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1.  Prevention of Diabetic Complications by Activation of Nrf2: Diabetic Cardiomyopathy and Nephropathy 
Experimental Diabetes Research  2012;2012:216512.
Diabetic cardiomyopathy and nephropathy are two major causes of death of patients with diabetes. Extra generation of reactive oxygen species (ROS), induced by hyperglycemia, is considered as the main reason for the development of these diabetic complications. Transcription factor, NFE2-related factor 2 (Nrf2), is a master regulator of cellular detoxification response and redox status, and also provides a protective action from various oxidative stresses and damages. Recently we have demonstrated its important role in determining the susceptibility of cells or tissues to diabetes-induced oxidative stress and/or damage. Therefore, this review will specifically summarize the information available regarding the effect of Nrf2 on the diabetic complications with a focus on diabetic cardiomyopathy and nephropathy. Given the feature that Nrf2 is easily induced by several compounds, we also discussed the role of different Nrf2 activators in the prevention or therapy of various diabetic complications. These findings suggest that Nrf2 has a potential application in the clinic setting for diabetic patients in the short future.
doi:10.1155/2012/216512
PMCID: PMC3356887  PMID: 22645602
2.  Prevention of Diabetic Nephropathy by Sulforaphane: Possible Role of Nrf2 Upregulation and Activation 
The present study was to investigate whether sulforaphane (SFN) can prevent diabetic nephropathy in type 1 diabetic mouse model induced by multiple low-dose streptozotocin. Diabetic and age-matched control mice were given SFN at 0.5 mg/kg body weight daily for 3 months. At the end of 3-month SFN treatment, the diabetic nephropathy, shown by renal inflammation, oxidative damage, fibrosis, and dysfunction, was significantly prevented along with an elevation of renal Nrf2 expression and transcription in diabetes/SFN group compared with diabetic group. However, this renal prevention by SFN was not seen when the 3-month SFN-treated diabetic mice were aged for additional 3 months without further SFN treatment. Nrf2-mediated renal protective effects in diabetes were evaluated in human renal tubular HK11 cells transfected with control and Nrf2 siRNA and treated with 27.5 mM mannitol or high glucose plus palmitate (300 μM). Blockade of Nrf2 expression completely abolished SFN prevention of the profibrotic effect induced by high glucose plus palmitate. These results support that renal Nrf2 expression and its transcription play important roles in SFN prevention of diabetes-induced renal damage. However, the SFN preventive effect on diabetes-induced renal pathogeneses is not sustained, suggesting the requirement of continual use of SFN for its sustained effect.
doi:10.1155/2012/821936
PMCID: PMC3461640  PMID: 23050040
3.  Enhanced Expression of Janus Kinase–Signal Transducer and Activator of Transcription Pathway Members in Human Diabetic Nephropathy 
Diabetes  2009;58(2):469-477.
OBJECTIVE—Glomerular mesangial expansion and podocyte loss are important early features of diabetic nephropathy, whereas tubulointerstitial injury and fibrosis are critical for progression of diabetic nephropathy to kidney failure. Therefore, we analyzed the expression of genes in glomeruli and tubulointerstitium in kidney biopsies from diabetic nephropathy patients to identify pathways that may be activated in humans but not in murine models of diabetic nephropathy that fail to progress to glomerulosclerosis, tubulointerstitial fibrosis, and kidney failure.
RESEARCH DESIGN AND METHODS—Kidney biopsies were obtained from 74 patients (control subjects, early and progressive type 2 diabetic nephropathy). Glomerular and tubulointerstitial mRNAs were microarrayed, followed by bioinformatics analyses. Gene expression changes were confirmed by real-time RT-PCR and immunohistological staining. Samples from db/db C57BLKS and streptozotocin-induced DBA/2J mice, commonly studied murine models of diabetic nephropathy, were analyzed.
