homeostasis, promoting resolution of inflammation, and facilitating repair. NRF2 activators such as sulforaphane and bardoxolone methyl are already in clinical trials. The safety and efficacy information of these modulators in humans, together with their well-documented cytoprotective and anti-inflammatory effects in preclinical models, highlight the potential of this armamentarium for deployment to the battlefield against COVID-19.

Plant-Based Chemical Moieties for Targeting Chronic Respiratory Diseases

Chapter

Jul 2021

The World Health Organization (WHO) has reported that chronic respiratory diseases such as asthma, chronic obstructive pulmonary disease, interstitial lung disease, and lung cancer are among the major chronic human diseases that posed a huge challenge to public health and socioeconomic growth. Pharmacotherapy is crucial in the management of these diseases; however, the utilization of conventional treatments is found to be futile, as most patients remained poorly controlled with low quality of life. This has prompted the discovery and development of novel therapeutic agents to improve treatment outcomes. Over the years, researchers have studied a vast range of natural products for their potential in managing chronic respiratory diseases. It has been demonstrated that chemical moieties obtained from plant sources improved pharmacokinetic and toxicological profiles, with a robust multi-prolonged action. Hence, they are held in high regard as possible replacements to address the limitations faced by current therapies. In this chapter, such a phytochemical approach with respect to their molecular mechanisms targeting signalling pathways involved in various chronic respiratory diseases will be discussed. We have also summarized some of the experimental evidence that supports the use of plant-based chemical moieties in chronic respiratory diseases.

Recent Insights Into the Protective Mechanisms of Paeoniflorin in Neurological, Cardiovascular, and Renal Diseases

Article

Apr 2021
J CARDIOVASC PHARM

The monoterpene glycoside paeoniflorin is the principal active constituent of the traditional Chinese herbal medicines, Radix Paeoniae Alba (RPA) and Radix Paeoniae Rubra (RPR), which have been used for millennia to treat cardiovascular diseases (e.g., hypertension, bleeding, and atherosclerosis) and neurological ailments (e.g., headaches, vertigo, dementia, and pain). Recent evidence has revealed that paeoniflorin exerts inhibitory effects on inflammation, fibrosis, and apoptosis by targeting several intracellular signaling cascades. In this review, we address the current knowledge about the pharmacokinetic properties of paeoniflorin and its molecular mechanisms of action. We also present results from recent pre-clinical studies supporting the utility of paeoniflorin for the treatment of pain, cerebral ischemic injury, and neurodegenerative diseases, such as Alzheimer's and Parkinson's diseases. Moreover, new evidence suggests a general protective role of paeoniflorin in heart attack, diabetic kidney, and atherosclerosis. Mechanistically, paeoniflorin exerts multiple anti-inflammatory actions by targeting Toll-like receptor (TLR)-mediated signaling in both parenchymal and immune cells (in particular, macrophages and dendritic cells). A better understanding of the molecular actions of paeoniflorin may lead to the expansion of its therapeutic uses.

Synthesis and Evaluation of Non-covalent Naphthalene-Based KEAP1-NRF2 Inhibitors

Article

Full-text available

Feb 2020

The oxidative stress response, gated by the protein-protein interaction of KEAP1 and NRF2, has garnered significant interest in the past decade. Misregulation in this pathway has been implicated in disease states such as multiple sclerosis, rheumatoid arthritis, and diabetic chronic wounds. Many of the known activators of NRF2 are electrophilic in nature and may operate through several biological pathways, rather than solely through the activation of the oxidative stress response. Recently, our lab has reported a non-electrophilic, monoacidic, naphthalene-based NRF2 activator which exhibited good potency in vitro. Herein, we report a detailed structure-activity relationship of naphthalene-based NRF2 activators, an X-ray crystal structure of our monoacidic KEAP1 inhibitor, and identification of an underexplored area of the NRF2 binding pocket of KEAP1.

Targeting the phosphoinositide-3-kinase/protein kinase B pathway in airway innate immunity

Article

Sep 2020

The airway innate immune system maintains the first line of defense against respiratory infections. The airway epithelium and associated immune cells protect the respiratory system from inhaled foreign organisms. These cells sense pathogens via activation of receptors like toll-like receptors and taste family 2 receptors (T2Rs) and respond by producing antimicrobials, inflammatory cytokines, and chemokines. Coordinated regulation of fluid secretion and ciliary beating facilitates clearance of pathogens via mucociliary transport. Airway cells also secrete antimicrobial peptides and radicals to directly kill microorganisms and inactivate viruses. The phosphoinositide-3-kinase/protein kinase B (Akt) kinase pathway regulates multiple cellular targets that modulate cell survival and proliferation. Akt also regulates proteins involved in innate immune pathways. Akt phosphorylates endothelial nitric oxide synthase (eNOS) enzymes expressed in airway epithelial cells. Activation of eNOS can have anti-inflammatory, anti-bacterial, and anti-viral roles. Moreover, Akt can increase the activity of the transcription factor nuclear factor erythroid 2 related factor-2 that protects cells from oxidative stress and may limit inflammation. In this review, we summarize the recent findings of non-cancerous functions of Akt signaling in airway innate host defense mechanisms, including an overview of several known downstream targets of Akt involved in innate immunity.

Modulation of cigarette smoke extract-induced human bronchial epithelial damage by eucalyptol and curcumin

Article

Full-text available

Mar 2021
HUM EXP TOXICOL

Smoking is one of the most important leading death cause worldwide. From a toxicological perspective, cigarette smoke serves hazards especially for the human being exposed to passive smoke. Over the last decades, the effects of natural compounds on smoking-mediated respiratory diseases such as COPD, asthma, and lung cancer have been under investigation, as well as the mechanistic aspects of disease progression. In the present study, the protective mechanism of eucalyptol (EUC), curcumin (CUR), and their combination on BEAS-2B cells were investigated in vitro to understand their impact on cell death, oxidative cell injury, and inflammatory response induced by 3R4F reference cigarette extract (CSE). According to the present findings, EUC, CUR, and their combination improved cell viability, attenuated CSE-induced apoptosis, and LC3B expression. Further, CSE-induced oxidative damage and inflammatory response in human bronchial epithelial cells were remarkably reduced by the combination treatment through modification of enzymatic antioxidant activity, GSH, MDA, and intracellular ROS levels as well as nitrite and IL-6 levels. In addition, nuclear transloca-tion of Nrf2, a regulatory protein involved in the indirect antioxidant response, was remarkably up-regulated with the combination pre-treatment. In conclusion, EUC and CUR in combination might be a potential therapeutic against smoking-induced lung diseases through antioxidant and inflammatory pathways and results represent valuable background for future in vivo pulmonary toxicity studies.

Cigarette smoke extract decreased basal and lipopolysaccharide‐induced expression of MARCO via degradation of p300

Article

Jun 2020

Background and objective Alveolar macrophages of patients with COPD display impaired cytokine release and diminished phagocytosis. COPD exacerbations exhibit immune dysfunction towards the respiratory pathogens. CS and CSE were reported to aggravate bacterial infections in COPD patients. Methods MARCO is highly expressed in lungs and is involved in pathogen clearance. We investigated the effect of CSE on MARCO expression and its regulatory mechanisms. After relevant siRNA transfection and treatment with CSE and/or LPS, we measured the levels of MARCO by q‐RT PCR, immunoblotting and flow cytometry. Immunofluorescence staining and immunoprecipitation were used to evaluate the mechanism. Results CSE decreased LPS‐induced expression of MARCO mRNA and protein. Upregulation of MARCO by LPS was Nrf2‐dependent. Nrf2 knockdown significantly suppressed LPS‐induced increase in MARCO transcripts. CSE did not block nuclear translocation of Nrf2 in LPS‐treated cells, but rather CSE itself strongly accumulated Nrf2 in the nucleus through the degradation of its cytoplasmic inhibitor, KEAP1. However, CSE markedly suppressed LPS‐induced Nrf2 acetylation. Histone acetyltransferase p300/CBP directly acetylates Nrf2, which augments promoter‐specific DNA binding of Nrf2. Our results reveal CSE‐induced polyubiquitinylation and subsequent degradation of p300 via the proteasome. Pretreatment with proteasome inhibitors completely blocked CSE‐induced degradation of p300 and suppression of MARCO expression. Conclusion These findings suggest that CSE decreases MARCO expression via the proteasomal degradation of p300 in macrophages, which may be in part responsible for impaired bacterial phagocytosis.

Bu-Shen-Fang-Chuan formula attenuates cigarette smoke-induced inflammation by modulating the PI3K/Akt-Nrf2 and NF-κB signalling pathways

Article

Jun 2020
J ETHNOPHARMACOL

Ethnopharmacological relevance Chronic obstructive pulmonary disease (COPD) is a respiratory inflammatory disease. Unlike asthma, COPD is insensitive to glucocorticoid treatment; thus, it is of great importance to find alternative medications, including Chinese medicine, to suppress inflammation. Bu-Shen-Fang-Chuan formula (BSFCF) is commonly used for the treatment of COPD in China. However, the mechanisms of BSFCF in COPD treatment are still unclear. Aim of the study To verify the anti-inflammatory efficacy of BSFCF in COPD and to explore the possible mechanisms underlying its anti-inflammatory efficacy based on the phosphatidylinositol 3 kinase (PI3K)/protein kinase B (Akt)-Nuclear factor erythroid 2-related factor 2 (Nrf2) and Nuclear factor (NF)-κB signalling pathways. Materials and methods A rat model of COPD was established by chronic exposure to cigarette smoke (CS) for 6 months. Bronchoalveolar lavage fluid (BALF) and blood were obtained to detect inflammatory cytokines. Lung samples were harvested, and part of each sample was fixed for subsequent H&E staining and immunohistochemical (IHC) analysis. The remaining lung tissues were used for RNA sequencing analysis and western blotting. Results BSFCF significantly reduced inflammatory infiltration in the lungs of CS-exposed rats and decreased the concentrations of tumor necrosis factor (TNF)-α and interleukin (IL)-6 in both the BALF and serum. Additionally, BSFCF evidently attenuated NF-κB activation and downregulation of glucocorticoid receptor (GR) caused by CS. Furthermore, BSFCF increased the activation of PI3K/Akt-Nrf2 signalling in response to CS. Conclusions BSFCF attenuated CS-induced inflammation in COPD, which was partially achieved through the PI3K/Akt-Nrf2 and NF-κB signalling pathways.