RESULTS—In human glomeruli and tubulointerstitial samples, the Janus kinase (Jak)-signal transducer and activator of transcription (Stat) pathway was highly and significantly regulated. Jak-1, -2, and -3 as well as Stat-1 and -3 were expressed at higher levels in patients with diabetic nephropathy than in control subjects. The estimated glomerular filtration rate significantly correlated with tubulointerstitial Jak-1, -2, and -3 and Stat-1 expression (R2 = 0.30–0.44). Immunohistochemistry found strong Jak-2 staining in glomerular and tubulointerstitial compartments in diabetic nephropathy compared with control subjects. In contrast, there was little or no increase in expression of Jak/Stat genes in the db/db C57BLKS or diabetic DBA/2J mice.
CONCLUSIONS—These data suggest a direct relationship between tubulointerstitial Jak/Stat expression and progression of kidney failure in patients with type 2 diabetic nephropathy and distinguish progressive human diabetic nephropathy from nonprogressive murine diabetic nephropathy.
doi:10.2337/db08-1328
PMCID: PMC2628622  PMID: 19017763
4.  Transcription Factor Nrf2-Mediated Antioxidant Defense System in the Development of Diabetic Retinopathy 
Purpose.
Increase in reactive oxygen species (ROS) is one of the major retinal metabolic abnormalities associated with the development of diabetic retinopathy. NF-E2–related factor 2 (Nrf2), a redox sensitive factor, provides cellular defenses against the cytotoxic ROS. In stress conditions, Nrf2 dissociates from its cytosolic inhibitor, Kelch like-ECH-associated protein 1 (Keap1), and moves to the nucleus to regulate the transcription of antioxidant genes including the catalytic subunit of glutamylcysteine ligase (GCLC), a rate-limiting reduced glutathione (GSH) biosynthesis enzyme. Our aim is to understand the role of Nrf2-Keap1-GCLC in the development of diabetic retinopathy.
Methods.
Effect of diabetes on Nrf2-Keap1-GCLC pathway, and subcellular localization of Nrf2 and its binding with Keap1 was investigated in the retina of streptozotocin-induced diabetic rats. The binding of Nrf2 at GCLC was quantified by chromatin immunoprecipitation technique. The results were confirmed in isolated retinal endothelial cells, and also in the retina from human donors with diabetic retinopathy.
Results.
Diabetes increased retinal Nrf2 and its binding with Keap1, but decreased DNA-binding activity of Nrf2 and also its binding at the promoter region of GCLC. Similar impairments in Nrf2-Keap1-GCLC were observed in the endothelial cells exposed to high glucose and in the retina from donors with diabetic retinopathy. In retinal endothelial cells, glucose-induced impairments in Nrf2-GCLC were prevented by Nrf2 inducer tBHQ and also by Keap1-siRNA.
Conclusions.
Due to increased binding of Nrf2 with Keap1, its translocation to the nucleus is compromised contributing to the decreased GSH levels. Thus, regulation of Nrf2-Keap1 by pharmacological or molecular means could serve as a potential adjunct therapy to combat oxidative stress and inhibit the development of diabetic retinopathy.
Diabetes increases retinal Nrf2 levels, but decreases its DNA binding activity. Due to increased binding of Nrf2 with its inhibitor, the recruitment of Nrf2 at the promoter of GCLC, a rate-limiting enzyme in GSH biosynthesis, is decreased, resulting in subnormal antioxidant defense system.
doi:10.1167/iovs.13-11598
PMCID: PMC3676188  PMID: 23633659
antioxidant defense; diabetic retinopathy; Nrf2
5.  Nrf2 protects against As(III)-induced damage in mouse liver and bladder 
Arsenic compounds are classified as toxicants and human carcinogens. Environmental exposure to arsenic imposes a big health issue worldwide. Arsenic elicits its toxic efforts through many mechanisms, including generation of reactive oxygen species (ROS). Nrf2 is the primary transcription factor that controls expression of a main cellular antioxidant response, which is required for neutralizing ROS and thus defending cells from exogenous insults. Previously, we demonstrated a protective role of Nrf2 against arsenic-induced toxicity using a cell culture model. In this report, we present evidence that Nrf2 protects against liver and bladder injury in response to six-weeks of arsenic exposure in a mouse model. Nrf2−/− mice displayed more severe pathological changes in the liver and bladder, compared to Nrf2+/+ mice. Furthermore, Nrf2−/− mice were more sensitive to arsenic-induced DNA hypomethylation, oxidative DNA damage, and apoptotic cell death. These results indicate a protective role of Nrf2 against arsenic toxicity in vivo. Hence, this work demonstrates the feasibility of using dietary compounds that target activation of the Nrf2 signaling pathway to alleviate arsenic-induced damage.
doi:10.1016/j.taap.2009.06.010
PMCID: PMC2739886  PMID: 19538980
6.  Targeting the Transcription Factor Nrf2 to Ameliorate Oxidative Stress and Inflammation in Chronic Kidney Disease 
Kidney international  2013;83(6):1029-1041.
Oxidative stress and inflammation are mediators in the development and progression of chronic kidney disease (CKD) and its complications, and they are inseparably linked as each begets and amplifies the other. CKD-associated oxidative stress is due to increased production of reactive oxygen species (ROS) and diminished antioxidant capacity. The latter is largely caused by impaired activation of Nrf2, the transcription factor that regulates genes encoding antioxidant and detoxifying molecules. Protective effects of Nrf2 are evidenced by amelioration of oxidative stress, inflammation, and kidney disease in response to natural Nrf2 activators in animal models, while Nrf2 deletion amplifies these pathogenic pathways and leads to autoimmune nephritis. Given the role of impaired Nrf2 activity in CKD-induced oxidative stress and inflammation, interventions aimed at restoring Nrf2 may be effective in retarding CKD progression. Clinical trials of the potent Nrf2 activator bardoxolone methyl showed significant improvement in renal function in CKD patients with type 2 diabetes. Results of the ongoing BEACON trial investigating the effect of this drug on time to end-stage renal disease or cardiovascular death will help further characterize the efficacy of Nrf2 pharmacological modulation in CKD. This article provides an overview of the role of impaired Nrf2 activity in the pathogenesis of CKD-associated oxidative stress and inflammation and the potential utility of targeting Nrf2 in the treatment of CKD.
doi:10.1038/ki.2012.439
PMCID: PMC3633725  PMID: 23325084
Oxidative stress; Inflammation; CKD progression; Cardiovascular disease; ESRD; Antioxidant therapy; bardoxolone methyl
7.  Therapeutic Potential of Nrf2 Activators in Streptozotocin-Induced Diabetic Nephropathy 
Diabetes  2011;60(11):3055-3066.
OBJECTIVE
To determine whether dietary compounds targeting NFE2-related factor 2 (Nrf2) activation can be used to attenuate renal damage and preserve renal function during the course of streptozotocin (STZ)-induced diabetic nephropathy.
RESEARCH DESIGN AND METHODS
Diabetes was induced in Nrf2+/+ and Nrf2−/− mice by STZ injection. Sulforaphane (SF) or cinnamic aldehyde (CA) was administered 2 weeks after STZ injection and metabolic indices and renal structure and function were assessed (18 weeks). Markers of diabetes including blood glucose, insulin, polydipsia, polyuria, and weight loss were measured. Pathological alterations and oxidative damage in glomeruli were also determined. Changes in protein expression of the Nrf2 pathway, as well as transforming growth factor-β1 (TGF-β1), fibronectin (FN), collagen IV, and p21/WAF1Cip1 (p21) were analyzed. The molecular mechanisms of Nrf2-mediated protection were investigated in an in vitro model using human renal mesangial cells (HRMCs).
RESULTS
SF or CA significantly attenuated common metabolic disorder symptoms associated with diabetes in Nrf2+/+ but not in Nrf2−/− mice, indicating SF and CA function through specific activation of the Nrf2 pathway. Furthermore, SF or CA improved renal performance and minimized pathological alterations in the glomerulus of STZ-Nrf2+/+ mice. Nrf2 activation reduced oxidative damage and suppressed the expression of TGF-β1, extracellular matrix proteins and p21 both in vivo and in HRMCs. In addition, Nrf2 activation reverted p21-mediated growth inhibition and hypertrophy of HRMCs under hyperglycemic conditions.