Discovery of 2-oxy-2-phenylacetic acid substituted naphthalene sulfonamide derivatives as potent KEAP1-NRF2 protein-protein interaction inhibitors for inflammatory conditions

Article

Aug 2020
EUR J MED CHEM

Nuclear factor erythroid 2-related factor 2 (NRF2) is a pleiotropic transcription factor which regulates the constitutive and inducible transcription of a wide array of genes and confers protection against a variety of pathologies. Directly disrupting Kelch-like ECH-associated protein 1 (KEAP1)-NRF2 protein-protein interaction (PPI) has been explored as a promising strategy to activate NRF2. We reported here the first identification of a series of 2-oxy-2-phenylacetic acid substituted naphthalene sulfonamide derivatives as potent KEAP1-NRF2 inhibitors. Our efforts led to the potent small molecule KEAP1-NRF2 inhibitor, 20c, which exhibited a Kd of 24 nM to KEAP1 and an IC50 of 75 nM in disrupting KEAP1-NRF2 interaction. Subsequent biological studies provided consistent evidence across mouse macrophage cell-based and in vivo models that 20c induced NRF2 target gene expression and enhanced downstream antioxidant and anti-inflammatory activities. Our study not only demonstrated that small molecule KEAP1-NRF2 PPI inhibitors can be potential preventive and therapeutic agents for diseases and conditions involving oxidative stress and inflammation but also enriched the chemical diversity of the KEAP1-NRF2 inhibitors.

Antioxidative effect of DJ-1 is enhanced in NG108-15 cells by DPMQ induced copper influx

Article

Dec 2020

Disruption of copper homeostasis is closely involved in neurodegenerative disorders. This study examined whether a hybrid copper binding compound, (E)-2-(4-(dimethylamino)phenylimino)methyl)quinolin-8-ol (DPMQ), is able to protect NG108-15 cells against oxidative stress. we found that treatment of cells with rotenone or hydrogen peroxide increased cellular oxidative stress and resulted in mitochondrial dysfunction and apoptosis. The cellular levels of Nrf2 and the Cu ²⁺ chaperone DJ-1 were also decreased. These oxidative detrimental effects were all inhibited when cells were co-treated with DPMQ. DPMQ increased cellular Cu ²⁺ content, DJ-1 protein level, superoxide dismutase (SOD) activity and Nrf2 nuclear translocation under basal state. The activity of SOD decreased under redox imbalance and this decrease was blocked by DPMQ treatment, while the protein level of SOD1 remained unaltered regardless of the oxidative stress and DPMQ treatment. Using endogenous proteins, co-immunoprecipitation showed that DJ-1 bound with SOD1 and Nrf2 individually. The amount of Nrf2, bound to DJ-1, consistently reflected its cellular level, while the amount of SOD1, bound to DJ-1, was potentiated by DPMQ, being greater in basal state than under redox imbalance. Simultaneous inclusion of non-permeable Cu ²⁺ chelator tetrathiomolybdate or triethylenetetramine during DPMQ treatment blocked all aforementioned effects of DPMQ, showing that the dependency of the effect of DPMQ on extracellular Cu ²⁺ . In addition, silencing of DJ-1 blocked the protection of DPMQ against oxidative stress. Taken all together, our results suggest that DPMQ stabilizes DJ-1 in a Cu ²⁺ dependent manner, which then brings about SOD1 activation and Nrf2 nuclear translocation; these together alleviate cellular oxidative stress.

Transcription Factor Nrf2 as a Potential Therapeutic Target for Prevention of Cytokine Storm in COVID-19 Patients

Article

Full-text available

Jul 2020

Nrf2 is a key transcription factor responsible for antioxidant defense in many tissues and cells, including alveolar epithelium, endothelium, and macrophages. Furthermore, Nrf2 functions as a transcriptional repressor that inhibits expression of the inflammatory cytokines in macrophages. Critically ill patients with COVID-19 infection often present signs of high oxidative stress and systemic inflammation – the leading causes of mortality. This article suggests rationale for the use of Nrf2 inducers to prevent development of an excessive inflammatory response in COVID-19 patients.

Classes of drugs that target the cellular components of inflammation under clinical development for COPD

Article

May 2021

Introduction : The persistent inflammation that characterizes COPD and affects its natural course also impacting on symptoms has prompted research to find molecules that can regulate the inflammatory process but still available anti-inflammatory therapies provide little or no benefit in COPD patients. Consequently, numerous anti-inflammatory molecules that are effective in animal models of COPD have been or are being evaluated in humans. Areas covered : In this article we describe several classes of drugs that target the cellular components of inflammation under clinical development for COPD. Expert opinion Although the results of many clinical trials with new molecules have often been disappointing, several studies are underway to investigate whether some of these molecules may be effective in treating specific subgroups of COPD patients. Indeed, the current perspective is to apply a more personalized treatment to the patient. This means being able to better define the patient’s inflammatory state and treat it in a targeted manner. Unfortunately, the difficulty in translating encouraging experimental data into human clinical trials, the redundancy in the effects induced by signal-transmitting substances and the non-specific effects of many classes that are undergoing clinical trials, do not yet allow specific inflammatory cell types to be targeted.

Detection of Human Cytomegalovirus (HCMV) and Porcine Endogenous Retrovirus (PERV) with One Step Extraction Method

Article

Full-text available

Jan 2009

Xenotransplantation is thought to be one of the alternative methods to overcome the shortage of human organs for transplantation. Recipients should be immunosuppressed for graft survival, and thus, there is a need for developing diagnostic modality that can detect diverse infections originating from animals and recipients rapidly, in the early stage, and with high sensitivity using small volume of samples. This study was carried out to develop a fast, simple, and robust technique for the preparation of HCMV DNA and PERV RNA using small volume of samples.

GSK-3beta Acts Upstream of Fyn Kinase in Regulation of Nuclear Export and Degradation of NF-E2 Related Factor 2

Article

Full-text available

Jun 2007

NF-E2-related factor 2 (Nrf2) regulates expression and coordinated induction of a battery of chemoprotective genes in response to oxidative and electrophilic stress. This leads to protection against oxidative stress and neoplastic diseases. Nuclear import and export of Nrf2 play a significant role in control of nuclear levels of Nrf2 and thus the expression of Nrf2 down-stream genes. Tyrosine kinase Fyn phosphorylates tyrosine 568 of Nrf2 that leads to the nuclear export of Nrf2. In this study, we investigated the upstream factor(s) in regulation of Fyn and Fyn-mediated nuclear export of Nrf2. The investigations shed light on a novel mechanism of Nrf2 regulation in response to oxidative stress. We demonstrate that GSK-3beta acts upstream of Fyn kinase in control of nuclear export of Nrf2. Chemical and short interfering RNA-mediated inhibition of GSK-3beta led to nuclear accumulation of Nrf2 and transcriptional activation of the Nrf2 downstream gene nqo1. Chemical and short interfering RNA inhibition of GSK-3beta and Fyn individually and in combination revealed that both kinases follow the same pathway to regulate nuclear export of Nrf2. We further demonstrate that hydrogen peroxide phosphorylates tyrosine 216 of GSK-3beta. This leads to activation of GSK-3beta. The activated GSK-3beta phosphorylates Fyn at threonine residue(s). Phosphorylated Fyn accumulates in the nucleus and phosphorylates Nrf2 at tyrosine 568. This leads to nuclear export, ubiquitination, and degradation of Nrf2.

SCF/ -TrCP Promotes Glycogen Synthase Kinase 3-Dependent Degradation of the Nrf2 Transcription Factor in a Keap1-Independent Manner

Article

Full-text available

Feb 2011
MOL CELL BIOL

Regulation of transcription factor Nrf2 (NF-E2-related factor 2) involves redox-sensitive proteasomal degradation via the E3 ubiquitin ligase Keap1/Cul3. However, Nrf2 is controlled by other mechanisms that have not yet been elucidated. We now show that glycogen synthase kinase 3 (GSK-3) phosphorylates a group of Ser residues in the Neh6 domain of mouse Nrf2 that overlap with an SCF/β-TrCP destruction motif (DSGIS, residues 334 to 338) and promotes its degradation in a Keap1-independent manner. Nrf2 was stabilized by GSK-3 inhibitors in Keap1-null mouse embryo fibroblasts. Similarly, an Nrf2ΔETGE mutant, which cannot be degraded via Keap1, accumulated when GSK-3 activity was blocked. Phosphorylation of a Ser cluster in the Neh6 domain of Nrf2 stimulated its degradation because a mutant Nrf2ΔETGE 6S/6A protein, lacking these Ser residues, exhibited a longer half-life than Nrf2ΔETGE. Moreover, Nrf2ΔETGE 6S/6A was insensitive to β-TrCP regulation and exhibited lower levels of ubiquitination than Nrf2ΔETGE. GSK-3β enhanced ubiquitination of Nrf2ΔETGE but not that of Nrf2ΔETGE 6S/6A. The Nrf2ΔETGE protein but not Nrf2ΔETGE 6S/6A coimmunoprecipitated with β-TrCP, and this association was enhanced by GSK-3β. Our results show for the first time that Nrf2 is targeted by GSK-3 for SCF/β-TrCP-dependent degradation. We propose a “dual degradation” model to describe the regulation of Nrf2 under different pathophysiological conditions.

Induction of Heme Oxygenase-1, Biliverdin Reductase and H-Ferritin in Lung Macrophage in Smokers with Primary Spontaneous Pneumothorax: Role of HIF-1α

Article

Full-text available

May 2010
PLOS ONE

Few data concern the pathophysiology of primary spontaneous pneumothorax (PSP), which is associated with alveolar hypoxia/reoxygenation. This study tested the hypothesis that PSP is associated with oxidative stress in lung macrophages. We analysed expression of the oxidative stress marker 4-HNE; the antioxidant and anti-inflammatory proteins heme oxygenase-1 (HO-1), biliverdin reductase (BVR) and heavy chain of ferritin (H-ferritin); and the transcription factors controlling their expression Nrf2 and HIF-1alpha, in lung samples from smoker and nonsmoker patients with PSP (PSP-S and PSP-NS), cigarette smoke being a risk factor of recurrence of the disease. mRNA was assessed by RT-PCR and proteins by western blot, immunohistochemistry and confocal laser analysis. 4-HNE, HO-1, BVR and H-ferritin were increased in macrophages from PSP-S as compared to PSP-NS and controls (C). HO-1 increase was associated with increased expression of HIF-1alpha mRNA and protein in alveolar macrophages in PSP-S patients, whereas Nrf2 was not modified. To understand the regulation of HO-1, BVR and H-ferritin, THP-1 macrophages were exposed to conditions mimicking conditions in C, PSP-S and PSP-NS patients: cigarette smoke condensate (CS) or air exposure followed or not by hypoxia/reoxygenation. Silencing RNA experiments confirmed that HIF-1alpha nuclear translocation was responsible for HO-1, BVR and H-ferritin induction mediated by CS and hypoxia/reoxygenation. PSP in smokers is associated with lung macrophage oxidative stress. The response to this condition involves HIF-1alpha-mediated induction of HO-1, BVR and H-ferritin.

The Ubiquitin-conjugating Enzyme UbcM2 Can Regulate the Stability and Activity of the Antioxidant Transcription Factor Nrf2

Article

Full-text available

Jul 2010
J BIOL CHEM

The transcription factor nuclear factor E2-related factor 2 (Nrf2) induces the expression of antioxidant gene products that neutralize reactive oxygen species and restore redox homeostasis. Nrf2 is constitutively degraded by the ubiquitin proteolytic system in unperturbed cells, but this turnover is arrested in response to oxidative stress, thereby leading to Nrf2 accumulation. Yet, a mechanistic understanding of how Nrf2 stabilization and transcriptional activation are coupled remains to be determined. We have discovered that the ubiquitin-conjugating enzyme UbcM2 is a novel regulator of Nrf2. Recombinant Nrf2 and UbcM2 form a complex upon alkylation of a non-catalytic cysteine in UbcM2, Cys-136. Substitution of this cysteine with a phenylalanine (C136F) to mimic cysteine oxidation/alkylation results in constitutive binding of UbcM2 to Nrf2 and an increased half-life of the transcription factor in vivo. We provide evidence that UbcM2 and Nrf2 form a nuclear complex utilizing the DNA binding, Neh1 domain, of Nrf2. Finally, we demonstrate that UbcM2 can enhance the transcriptional activity of endogenous Nrf2 and that Cys-136 and the active-site cysteine, Cys-145, jointly contribute to this regulation. Collectively, these data identify UbcM2 as a novel component of the Nrf2 regulatory circuit and position cysteine 136 as a putative redox sensor in this signaling pathway. This work implicates UbcM2 in the restoration of redox homeostasis following oxidative stress.

Global mapping of binding sites for Nrf2 identifies novel targets in cell survival response through ChIP-Seq profiling and network analysis

Article

Full-text available

May 2010
NUCLEIC ACIDS RES

The Nrf2 (nuclear factor E2 p45-related factor 2) transcription factor responds to diverse oxidative and electrophilic environmental stresses by circumventing repression by Keap1, translocating to the nucleus, and activating cytoprotective genes. Nrf2 responses provide protection against chemical carcinogenesis, chronic inflammation, neurodegeneration, emphysema, asthma and sepsis in murine models. Nrf2 regulates the expression of a plethora of genes that detoxify oxidants and electrophiles and repair or remove damaged macromolecules, such as through proteasomal processing. However, many direct targets of Nrf2 remain undefined. Here, mouse embryonic fibroblasts (MEF) with either constitutive nuclear accumulation (Keap1(-/-)) or depletion (Nrf2(-/-)) of Nrf2 were utilized to perform chromatin-immunoprecipitation with parallel sequencing (ChIP-Seq) and global transcription profiling. This unique Nrf2 ChIP-Seq dataset is highly enriched for Nrf2-binding motifs. Integrating ChIP-Seq and microarray analyses, we identified 645 basal and 654 inducible direct targets of Nrf2, with 244 genes at the intersection. Modulated pathways in stress response and cell proliferation distinguish the inducible and basal programs. Results were confirmed in an in vivo stress model of cigarette smoke-exposed mice. This study reveals global circuitry of the Nrf2 stress response emphasizing Nrf2 as a central node in cell survival response.

The Flavanol (−)-Epicatechin Prevents Stroke Damage through the Nrf2/HO1 Pathway

Article

Full-text available

May 2010
J CEREBR BLOOD F MET

Correction to: Journal of Cerebral Blood Flow & Metabolism (2010) 30, 1951–1961; doi: 10.1038/jcbfm.2010.53 After the publication of this article, the following errors in the Materials and Methods section were noticed: On page 1953 in the Materials and Methods section it is erroneously stated that ‘Cells were washed and incubated with rhodamine-conjugated, affinity-purified donkey anti-rat IgG (H+L) and fluorescein isothiocyanate (FITC)-conjugated, affinity-purified goat anti-rabbit IfG (H+L)’. Correction: Cells were washed and incubated with rhodamine-conjugated, affinity-purified donkey anti-rabbit IgG (H+L) and fluorescein isothiocyanate (FITC)-conjugated, affinity-purified goat anti-mouse IgG (H+L). In Figure 5B the +and – representation is incorrect as published. The corrected appears below. [Figure: see text]

Nrf2-regulated PPARγ Expression Is Critical to Protection against Acute Lung Injury in Mice

Article

Full-text available

Mar 2010
AM J RESP CRIT CARE

The NF-E2 related factor 2 (Nrf2)-antioxidant response element (ARE) pathway is essential for protection against oxidative injury and inflammation including hyperoxia-induced acute lung injury. Microarray expression profiling revealed that lung peroxisome proliferator activated receptor gamma (PPARgamma) induction is suppressed in hyperoxia-susceptible Nrf2-deficient (Nrf2(-/-)) mice compared with wild-type (Nrf2(+/+)) mice. PPARgamma has pleiotropic beneficial effects including antiinflammation in multiple tissues. We tested the hypothesis that PPARgamma is an important determinant of pulmonary responsivity to hyperoxia regulated by Nrf2. A computational bioinformatic method was applied to screen potential AREs in the Pparg promoter for Nrf2 binding. The functional role of a potential ARE was investigated by in vitro promoter analysis. A role for PPARgamma in hyperoxia-induced acute lung injury was determined by temporal silencing of PPARgamma via intranasal delivery of PPARgamma-specific interference RNA and by administration of a PPARgamma ligand 15-deoxy-Delta(12,14)-prostaglandin J(2) in mice. Deletion or site-directed mutagenesis of a potential ARE spanning -784/-764 sequence significantly attenuated hyperoxia-increased Pparg promoter activity in airway epithelial cells overexpressing Nrf2, indicating that the -784/-764 ARE is critical for Nrf2-regulated PPARgamma expression. Mice with decreased lung PPARgamma by specific interference RNA treatment had significantly augmented hyperoxia-induced pulmonary inflammation and injury. 15 Deoxy-Delta(12,14)-prostaglandin J(2) administration significantly reduced hyperoxia-induced lung inflammation and edema in Nrf2(+/+), but not in Nrf2(-/-) mice. Results indicate for the first time that Nrf2-driven PPARgamma induction has an essential protective role in pulmonary oxidant injury. Our observations provide new insights into the therapeutic potential of PPARgamma in airway oxidative inflammatory disorders.

Oxidative stress targets in pulmonary emphysema: Focus on the Nrf2 pathway

Article

Full-text available

Mar 2010
EXPERT OPIN THER TAR

Oxidative stress has been implicated in the pathogenesis of pulmonary emphysema. Nuclear factor erythroid-2-related factor 2 (Nrf2) a major antioxidant transcription factor could play a protective role in pulmonary emphysema. Nrf2 is ubiquitously expressed throughout the lung, but is predominantly found in epithelium and alveolar macrophages. Evidence suggests that Nrf2 and several Nrf2 downstream genes have an essential protective role in the lung against oxidative stress from environmental pollutants and toxicants such as cigarette smoke, a major causative factor for the development and progression of pulmonary emphysema. Application of Nrf2-deficient mice identified an extensive range of protective roles for Nrf2 against the pathogenesis of pulmonary emphysema. Therefore, Nrf2 promises to be an attractive therapeutic target for intervention and prevention strategies. In this review, we discuss recent findings on the association of oxidative stress with pulmonary emphysema. We also address the mechanisms of Nrf2 lung protection against oxidative stress based on emerging evidence from experimental oxidative disease models and human studie. The current literature suggests that among oxidative stress targets, Nrf2 is a valuable therapeutic target in pulmonary emphysema.

Clinical COPD phenotypes: A novel approach using principal component and cluster analyses

Article

Full-text available

Sep 2010
EUR RESPIR J

Classification of chronic obstructive pulmonary disease (COPD) is usually based on the severity of airflow limitation, which may not reflect phenotypic heterogeneity. Here, we sought to identify COPD phenotypes using multiple clinical variables. COPD subjects recruited in a French multicentre cohort were characterised using a standardised process. Principal component analysis (PCA) was performed using eight variables selected for their relevance to COPD: age, cumulative smoking, forced expiratory volume in 1 s (FEV(1)) (% predicted), body mass index, exacerbations, dyspnoea (modified Medical Research Council scale), health status (St George's Respiratory Questionnaire) and depressive symptoms (hospital anxiety and depression scale). Patient classification was performed using cluster analysis based on PCA-transformed data. 322 COPD subjects were analysed: 77% were male; median (interquartile range) age was 65.0 (58.0-73.0) yrs; FEV(1) was 48.9 (34.1-66.3)% pred; and 21, 135, 107 and 59 subjects were classified in Global Initiative for Chronic Obstructive Lung Disease (GOLD) stages 1, 2, 3 and 4, respectively. PCA showed that three independent components accounted for 61% of variance. PCA-based cluster analysis resulted in the classification of subjects into four clinical phenotypes that could not be identified using GOLD classification. Importantly, subjects with comparable airflow limitation (FEV(1)) belonged to different phenotypes and had marked differences in age, symptoms, comorbidities and predicted mortality. These analyses underscore the need for novel multidimensional COPD classification for improving patient care and quality of clinical trials.

Dihydro-CDDO-Trifluoroethyl Amide (dh404), a Novel Nrf2 Activator, Suppresses Oxidative Stress in Cardiomyocytes

Article

Full-text available

Dec 2009
PLOS ONE

Targeting Nrf2 signaling appears to be an attractive approach for the treatment of maladaptive cardiac remodeling and dysfunction; however, pharmacological modulation of the Nrf2 pathway in the cardiovascular system remains to be established. Herein, we report that a novel synthetic triterpenoid derivative, dihydro-CDDO-trifluoroethyl amide (dh404), activates Nrf2 and suppresses oxidative stress in cardiomyocytes. Dh404 interrupted the Keap1-Cul3-Rbx1 E3 ligase complex-mediated Nrf2 ubiquitination and subsequent degradation saturating the binding capacity of Keap1 to Nrf2, thereby rendering more Nrf2 to be translocated into the nuclei to activate Nrf2-driven gene transcription. A mutant Keap1 protein containing a single cysteine-to-serine substitution at residue 151 within the BTB domain of Keap1 was resistant to dh404-induced stabilization of Nrf2 protein. In addition, dh404 did not dissociate the interaction of Nrf2 with the Keap1-Cul3-Rbx1 E3 ligase complex. Thus, it is likely that dh404 inhibits the ability of Keap1-Cul3-Rbx1 E3 ligase complex to target Nrf2 for ubiquitination and degradation via modifying Cys-151 of Keap1 to change the conformation of the complex. Moreover, dh404 was able to stabilize Nrf2 protein, to enhance Nrf2 nuclear translocation, to activate Nrf2-driven transcription, and to suppress angiotensin II (Ang II)-induced oxidative stress in cardiomyocytes. Knockdown of Nrf2 almost blocked the anti-oxidative effect of dh404. Dh404 activated Nrf2 signaling in the heart. Taken together, dh404 appears to be a novel Nrf2 activator with a therapeutic potential for cardiac diseases via suppressing oxidative stress.