CONCLUSIONS
We provide experimental evidence indicating that dietary compounds targeting Nrf2 activation can be used therapeutically to improve metabolic disorder and relieve renal damage induced by diabetes.
doi:10.2337/db11-0807
PMCID: PMC3198067  PMID: 22025779
8.  Boldine Prevents Renal Alterations in Diabetic Rats 
Journal of Diabetes Research  2013;2013:593672.
Diabetic nephropathy alters both structure and function of the kidney. These alterations are associated with increased levels of reactive oxygen species, matrix proteins, and proinflammatory molecules. Inflammation decreases gap junctional communication and increases hemichannel activity leading to increased membrane permeability and altering tissue homeostasis. Since current treatments for diabetic nephropathy do not prevent renal damage, we postulated an alternative treatment with boldine, an alkaloid obtained from boldo with antioxidant, anti-inflammatory, and hypoglycemic effects. Streptozotocin-induced diabetic and control rats were treated or not treated with boldine (50 mg/Kg/day) for ten weeks. In addition, mesangial cells were cultured under control conditions or in high glucose concentration plus proinflammatory cytokines, with or without boldine (100 µmol/L). Boldine treatment in diabetic animals prevented the increase in glycemia, blood pressure, renal thiobarbituric acid reactive substances and the urinary protein/creatinine ratio. Boldine also reduced alterations in matrix proteins and markers of renal damage. In mesangial cells, boldine prevented the increase in oxidative stress, the decrease in gap junctional communication, and the increase in cell permeability due to connexin hemichannel activity induced by high glucose and proinflammatory cytokines but did not block gap junction channels. Thus boldine prevented both renal and cellular alterations and could be useful for preventing tissue damage in diabetic subjects.
doi:10.1155/2013/593672
PMCID: PMC3876708  PMID: 24416726
9.  High levels of Nrf2 determine chemoresistance in type II endometrial cancer 
Cancer research  2010;70(13):5486-5496.
Type II endometrial cancer, which mainly presents as serous and clear cell types, has proved to be the most malignant and recurrent carcinoma among various female genital malignancies. The transcription factor, Nrf2, was first described as having chemopreventive activity. Activation of the Nrf2-mediated cellular defense response protects cells against the toxic and carcinogenic effects of environmental insults by upregulating an array of genes that detoxify reactive oxygen species (ROS) and restore cellular redox homeostasis. However, the cancer-promoting role of Nrf2 has recently been revealed. Nrf2 is constitutively upregulated in several types of human cancer tissues and cancer cell lines. Furthermore, inhibition of Nrf2 expression sensitizes cancer cells to chemotherapeutic drugs. In this study, the constitutive level of Nrf2 was compared in different types of human endometrial tumors. It was found that Nrf2 was highly expressed in endometrial serous carcinoma (ESC), whereas complex hyperplasia (CH) and endometrial endometrioid carcinoma (EEC) had no or marginal expression of Nrf2. Likewise, the ESC derived SPEC-2 cell line had a higher level of Nrf2 expression and was more resistant to the toxic effects of cisplatin and paclitaxel than that of the Ishikawa cell line, which was generated from EEC. Silencing of Nrf2 rendered SPEC-2 cells more susceptible to chemotherapeutic drugs while it had a limited effect on Ishikawa cells. Inhibition of Nrf2 expression by overexpressing Keap1 sensitized SPEC-2 cells or SPEC-2-derived xenografts to chemotherapeutic treatments using both cell culture and SCID mouse models. Collectively, we provide a molecular basis for the use of Nrf2 inhibitors to increase the efficacy of chemotherapeutic drugs and to combat chemoresistance, the biggest obstacle in chemotherapy.
doi:10.1158/0008-5472.CAN-10-0713
PMCID: PMC2896449  PMID: 20530669
Nrf2; chemoresistance; and endometrial cancer
10.  Nrf2 protects human bladder urothelial cells from arsenite and monomethylarsonous acid toxicity 
Toxicology and applied pharmacology  2007;225(2):206-213.