Cigarette Smoking Blocks the Protective Expression of Nrf2/ARE Pathway in Peripheral Mononuclear Cells of Young Heavy Smokers Favouring Inflammation

Article

Full-text available

Dec 2009
PLOS ONE

Cigarette smoking is an important risk factor for atherosclerosis, a chronic inflammatory disease. However the underlying factors of this effect are unclear. It has been hypothesized that water-soluble components of cigarette smoke can directly promote oxidative stress in vasculature and blood cells. Aim of this study was to study the relationship between oxidative stress and inflammation in a group of young smokers. To do this we evaluated: 1) the oxidation products of phospholipids (oxPAPC) in peripheral blood mononuclear cells (PBMC); 2) their role in causing PBMC reactive oxygen species (ROS) generation and changes in GSH; 3) the expression of the transcription factor NF-E2-related factor 2 (Nrf2) and of related antioxidant genes (ARE); 4) the activation of NF-kB and C-reactive protein (CRP) values. We studied 90 healthy volunteers: 32 non-smokers, 32 moderate smokers (5-10 cigarettes/day) and 26 heavy smokers (25-40 cigarettes/day). OxPAPC and p47phox expression, that reasonably reflects NADPH oxidase activity, were higher in moderate smokers and heavy smokers than in non-smokers (p

Heightened Endoplasmic Reticulum Stress in the Lungs of Patients with Chronic Obstructive Pulmonary Disease

Article

Full-text available

Oct 2009
AM J RESP CRIT CARE

Nuclear factor erythroid 2-related factor 2 (Nrf2), an important regulator of lung antioxidant defenses, declines in chronic obstructive pulmonary disease (COPD). However, Nrf2 also regulates the proteasome system that degrades damaged and misfolded proteins. Because accumulation of misfolded proteins in the endoplasmic reticulum (ER) causes ER stress and ER stress-induced apoptosis, Nrf2 may potentially prevent ER stress-mediated apoptosis in COPD. To determine whether Nrf2-regulated proteasome function affects ER stress-mediated apoptosis in COPD. We assessed the expression of Nrf2, Nrf2-dependent proteasomal subunits, proteasomal activity, markers of ER stress, and apoptosis in emphysematous lungs of mice exposed to cigarette smoke (CS) as well as peripheral lung tissues from normal control subjects and patients with COPD. Compared with wild-type mice, emphysematous lungs of CS-exposed Nrf2-deficient mice exhibited markedly lower proteasomal activity and elevated markers of ER stress and apoptosis. Furthermore, compared with normal control subjects, lungs of patients with mild and advanced COPD showed a marked decrease in the expression of Nrf2-regulated proteasomal subunits and total proteasomal activity. However, they were associated with greater levels of ER stress and apoptosis markers. In vitro studies have demonstrated that enhancing proteasomal activity in Beas2B cells either by sulforaphane, an activator of Nrf2, or overexpression of Nrf2-regulated proteasomal subunit PSMB6, significantly inhibited cigarette smoke condensate (CSC)-induced ER stress and cell death. Impaired Nrf2 signaling causes significant decline in proteasomal activity and heightens ER stress response in lungs of patients with COPD and CS-exposed mice. Accordingly, pharmacological approaches that augment Nrf2 activity may protect against COPD progression by both up-regulating antioxidant defenses and relieving ER stress.

Targeted treatment in COPD: a multi-system approach for a multi-system disease

Article

Full-text available

Sep 2009

Chronic obstructive pulmonary disease is a varied condition when examined from a number of different perspectives including factors which influence disease development, pathological process and clinical features. There may be a complex interaction between the degree by which each of these processes influences the development of COPD and the subsequent clinical phenotype with which the patient presents. The varied host response and subsequent clinical phenotype has generated much interest in recent years. It is possible that failure of treatment to impact on mortality and reverse the disease process is because of the heterogeneous nature of the condition. Identification and targeted treatment of clinical and pathological phenotypes within the broad spectrum of COPD may therefore improve outcome. This article will review previous work which has attempted to phenotype COPD and identify if specific treatment for these phenotypes has been shown to be of benefit. It will examine the work on pathological processes and clinical manifestations, both pulmonary and systemic, and will focus on pharmacological therapies.

Level and course of FEV1 in relation to polymorphisms in NFE2L2 and KEAP1 in the general population

Article

Full-text available

Sep 2009

The metabolism of xenobiotics plays an essential role in smoking related lung function loss and development of Chronic Obstructive Pulmonary Disease. Nuclear Factor Erythroid 2-Like 2 (NFE2L2 or NRF2) and its cytosolic repressor Kelch-like ECH-associated protein-1 (KEAP1) regulate transcription of enzymes involved in cellular detoxification processes and Nfe2l2-deficient mice develop tobacco-induced emphysema. We assessed the impact of Single Nucleotide Polymorphisms (SNPs) in both genes on the level and longitudinal course of Forced Expiratory Volume in 1 second (FEV1) in the general population. Five NFE2L2 and three KEAP1 tagging SNPs were genotyped in the population-based Doetinchem cohort (n = 1,152) and the independent Vlagtwedde-Vlaardingen cohort (n = 1,390). On average 3 FEV1 measurements during 3 surveys, respectively 7 FEV1 measurements during 8 surveys were present. Linear Mixed Effect models were used to test cross-sectional and longitudinal genetic effects on repeated FEV1 measurements. In the Vlagtwedde-Vlaardingen cohort SNP rs11085735 in KEAP1 was associated with a higher FEV1 level (p = 0.02 for an additive effect), and SNP rs2364723 in NFE2L2 was associated with a lower FEV1 level (p = 0.06). The associations were even more significant in the pooled cohort analysis. No significant association of KEAP1 or NFE2L2 SNPs with FEV1 decline was observed. This is the first genetic study on variations in key antioxidant transcriptional regulators KEAP1 and NFE2L2 and lung function in a general population. It identified 2 SNPs in NFE2L2 and KEAP1 which affect the level of FEV1 in the general population. It additionally shows that NFE2L2 and KEAP1 variations are unlikely to play a role in the longitudinal course of FEV1 in the general population.

Neuroprotective Effects of the Triterpenoid, CDDO Methyl Amide, a Potent Inducer of Nrf2-Mediated Transcription

Article

Full-text available

Feb 2009
PLOS ONE

The NF-E2-related factor-2 (Nrf2)/antioxidant response element (ARE) signaling pathway regulates phase 2 detoxification genes, including a variety of antioxidative enzymes. We tested neuroprotective effects of the synthetic triterpenoid CDDO-MA, a potent activator of the Nrf2/ARE signaling. CDDO-MA treatment of neuroblastoma SH-SY5Y cells resulted in Nrf2 upregulation and translocation from cytosol to nucleus and subsequent activation of ARE pathway genes. CDDO-MA blocked t-butylhydroperoxide-induced production of reactive oxygen species (ROS) by activation of ARE genes only in wild type, but not Nrf2 knockout mouse embryonic fibroblasts. Oral administration of CDDO-MA resulted in significant protection against MPTP-induced nigrostriatal dopaminergic neurodegeneration, pathological alpha-synuclein accumulation and oxidative damage in mice. Additionally, CDDO-MA treatment in rats produced significant rescue against striatal lesions caused by the neurotoxin 3-NP, and associated increases in the oxidative damage markers malondialdehyde, F(2)-Isoprostanes, 8-hydroxy-2-deoxyguanosine, 3-nitrotyrosine, and impaired glutathione homeostasis. Our results indicate that the CDDO-MA renders its neuroprotective effects through its potent activation of the Nrf2/ARE pathway, and suggest that triterpenoids may be beneficial for the treatment of neurodegenerative diseases like Parkinson's disease and Huntington's disease.

Hayes JD, McMahon M.. NRF2 and KEAP1 mutations: permanent activation of an adaptive response in cancer. Trends Biochem Sci 34: 176-188

Article

Full-text available

Apr 2009
TRENDS BIOCHEM SCI

Transcription factor nuclear factor-erythroid 2-related factor 2 (NRF2) controls cellular adaptation to oxidants and electrophiles by inducing antioxidant and detoxification genes in response to redox stress. NRF2 is negatively regulated by Kelch-like ECH-associated protein 1 (KEAP1). Tumours from approximately 15% of patients with lung cancer harbour somatic mutations in KEAP1 that prevent effective NRF2 repression. Recently, two NRF2 mutation 'hot-spots' were identified in approximately 10% of patients with lung cancer, enabling the transcription factor to evade KEAP1-mediated repression. Somatic mutations in KEAP1 and NRF2 provide an insight into the molecular mechanisms by which NRF2 is regulated. Moreover, constitutive NRF2 activation might cause drug resistance in tumours, and an understanding of how the transcription factor is regulated indicates ways in which this could be overcome.

Curcumin attenuates elastase- and cigarette smoke-induced pulmonary emphysema in mice

Article

Full-text available

Feb 2009

Curcumin, a yellow pigment obtained from turmeric (Curcumina longa), is a dietary polyphenol that has been reported to possess anti-inflammatory and antioxidant properties. The effect of curcumin against the development of pulmonary emphysema in animal models is unknown. The aim of this study was to determine whether curcumin is able to attenuate the development of pulmonary emphysema in mice. Nine-week-old male C57BL/6J mice were treated with intratracheal porcine pancreatic elastase (PPE) or exposed to mainstream cigarette smoke (CS) (60 min/day for 10 consecutive days or 5 days/wk for 12 wk) to induce pulmonary inflammation and emphysema. Curcumin (100 mg/kg) or vehicle was administrated daily by oral gavage 1 h and 24 h before intratracheal PPE treatment and daily thereafter throughout a 21-day period in PPE-exposed mice and 1 h before each CS exposure in CS-exposed mice. As a result, curcumin treatment significantly inhibited PPE-induced increase of neutrophils in bronchoalveolar lavage fluid at 6 h and on day 1 after PPE administration, with an increase in antioxidant gene expression at 6 h and significantly attenuated PPE-induced air space enlargement on day 21. It was also found that curcumin treatment significantly inhibited CS-induced increase of neutrophils and macrophages in bronchoalveolar lavage fluid after 10 consecutive days of CS exposure and significantly attenuated CS-induced air space enlargement after 12 wk of CS exposure. In conclusion, oral curcumin administration attenuated PPE- and CS-induced pulmonary inflammation and emphysema in mice.

Review: Antioxidant therapeutic advances in COPD

Article

Full-text available

Jan 2009

Irfan Rahman

Chronic obstructive pulmonary disease (COPD) is associated with a high incidence of morbidity and mortality. Cigarette smoke-induced oxidative stress is intimately associated with the progression and exacerbation of COPD and therefore targeting oxidative stress with antioxidants or boosting the endogenous levels of antioxidants is likely to have beneficial outcome in the treatment of COPD. Among the various antioxidants tried so far, thiol antioxidants and mucolytic agents, such as glutathione, N-acetyl-L-cysteine, N-acystelyn, erdosteine, fudosteine and carbocysteine; Nrf2 activators; and dietary polyphenols (curcumin, resveratrol, and green tea catechins/quercetin) have been reported to increase intracellular thiol status along with induction of GSH biosynthesis. Such an elevation in the thiol status in turn leads to detoxification of free radicals and oxidants as well as inhibition of ongoing inflammatory responses. In addition, specific spin traps, such as alpha-phenyl-N-tert-butyl nitrone, a catalytic antioxidant (ECSOD mimetic), porphyrins (AEOL 10150 and AEOL 10113), and a SOD mimetic M40419 have also been reported to inhibit cigarette smoke-induced inflammatory responses in vivo in the lung. Since a variety of oxidants, free radicals and aldehydes are implicated in the pathogenesis of COPD, it is possible that therapeutic administration of multiple antioxidants and mucolytics will be effective in management of COPD. However, a successful outcome will critically depend upon the choice of antioxidant therapy for a particular clinical phenotype of COPD, whose pathophysiology should be first properly understood. This article will review the various approaches adopted to enhance lung antioxidant levels, antioxidant therapeutic advances and recent past clinical trials of antioxidant compounds in COPD.

Identifying targets for COPD treatment through gene expression analyses

Article

Full-text available

Feb 2008

Despite the status of chronic obstructive pulmonary disease (COPD) as a major global health problem, no currently available therapies can limit COPD progression. Therefore, an urgent need exists for the development of new and effective treatments for COPD. An improved understanding in the molecular pathogenesis of COPD can potentially identify molecular targets to facilitate the development of new therapeutic modalities. Among the best approaches for understanding the molecular basis of COPD include gene expression profiling techniques, such as serial analysis of gene expression or microarrays. Using these methods, recent studies have mapped comparative gene expression profiles of lung tissues from patients with different stages of COPD relative to healthy smokers or non-smokers. Such studies have revealed a number of differentially-regulated genes associated with COPD progression, which include genes involved in the regulation of inflammation, extracellular matrix, cytokines, chemokines, apoptosis, and stress responses. These studies have shed new light on the molecular mechanisms of COPD, and suggest novel targets for clinical treatments.

Article

Full-text available

Oct 2008
P NATL ACAD SCI USA

The nuclear factor E2-related factor 2 (Nrf2) is a master transcriptional activator of genes encoding numerous cytoprotective enzymes that are induced in response to environmental and endogenously derived oxidative/electrophilic agents. Under normal, nonstressed circumstances, low cellular concentrations of Nrf2 are maintained by proteasomal degradation through a Keap1-Cul3-Roc1-dependent mechanism. A model for Nrf2 activation has been proposed in which two amino-terminal motifs, DLG and ETGE, promote efficient ubiquitination and rapid turnover; known as the two-site substrate recognition/hinge and latch model. Here, we show that in human cancer, somatic mutations occur in the coding region of NRF2, especially among patients with a history of smoking or suffering from squamous cell carcinoma; in the latter case, this leads to poor prognosis. These mutations specifically alter amino acids in the DLG or ETGE motifs, resulting in aberrant cellular accumulation of Nrf2. Mutant Nrf2 cells display constitutive induction of cytoprotective enzymes and drug efflux pumps, which are insensitive to Keap1-mediated regulation. Suppression of Nrf2 protein levels by siRNA knockdown sensitized cancer cells to oxidative stress and chemotherapeutic reagents. Our results strongly support the contention that constitutive Nrf2 activation affords cancer cells with undue protection from their inherently stressed microenvironment and anti-cancer treatments. Hence, inactivation of the Nrf2 pathway may represent a therapeutic strategy to reinforce current treatments for malignancy. Congruously, the present study also provides in vivo validation of the two-site substrate recognition model for Nrf2 activation by the Keap1-Cul3-based E3 ligase. • cancer cell microenvironment • multidrug resistant-associated protein • oxidative stress • somatic mutation • ubiquitin-proteasome system

Kang MI, Kobayashi A, Wakabayashi N, Kim SG, Yamamoto M.. Scaffolding of Keap1 to the actin cytoskeleton controls the function of Nrf2 as key regulator of cytoprotective phase 2 genes. Proc Natl Acad Sci USA 101: 2046-2051

Article

Full-text available

Mar 2004

Transcription factor Nrf2 regulates basal and inducible expression of phase 2 proteins that protect animal cells against the toxic effects of electrophiles and oxidants. Under basal conditions, Nrf2 is sequestered in the cytoplasm by Keap1, a multidomain, cysteinerich protein that is bound to the actin cytoskeleton. Keap1 acts both as a repressor of the Nrf2 transactivation and as a sensor of phase 2 inducers. Electrophiles and oxidants disrupt the Keap1-Nrf2 complex, resulting in nuclear accumulation of Nrf2, where it enhances the transcription of phase 2 genes via a common upstream regulatory element, the antioxidant response element. Reporter cotransfection-transactivation analyses with a series of Keap1 deletion mutants revealed that in the absence of the double glycine repeat domain Keap1 does not bind to Nrf2. In addition, deletion of either the intervening region or the C-terminal region also abolished the ability of Keap1 to sequester Nrf2, indicating that all of these domains contribute to the repressor activity of Keap1. Immunocytochemical and immunoprecipitation analyses demonstrated that Keap1 associates with actin filaments in the cytoplasm through its double glycine repeat domain. Importantly, disruption of the actin cytoskeleton promotes nuclear entry of an Nrf2 reporter protein. The actin cytoskeleton therefore provides scaffolding that is essential for the function of Keap1, which is the sensor for oxidative and electrophilic stress.

Genetic ablation of Nrf2 enhances susceptibility to cigarette smoke-induced emphysema in mice

Article

Full-text available

Dec 2004

Although inflammation and protease/antiprotease imbalance have been postulated to be critical in cigarette smoke-induced (CS-induced) emphysema, oxidative stress has been suspected to play an important role in chronic obstructive pulmonary diseases. Susceptibility of the lung to oxidative injury, such as that originating from inhalation of CS, depends largely on its upregulation of antioxidant systems. Nuclear factor, erythroid-derived 2, like 2 (Nrf2) is a redox-sensitive basic leucine zipper protein transcription factor that is involved in the regulation of many detoxification and antioxidant genes. Disruption of the Nrf2 gene in mice led to earlier-onset and more extensive CS-induced emphysema than was found in wild-type littermates. Emphysema in Nrf2-deficient mice exposed to CS for 6 months was associated with more pronounced bronchoalveolar inflammation; with enhanced alveolar expression of 8-oxo-7,8-dihydro-2'-deoxyguanosine, a marker of oxidative stress; and with an increased number of apoptotic alveolar septal cells--predominantly endothelial and type II epithelial cells--as compared with wild-type mice. Microarray analysis identified the expression of nearly 50 Nrf2-dependent antioxidant and cytoprotective genes in the lung that may work in concert to counteract CS-induced oxidative stress and inflammation. The responsiveness of the Nrf2 pathway may act as a major determinant of susceptibility to tobacco smoke-induced emphysema by upregulating antioxidant defenses and decreasing lung inflammation and alveolar cell apoptosis.

Relation between duration of smoking cessation and bronchial inflammation in COPD

Article

Full-text available

Mar 2006

Chronic obstructive pulmonary disease (COPD) is associated with airway inflammation. Although smoking cessation improves symptoms and the decline in lung function in COPD, it is unknown whether bronchial inflammation in patients with established COPD varies with the duration of smoking cessation. 114 patients (99 men) with COPD of mean (SD) age 62 (8) years, a median (IQR) smoking history of 42 (31-55) pack years, no inhaled or oral corticosteroids, all current or ex-smokers (n = 42, quit >1 month, median cessation duration 3.5 years), post-bronchodilator FEV(1) 63 (9)% predicted, and FEV(1)/IVC 48 (9)% were studied cross sectionally. The numbers of subepithelial T lymphocytes (CD3, CD4, CD8), neutrophils, macrophages, eosinophils, mast cells, and plasma cells were measured in bronchial biopsy specimens (median (IQR)/0.1 mm(2)) using fully automated image analysis. Ex-smokers with COPD had higher CD3+, CD4+, and plasma cell numbers than current smokers with COPD (149 (88-225) v 108 (61-164), p = 0.036; 58 (32-90) v 40 (25-66), p = 0.023; and 9.0 (5.5-20) v 7.5 (3.1-14), p = 0.044, respectively), but no difference in other inflammatory cells. Short term ex-smokers (<3.5 years) had higher CD4+ and CD8+ cell numbers than current smokers (p = 0.017, p = 0.023; respectively). Conversely, long term ex-smokers (quit > or =3.5 years) had lower CD8+ cell numbers than short term ex-smokers (p = 0.009), lower CD8/CD3 ratios than both current smokers and short-term ex-smokers (p = 0.012, p = 0.003; respectively), and higher plasma cell numbers than current smokers (p = 0.003). With longer duration of smoking cessation, CD8 cell numbers decrease and plasma cell numbers increase. This indicates that bronchial T lymphocyte and plasma cell counts, but not other inflammatory cells, are related to duration of smoking cessation in patients with COPD.

Transcription Factor Nrf2 Plays a Pivotal Role in Protection against Elastase-Induced Pulmonary Inflammation and Emphysema

Article

Full-text available

Dec 2005

Emphysema is one of the major pathological abnormalities associated with chronic obstructive pulmonary disease. The protease/antiprotease imbalance and inflammation resulting from oxidative stress have been attributed to the pathogenesis of emphysema. Nrf2 is believed to protect against oxidative tissue damage through the transcriptional activation of a battery of antioxidant enzymes. In this study, we investigated the protective role of Nrf2 in the development of emphysema using elastase-induced emphysema as our model system. We found that elastase-provoked emphysema was markedly exacerbated in Nrf2-knockout (KO) mice compared with wild-type mice. The severity of emphysema in Nrf2-KO mice correlated intimately with the degree of lung inflammation in the initial stage of elastase treatment. The highly inducible expression of antioxidant and antiprotease genes observed in wild-type alveolar macrophages was significantly attenuated in the lungs of Nrf2-KO mice. Interestingly, transplantation of wild-type bone marrow cells into Nrf2-KO mice retarded the development of initial lung inflammation and subsequent emphysema, and this improvement correlated well with the appearance of macrophages expressing Nrf2-regulated antiprotease and antioxidant genes. Thus, Nrf2 appears to exert its protective effects through the transcriptional activation of antiprotease and antioxidant genes in alveolar macrophages.

Important role of abnormal glycogen structure in the development of liver cirrhosis and progression to hepatocellular carcinoma in patients with glycogen storage disease type-III

Article

Aug 2007
J HEPATOL

Nrf2 Deficiency Influences Susceptibility to Steroid Resistance via HDAC2 reduction

Article

Dec 2010
BIOCHEM BIOPH RES CO

Abnormal lung inflammation and oxidant burden are associated with a significant reduction in histone deacetylase 2 (HDAC2) abundance and steroid resistance. We hypothesized that Nrf2 regulates steroid sensitivity via HDAC2 in response to inflammation in mouse lung. Furthermore, HDAC2 deficiency leads to steroid resistance in attenuating lung inflammatory response, which may be due to oxidant/antioxidant imbalance. Loss of antioxidant transcription factor Nrf2 resulted in decreased HDAC2 level in lung, and increased inflammatory lung response which was not reversed by steroid. Thus, steroid resistance or inability of steroids to control lung inflammatory response is dependent on Nrf2-HDAC2 axis. These findings have implications in steroid resistance, particularly during the conditions of oxidative stress when the lungs are more susceptible to inflammatory response, which is seen in patients with chronic obstructive pulmonary disease, asthma, rheumatoid arthritis, and inflammatory bowel disease.

Discovery of the Negative Regulator of Nrf2, Keap1: A Historical Overview

Article

May 2010
ANTIOXID REDOX SIGN

An antioxidant response element (ARE) or an electrophile responsive element (EpRE) regulate the transcriptional induction of a battery of drug-detoxifying enzymes that are protective against electrophiles. Based on the high similarity of the ARE consensus sequence to an erythroid gene regulatory element NF-E2 binding site, we have found that the transcription factor Nrf2 is indispensable for the ARE-mediated induction of drug-metabolizing enzymes. Recent genome-wide analysis demonstrated that Nrf2 regulates hundreds of genes that are involved in the cytoprotective response against oxidative stress. In-depth analysis of Nrf2 regulatory mechanisms has led us to the discovery of a novel protein, which we have named Keap1. Keap1 suppresses Nrf2 activity by specifically binding to its evolutionarily conserved N-terminal Neh2 regulatory domain. In this review article, we summarize the findings and observations that have lead to the discovery of the Nrf2-Keap1 system. Furthermore, we briefly discuss the function of the Nrf2-Keap1 system under the regulation of the endogenous electrophilic compound 15-deoxy-Δ¹²(,)¹⁴-prostaglandin J₂. We propose that Nrf2-Keap1 plays a significant physiological role in the response to endogenous, environmental, and pharmacological electrophiles.

Outpatient management of severe COPD

Article

Apr 2010
NEW ENGL J MED

Dennis E. Niewoehner

A 67-year-old man presents with a history of dyspnea, which has progressed for the past several years. He began smoking cigarettes at 15 years of age and continues to smoke one pack per day. Worsening breathlessness forced him to retire as a laborer, and he has sought emergency care for what he calls bronchitis twice in the past year. His physical examination is notable for diminished breath sounds on auscultation, with a prolonged expiratory phase. Spirometry reveals severe airflow obstruction (ratio of forced expiratory volume in 1 second [FEV1] to forced vital capacity [FVC], 0.43; FEV 1, 34% of the predicted value). How should this case be managed?

Update in Chronic Obstructive Pulmonary Disease 2009

Article

Apr 2010
AM J RESP CRIT CARE

Activation of apoptosis signal-regulating kinase 1 is a key factor in paraquat-induced cell death: Modulation by the Nrf2/Trx axis

Article

Mar 2010

Although oxidative stress is fundamental to the etiopathology of Parkinson disease, the signaling molecules involved in transduction after oxidant exposure to cell death are ill-defined, thus making it difficult to identify molecular targets of therapeutic relevance. We have addressed this question in human dopaminergic neuroblastoma SH-SY5Y cells exposed to the parkinsonian toxin paraquat (PQ). This toxin elicited a dose-dependent increase in reactive oxygen species and cell death that correlated with activation of ASK1 and the stress kinases p38 and JNK. The relevance of these kinases in channeling PQ neurotoxicity was demonstrated with the use of interference RNA for ASK1 and two well-established pharmaceutical inhibitors for JNK and p38. The toxic effect of PQ was substantially attenuated by preincubation with vitamin E, blocking ASK1 pathways and preventing oxidative stress and cell death. In a search for a physiological pathway that might counterbalance PQ-induced ASK1 activation, we analyzed the role of the transcription factor Nrf2, master regulator of redox homeostasis, and its target thioredoxin (Trx), which binds and inhibits ASK1. Trx levels were undetectable in Nrf2-deficient mouse embryo fibroblasts (MEFs), whereas they were constitutively high in Keap1-deficient MEFs as well as in SH-SY5Y cells treated with sulforaphane (SFN). Consistent with these data, Nrf2-deficient MEFs were more sensitive and Keap1-deficient MEFs and SH-SY5Y cells incubated with SFN were more resistant to PQ-induced cell death. This study identifies ASK1/JNK and ASK1/p38 as two critical pathways involved in the activation of cell death under oxidative stress conditions and identifies the Nrf2/Trx axis as a new target to block these pathways and protect from oxidant exposure such as that found in Parkinson and other neurodegenerative diseases.

Molecular Targets of Dietary Phenethyl Isothiocyanate and Sulforaphane for Cancer Chemoprevention

Article

Dec 2009
AAPS J

Development of cancer is a long-term and multistep process which comprises initiation, progression, and promotion stages of carcinogenesis. Conceivably, it can be targeted and interrupted along these different stages. In this context, many naturally occurring dietary compounds from our daily consumption of fruits and vegetables have been shown to possess cancer preventive effects. Phenethyl isothiocyanate (PEITC) and sulforaphane (SFN) are two of the most widely investigated isothiocyanates from the crucifers. Both have been found to be very potent chemopreventive agents in numerous animal carcinogenesis models as well as cell culture models. They exert their chemopreventive effects through regulation of diverse molecular mechanisms. In this review, we will discuss the molecular targets of PEITC and SFN potentially involved in cancer chemoprevention. These include the regulation of drug-metabolizing enzymes phase I cytochrome P450s and phase II metabolizing enzymes. In addition, the signaling pathways including Nrf2-Keap 1, anti-inflammatory NFkappaB, apoptosis, and cell cycle arrest as well as some receptors will also be discussed. Furthermore, we will also discuss the similarities and their potential differences in the regulation of these molecular targets by PEITC and SFN.

Prolonged cigarette smoke exposure decreases heme oxygenase-1 and alters Nrf2 and Bach1 expression in human macrophages: Roles of the MAP kinases ERK1/2 and JNK

Article

Oct 2009
FEBS LETT

Tobacco may be involved in the decreased macrophage heme oxygenase-1 (HO-1) expression described in smoking-induced severe emphysema, via the nuclear factor erythroid 2-related factor 2 (Nrf2)/Kelch-like ECH-associated protein 1 (Keap1)-BTB and CNC homology 1, basic leucine zipper transcription factor 1 (Bach1) pathway. We assessed in vitro effects of cigarette smoke condensate (CS) in the human monocyte/macrophage cell line (THP-1). CS exposure led to increased HO-1 and nuclear Nrf2 expression (6 h) followed by decreased HO-1 expression concomitantly with nuclear Nrf2/Bach1 ratio decrease (72h). CS-induced mitogen-activated protein kinase (MAPK) phosphorylation. Extracellular-signal-regulated kinase(1/2) (ERK(1/2)) and c-Jun NH2-terminal kinase (JNK) inhibition completely abrogated CS effects on HO-1 expression and nuclear Nrf2/Bach1 translocation. These results suggest that ERK(1/2) and JNK are involved in CS-induced biphasic HO-1 expression by a specific regulation of Nrf2/Keap1-Bach1.

Nrf2:INrf2(Keap1) Signaling in Oxidative Stress

Article

Sep 2009

Nrf2:INrf2 (Keap1) are cellular sensors of chemical- and radiation-induced oxidative and electrophilic stress. Nrf2 is a nuclear transcription factor that controls the expression and coordinated induction of a battery of defensive genes encoding detoxifying enzymes and antioxidant proteins. This is a mechanism of critical importance for cellular protection and cell survival. Nrf2 is retained in the cytoplasm by an inhibitor, INrf2 which functions as an adapter for Cul3/Rbx1-mediated degradation of Nrf2. In response to oxidative/electrophilic stress, Nrf2 is switched on and then off by distinct early and delayed mechanisms. Oxidative/electrophilic modification of INrf2 cysteine 151 and/or protein kinase C phosphorylation of Nrf2 serine 40 results in the escape or release of Nrf2 from INrf2. Nrf2 is stabilized and translocates to the nucleus, forms heterodimers with unknown proteins, and binds the antioxidant response element, which leads to coordinated activation of gene expression. It takes less than 15 min from the time of exposure to switch on nuclear import of Nrf2. This is followed by activation of a delayed mechanism that controls the switching off of Nrf2 activation of gene expression. GSK3beta phosphorylates Fyn at an unknown threonine residue(s), leading to the nuclear localization of Fyn. Fyn phosphorylates Nrf2 tyrosine 568, resulting in the nuclear export of Nrf2, binding with INrf2, and degradation of Nrf2. The switching on and off of Nrf2 protects cells against free radical damage, prevents apoptosis, and promotes cell survival.

Molecular pathogenesis of Parkinson disease: Insights from genetic studies

Article

Feb 2009
Expet Rev Mol Med

Thomas Gasser

Over the past few years, genetic findings have changed our views on the molecular pathogenesis of Parkinson disease (PD), as mutations in a growing number of genes have been found to cause monogenic forms of the disorder. These mutations cause neuronal dysfunction and neurodegeneration either by a toxic gain of function, as in the case of the dominant forms of monogenic PD caused by mutations in the genes for alpha-synuclein or LRRK2, or by a loss of an intrinsic protective function, as is likely for the recessive PD genes parkin (PRKN), PINK1 and DJ-1. Evidence is emerging that at least some of the pathways uncovered in the rare monogenic forms of PD may play a direct role in the aetiology of the common sporadic disorder and that variants of the respective genes contribute to the risk of developing the disease. These findings will allow the search for new treatment strategies that focus on the underlying molecular pathophysiology, rather than simply on ameliorating symptoms.

Meta-analyses on Suspected Chronic Obstructive Pulmonary Disease Genes: A Summary of 20 Years' Research

Article

Aug 2009
AM J RESP CRIT CARE

Chronic obstructive pulmonary disease (COPD) is a complex disorder with high mortality worldwide. Studies on the role of candidate genes and their polymorphisms in COPD development have so far produced ambiguous results. The aim of this study was to reveal the role of COPD candidate genes using data collected in previous research. We performed meta-analyses on 20 polymorphisms in 12 genes, after searching the PubMed and Embase databases for publications on COPD. These genes involve three main pathways associated with COPD development: the inflammatory, protease-antiprotease balance, and antioxidant pathways. We obtained significant results for three TGFB1 polymorphisms, although these were based only on a few studies. The IL1RN VNTR polymorphism increases the risk for COPD (odds ratio [OR], 1.7; 95% confidence interval [CI], 1.09-2.65), whereas the TNFA -308 G/A polymorphism does so only in Asian populations (OR, 2.01; 95% CI, 1.21-3.31). The GSTP1 I105V polymorphism was protective for COPD in Asian populations only (OR, 0.69; 95% CI, 0.56-0.85). These results demonstrate the importance of ethnicity in identifying specific COPD genes.

Coordinate Control of Expression of Nrf2-Modulated Genes in the Human Small Airway Epithelium Is Highly Responsive to Cigarette Smoking

Article

Nov 2008
MOL MED

Nuclear factor erythroid 2-related factor 2 (Nrf2) is an oxidant-responsive transcription factor known to induce detoxifying and antioxidant genes. Cigarette smoke, with its large oxidant content, is a major stress on the cells of small airway epithelium, which are vulnerable to oxidant damage. We assessed the role of cigarette smoke in activation of Nrf2 in the human small airway epithelium in vivo. Fiberoptic bronchoscopy was used to sample the small airway epithelium in healthy-nonsmoker and healthy-smoker, and gene expression was assessed using microarrays. Relative to nonsmokers, Nrf2 protein in the small airway epithelium of smokers was activated and localized in the nucleus. The human homologs of 201 known murine Nrf2-modulated genes were identified, and 13 highly smoking-responsive Nrf2-modulated genes were identified. Construction of an Nrf2 index to assess the expression levels of these 13 genes in the airway epithelium of smokers showed coordinate control, an observation confirmed by quantitative PCR. This coordinate level of expression of the 13 Nrf2-modulated genes was independent of smoking history or demographic parameters. The Nrf2 index was used to identify two novel Nrf2-modulated, smoking-responsive genes, pirin (PIR) and UDP glucuronosyltransferase 1-family polypeptide A4 (UGT1A4). Both genes were demonstrated to contain functional antioxidant response elements in the promoter region. These observations suggest that Nrf2 plays an important role in regulating cellular defenses against smoking in the highly vulnerable small airway epithelium cells, and that there is variability within the human population in the Nrf2 responsiveness to oxidant burden.

Nrf2 Signaling and Cell Survival

Article

Jul 2009
TOXICOL APPL PHARM

Nrf2:INrf2 acts as a sensor for oxidative/electrophilic stress. INrf2 serves as an adaptor to link Nrf2 to the ubiquitin ligase Cul3-Rbx1 complex that ubiquitinate and degrade Nrf2. Under basal conditions, cytosolic INrf2/Cul3-Rbx1 is constantly degrading Nrf2. When a cell encounters stress Nrf2 dissociates from the INrf2 and translocates into the nucleus. Oxidative/electrophilic stress induced modification of INrf2Cysteine151 and/or protein kinase C (PKC)-mediated phosphorylation of Nrf2Serine40 controls Nrf2 release from INrf2 followed by stabilization and nuclear translocation of Nrf2. Nrf2 binds to the antioxidant response element (ARE) and activates a myriad of genes that protect cells against oxidative/electrophilic stress and neoplasia. A delayed response of oxidative/electrophilic stress activates GSK-3beta that phosphorylates Fyn at unknown threonine residue(s). Phosphorylated Fyn translocates to the nucleus and phosphorylates Nrf2Tyrosine568 that leads to nuclear export and degradation of Nrf2. Prothymosin-alpha mediated nuclear translocation of INrf2 also degrades nuclear Nrf2. The degradation of Nrf2 both in cytosol and nuclear compartments rapidly brings down its levels to normal resulting in suppression of Nrf2 downstream gene expression. An auto-regulatory loop between Nrf2 and INrf2 controls their cellular abundance. Nrf2 regulates INrf2 by controlling its transcription, and INrf2 controls Nrf2 by degrading it. In conclusion, switching on and off of Nrf2 combined with promoting an auto-regulatory loop between them regulates activation/deactivation of defensive genes leading to protection of cells against adverse effects of oxidative and electrophilic stress and promote cell survival.

Deletion of Keap1 in the Lung Attenuates Acute Cigarette Smoke-Induced Oxidative Stress and Inflammation

Article

Jul 2009
AM J RESP CELL MOL

Exposure to cigarette smoke (CS) is the primary factor associated with the development of chronic obstructive pulmonary disease (COPD). CS increases the level of oxidants in the lungs, resulting in a depletion of antioxidants, which promotes oxidative stress and the destruction of alveolar tissue. In response to CS, pulmonary epithelial cells counteract increased levels of oxidants by activating Nrf2-dependent pathways to augment the expression of detoxification and antioxidant enzymes, thereby protecting the lung from injury. We hypothesize that increasing the pathways activated by Nrf2 will afford protection against CS-induced lung damage. To this end we have developed a novel mouse model in which the cytosolic inhibitor of Nrf2, Keap1, is genetically deleted in Clara cells, which predominate in the upper airways in mice. Deletion of Keap1 in Clara cells resulted in increased expression of Nrf2-dependent genes, such as Nqo1 and Gclm, as determined by microarray analysis and quantitative PCR. Deletion of Keap1 in airway epithelium decreased Keap1 protein levels and significantly increased the total level of glutathione in the lungs. Increased Nrf2 activation protected Clara cells against oxidative stress ex vivo and attenuated oxidative stress and CS-induced inflammation in vivo. Expression of KEAP1 was also decreased in human epithelial cells through siRNA transfection, which increased the expression of Nrf2-dependent genes and attenuated oxidative stress. In conclusion, activating Nrf2 pathways in tissue-specific Keap1 knockout mice represents an important genetic approach against oxidant-induced lung damage.

Ginkgo biloba extract confers protection from cigarette smoke extract-induced apoptosis in human lung endothelial cells: Role of heme oxygenase-1

Article

Mar 2009
PULM PHARMACOL THER

Cigarette smoking is the major cause of chronic obstructive pulmonary disease, which is associated with increased oxidative stress and numbers of apoptotic endothelial cells in the lungs. Ginkgo biloba extract (EGb) is a therapeutic agent for disorders such as vascular insufficiency and Alzheimer's disease. Although EGb is known to possess antioxidant functions, its ability to alleviate cigarette smoke-induced pathophysiological consequences has not been elucidated. We investigated the cytoprotective effects and therapeutic mechanisms of EGb against oxidative stress and apoptosis induced by cigarette smoke extract (CSE) in human pulmonary artery endothelial cells (HPAECs). Challenge with CSE (160 microg/ml) caused a reduction in cell viability, an increase in intracellular reactive oxygen species and an acceleration of caspase-dependent apoptosis in HPAECs, all of which were alleviated by pretreatment with EGb (100 microg/ml). N-acetylcysteine (an antioxidant) also reduced both the CSE-induced oxidative stress and apoptosis, indicating that the former response triggered the latter. Additionally, EGb produced activation of ERK, JNK and p38 [three major mitogen-activated protein kinases (MAPKs)], an increase in the nuclear level of nuclear factor erythroid-2-related factor 2 (Nrf2) and upregulation of heme oxygenase-1 (HO-1, a stress-responsive protein with antioxidant function). Pretreatment with inhibitors of MAPKs abolished both EGb-induced Nrf2 nuclear translocation and HO-1 upregulation. Small interfering RNAs targeting HO-1 prevented EGb-induced HO-1 upregulation and also abolished the antioxidant, anti-apoptotic and cytoprotective effects of EGb in HPAECs insulted with CSE. We conclude that EGb confers protection from oxidative stress-related apoptosis induced by CSE in HPAECs and its therapeutic effects depend on transcriptional upregulation of HO-1 by EGb via the MAPKs/Nrf2 pathway.

Targeting the redox sensitive Nrf2-Keap1 defense pathway in cardiovascular disease: Protection afforded by dietary isoflavones

Article

Feb 2009
CURR OPIN PHARMACOL

Cells have evolved highly regulated defense systems, including the redox sensitive Nrf2-Keap1 signaling pathway involved in the transcriptional activation of phase II defense and antioxidant genes in oxidative stress. Increased generation of reactive oxygen species (ROS) in cardiovascular disease (CVD) leads to impaired endothelial function and reduced nitric oxide (NO) bioavailability. Although epidemiological evidence suggests that diets containing plant-derived isoflavones (phytoestrogens) afford protection against CVDs, supplementation trials have largely reported only marginal health benefits. The molecular mechanisms by which soy isoflavones (genistein, daidzein, and equol) afford protection against oxidative stress in CVD remain to be investigated in large-scale clinical trials. Studies in animal models and cultured vascular cells have established that isoflavones increase eNOS activity and expression and activate the Nrf2-Keap1 signaling pathway, leading to an upregulation of detoxifying and antioxidant defense genes. We review recent advances in the understanding of the signal transduction pathways involved in the activation of endothelial NO production and Nrf2-Keap1-mediated antioxidant gene expression by dietary isoflavones.

From the Cover: Targeting Nrf2 with the triterpenoid CDDO- imidazolide attenuates cigarette smoke-induced emphysema and cardiac dysfunction in mice

Article

Jan 2009
P NATL ACAD SCI USA

Chronic obstructive pulmonary disease (COPD), which comprises emphysema and chronic bronchitis resulting from prolonged exposure to cigarette smoke (CS), is a major public health burden with no effective treatment. Emphysema is also associated with pulmonary hypertension, which can progress to right ventricular failure, an important cause of morbidity and mortality among patients with COPD. Nuclear erythroid 2 p45 related factor-2 (Nrf2) is a redox-sensitive transcription factor that up-regulates a battery of antioxidative genes and cytoprotective enzymes that constitute the defense against oxidative stress. Recently, it has been shown that patients with advanced COPD have a decline in expression of the Nrf2 pathway in lungs, suggesting that loss of this antioxidative protective response is a key factor in the pathophysiological progression of emphysema. Furthermore, genetic disruption of Nrf2 in mice causes early-onset and severe emphysema. The present study evaluated whether the strategy of activation of Nrf2 and its downstream network of cytoprotective genes with a small molecule would attenuate CS-induced oxidative stress and emphysema. Nrf2+/+ and Nrf2−/− mice were fed a diet containing the potent Nrf2 activator, 1-[2-cyano-3-,12-dioxooleana-1,9(11)-dien-28-oyl]imidazole (CDDO-Im), while being exposed to CS for 6 months. CDDO-Im significantly reduced lung oxidative stress, alveolar cell apoptosis, alveolar destruction, and pulmonary hypertension in Nrf2+/+ mice caused by chronic exposure to CS. This protection from CS-induced emphysema depended on Nrf2, as Nrf2−/− mice failed to show significant reduction in alveolar cell apoptosis and alveolar destruction after treatment with CDDO-Im. These results suggest that targeting the Nrf2 pathway during the etiopathogenesis of emphysema may represent an important approach for prophylaxis against COPD. • chronic obstructive pulmonary disease • oxidative stress

Nrf2 Dependent Sulfiredoxin-1 Expression Protects Against Cigarette Smoke-induced Oxidative Stress in Lungs

Article

Dec 2008

Oxidative stress results in protein oxidation and is involved in the pathogenesis of lung diseases such as chronic obstructive pulmonary disorder (COPD). Sulfiredoxin-1 (Srx1) catalyzes the reduction of cysteine sulfinic acid to sulfenic acid in oxidized proteins and protects them from inactivation. This study examined the mechanism of transcriptional regulation of Srx1 and its possible protective role during oxidative stress associated with COPD. Nrf2, a transcription factor known to influence susceptibility to pulmonary diseases, upregulates Srx1 expression during oxidative stress caused by cigarette smoke exposure in the lungs of mice. Disruption of Nrf2 signaling by genetic knockout in mice or RNAi in cells downregulated the expression of Srx1. In silico analysis of the 5'-promoter-flanking region of Srx1 identified multiple antioxidant-response elements (AREs) that are highly conserved. Reporter and chromatin-immunoprecipitation assays demonstrated that ARE1 at -228 is critical for the Nrf2-mediated response. Attenuation of Srx1 expression with RNAi potentiated the toxicity of hydrogen peroxide (H2O2), whereas overexpression of Srx1 protected against H2O2-mediated cell death in vitro. Immunoblot analysis revealed dramatic decreases in Srx1 expression in lungs from patients with COPD relative to nonemphysematous lungs together with a decline in Nrf2 protein. Thus, Srx1, a key Nrf2-regulated gene, contributes to protection against oxidative injury in the lung.

The Pathology of Chronic Obstructive Pulmonary Disease

Article

Nov 2008
Annu Rev Pathol

The pathogenesis of chronic obstructive pulmonary disease (COPD) is based on the innate and adaptive inflammatory immune response to the inhalation of toxic particles and gases. Although tobacco smoking is the primary cause of this inhalation injury, many other environmental and occupational exposures contribute to the pathology of COPD. The immune inflammatory changes associated with COPD are linked to a tissue-repair and -remodeling process that increases mucus production and causes emphysematous destruction of the gas-exchanging surface of the lung. The common form of emphysema observed in smokers begins in the respiratory bronchioles near the thickened and narrowed small bronchioles that become the major site of obstruction in COPD. The mechanism(s) that allow small airways to thicken in such close proximity to lung tissue undergoing emphysematous destruction remains a puzzle that needs to be solved.

Nrf2 as a Master Redox Switch in Turning on the Cellular Signaling Involved in the Induction of Cytoprotective Genes by Some Chemopreventive Phytochemicals

Article

Nov 2008

A wide array of dietary phytochemicals have been reported to induce the expression of enzymes involved in both cellular antioxidant defenses and elimination/inactivation of electrophilic carcinogens. Induction of such cytoprotective enzymes by edible phytochemicals largely accounts for their cancer chemopreventive and chemoprotective activities. Nuclear factor-erythroid-2-related factor 2 (Nrf2) plays a crucial role in the coordinated induction of those genes encoding many stress-responsive and cytoptotective enzymes and related proteins. These include NAD(P)H:quinone oxidoreductase-1, heme oxygenase-1, glutamate cysteine ligase, glutathione S-transferase, glutathione peroxidase, thioredoxin, etc. In resting cells, Nrf2 is sequestered in the cytoplasm as an inactive complex with the repressor Kelch-like ECH-associated protein 1 (Keap1). The release of Nrf2 from its repressor is most likely to be achieved by alterations in the structure of Keap1. Keap1 contains several reactive cysteine residues that function as sensors of cellular redox changes. Oxidation or covalent modification of some of these critical cysteine thiols would stabilize Nrf2, thereby facilitating nuclear accumulation of Nrf2. After translocation into nucleus, Nrf2 forms a heterodimer with other transcription factors, such as small Maf, which in turn binds to the 5'-upstream CIS-acting regulatory sequence, termed antioxidant response elements (ARE) or electrophile response elements (EpRE), located in the promoter region of genes encoding various antioxidant and phase 2 detoxifying enzymes. Certain dietary chemopreventive agents target Keap1 by oxidizing or chemically modifying one or more of its specific cysteine thiols, thereby stabilizing Nrf2. In addition, phosphorylation of specific serine or threonine residues present in Nrf2 by upstream kinases may also facilitate the nuclear localization of Nrf2. Multiple mechanisms of Nrf2 activation by signals mediated by one or more of the upstream kinases, such as mitogen-activated protein kinases, phosphatidylionositol-3-kinase/Akt, protein kinase C, and casein kinase-2 have recently been proposed. This review highlights the cytoprotective gene expression induced by some representative dietary chemopreventive phytochemicals with the Nrf2-Keap1 system as a prime molecular target.

Role of HDAC2 in the pathophysiology of COPD

Article

Oct 2008
ANNU REV PHYSIOL

Peter J Barnes

Chronic obstructive pulmonary disease (COPD), characterized by progressive inflammation in the small airways and lung parenchyma, is mediated by the increased expression of multiple inflammatory genes. The increased expression of these genes is regulated by acetylation of core histones, whereas histone deacetylase 2 (HDAC2) suppresses inflammatory gene expression. In COPD, HDAC2 activity and expression are reduced in peripheral lung and in alveolar macrophages, resulting in amplification of the inflammatory response. Corticosteroid resistance in COPD occurs because corticosteroids use HDAC2 to switch off activated inflammatory genes. The reduction in HDAC2 appears to be secondary to the increased oxidative and nitrative stress in COPD lungs. Antioxidants and inhibitors of nitric oxide synthesis may therefore restore corticosteroid sensitivity in COPD, but this can also be achieved by low concentrations of theophylline and curcumin, which act as HDAC activators.

Identification of NRF2, a member of the NF-E2 family of transcription factors, as a substrate for caspase-3(-like) proteases

Article

Oct 1999
CELL DEATH DIFFER

Apoptosis is mediated by members of the interleukin-1beta converting enzyme (ICE) family of proteases (caspases), which are activated by diverse stimuli, although the downstream molecular targets of caspases are still poorly understood. Using the modified yeast two-hybrid system, which we recently established to clone genes for caspase substrates, we identified NRF2 as a novel caspase substrate. NRF2 is a member of the NF-E2 family of basic region leucine-zipper transcription factors and has been shown to induce phase II detoxifying enzymes through anti-oxidant response elements. NRF2 was cleaved at two sites by recombinant caspase-3 in vitro as well as in HeLa cells during TNFalpha-mediated apoptosis. Overexpression of the C-terminal cleavage fragment containing the DNA binding and leucine-zipper domains induced apoptosis in HeLa cells. These observations suggest that NRF2 might have some role in the induction of apoptosis after cleavage by caspases.

Microsatellite Polymorphism in the Heme Oxygenase-1 Gene Promoter Is Associated with Susceptibility to Emphysema

Article

Feb 2000

Cigarette smoke, containing reactive oxygen species, is the most important risk factor for chronic pulmonary emphysema (CPE). Heme oxygenase-1 (HO-1) plays a protective role as an antioxidant in the lung. A (GT)n dinucleotide repeat in the 5'-flanking region of human HO-1 gene shows length polymorphism and could modulate the level of gene transcription. To investigate the correlation between the length of the (GT)n repeat and susceptibility to the development of CPE, we screened the frequencies of alleles with varying numbers of (GT)n repeats in the HO-1 gene in 101 smokers with CPE and in 100 smokers without CPE. Polymorphisms of the (GT)n repeat were grouped into three classes: class S alleles (<25 repeats), class M alleles (25-29 repeats), and class L alleles (>/=30 repeats). The proportion of allele frequencies in class L, as well as the proportion of genotypic frequencies in the group with class L alleles (L/L, L/M, and L/S), was significantly higher in the smokers with CPE than in smokers without CPE. Moreover, we analyzed the promoter activities of the HO-1 gene carrying different (GT)n repeats (n=16, 20, 29, and 38), by transient-transfection assay in cultured cell lines. H2O2 exposure up-regulated the transcriptional activity of the HO-1 promoter/luciferase fusion genes with (GT)16 or (GT)20 but did not do so with (GT)29 or (GT)38. These findings suggest that the large size of a (GT)n repeat in the HO-1 gene promoter may reduce HO-1 inducibility by reactive oxygen species in cigarette smoke, thereby resulting in the development of CPE.

Increased Expression of Heme Oxygenase (HO)-1 in Alveolar Spaces and HO-2 in Alveolar Walls of Smokers

Article

Nov 2001

It has been suggested that oxidative stress protein heme oxygenase (HO)-1 plays a role in chronic airway diseases including chronic obstructive pulmonary disease (COPD). The inducible isoform HO-1 and the constitutive HO-2 catalyze the same reaction. Their distribution in situ was studied in lungs of 10 nonsmoking subjects, 6 healthy smokers, and 10 smokers with COPD. Paraffin-embedded sections of surgical lung specimens were immunostained with antibodies against HO-1 and HO-2. HO-1 immunoreactivity was observed mainly in alveolar macrophages. HO-1-positive macrophages were increased in smokers with COPD (median: 36%) as compared with nonsmoking subjects (13%; p < 0.02), whereas no differences were observed between patients with COPD and healthy smokers (34%). HO-2 had a more widespread distribution in cells of the alveolar wall, in adventitia of pulmonary arteries and bronchioles, and in vascular smooth muscle. Lower percentages of alveolar macrophages exhibited positive staining for HO-2 without significant differences between the three groups. HO-2(+) cells in the alveolar wall were increased in smokers with (15/mm) and without COPD (12/mm) as compared with nonsmokers (8/mm, p < 0.01). In conclusion, inducible HO-1 and constitutive HO-2 are detectable in human lung tissue and their expression is increased in smokers, suggesting that oxidative stress due to cigarette smoke may increase lung cells expressing HO-1 and HO-2.

Resveratrol upregulates heme oxygenase-1 expression via activation of NF-E2-related factor 2 in PC12 cells

Article

Jul 2005

Resveratrol (3,4',5-trihydroxy stilbene), a phytoalexin found in the skin and seeds of grapes, has been reported to possess anti-inflammatory, anticarcinogenic, and antioxidant activities. In this work, we assessed the ability of resveratrol to upregulate heme oxygenase-1 (HO-1) gene expression via activation of NF-E2-related factor 2 (Nrf2) in cultured PC12 cells. Nrf2 is a transcription factor involved in the cellular protection against oxidative stress through antioxidant response element (ARE)-directed induction of several phase 2 detoxifying and antioxidant enzymes, such as HO-1. Here, we report that resveratrol induces HO-1 expression via the ARE-mediated transcriptional activation of Nrf2. Moreover, PC12 cells treated with resveratrol exhibited transient activation of Akt/protein kinase B and extracellular signal-regulated protein kinase 1/2 (ERK1/2). LY294002 and U0126, pharmacological inhibitors of phosphatidylinositol 3-kinase and MEK1/2 which are upstream of Akt and ERK1/2, respectively, attenuated resveratrol-induced HO-1 expression and exhibited antioxidant effects. Taken together, the above findings suggest that resveratrol augments cellular antioxidant defense capacity through induction of HO-1 via Nrf2-ARE signaling, thereby protecting PC12 cells from oxidative stress.

NRF2 targeting: A promising therapeutic strategy in chronic obstructive pulmonary disease

Abstract

No full-text available

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