Arsenic is widely spread in our living environment and imposes a big challenge on human health worldwide. Arsenic damages biological systems through multiple mechanisms including the generation of reactive oxygen species. The transcription factor Nrf2 regulates the cellular antioxidant response that protects cells from various insults. In this study, the protective role of Nrf2 in arsenic toxicity was investigated in a human bladder urothelial cell line, UROtsa. Using an UROtsa cell line stably infected with Nrf2-siRNA, we clearly demonstrate that compromised Nrf2 expression sensitized the cells to As(III)- and MMA(III)-induced toxicity. On the other hand, the activation of the Nrf2 pathway by tert-butylhydroquinone (tBHQ) and sulforaphane (SF), the known Nrf2-inducers, rendered UROtsa cells more resistant to As(III)- and MMA(III). Furthermore, the wild type mouse embryo fibroblast (WT-MEF) cells were protected from As(III)- and MMA(III)-induced toxicity following Nrf2 activation by tBHQ or SF whereas neither tBHQ nor SF conferred protection in the Nrf2−/−-MEF cells, demonstrating that tBHQ- or SF-mediated protection against As(III)- and MMA(III)-induced toxicity depends on Nrf2 activation. These results, obtained by both loss of function and gain of function analyses, clearly demonstrate the protective role of Nrf2 in arsenic-induced toxicity. The current work lays the groundwork for using Nrf2 activators for therapeutic and dietary interventions against adverse effects of arsenic.
doi:10.1016/j.taap.2007.07.016
PMCID: PMC2610476  PMID: 17765279
Nrf2; Keap1; arsenic; arsenite; MMA(III); UROtsa
11.  The protective effect of α-Lipoic acid on mitochondria in the kidney of diabetic rats 
Background: Diabetic nephropathy (DN) is one of the most common chronic complications of diabetes and the leading cause of end-stage renal disease. Recent research has found that oxidative stress participates in the development of diabetic nephropathy. α-lipoic acid (α-LA), a powerful antioxidant, plays an important role in renal protection against DN, but the underlying mechanism remains unknown. This study modeled the renal protective effects of α-lipoic acid in streptozotocin (STZ) induced diabetic rats and explore the underlying mechanism, which provides new theoretical bases for clinical treatment of diabetic nephropathy. Methods: The diabetic model was induced by intraperitoneal injection of STZ on Male SD and then the diabetic rats were randomly divided into two groups: untreated-diabetic group (DM group), α-LA treated-diabetic group (α-LA group), and the normal rats served as control group (NC group). After 8 weeks of STZ induction, Blood glucose (BG), Blood Urea Nitrogen (BUN), Serum Creatinine (SCr) and urinary albumin excretion rate (UAER) were examined, and morphological changes were assessed by histology. The levels of malondialdehyde (MDA) and the activities of superoxide dismutase (SOD) were also evaluated in serum and renal cortex. Additionally, kidney mitochondrial membrane potential and mitochondrial swelling were measured for different groups. The expression of voltage-dependent anion channel (VDAC) on mitochondria were evaluated by both Western blotting and Immunohistochemistry. Results: After 8 weeks induction of STZ, significant reductions in BUN, SCr, UAER (P<0.01 or P<0.05) and histological improvement were observed in the α-LA group compared to the DM group. In the serum and renal cortex of α-LA group, the content of MDA and the activities of SOC were both significantly decreased (P<0.05). Compared to the DM group, the mitochondrial membrane potential in the α-LA group was significantly increased (P<0.05) and mitochondrial swelling was reduced. Meanwhile, the expression of VDAC on mitochondrial was significantly increased (P<0.05) in the α-LA group. Conclusion: Our findings indicate that antioxidant α-LA exerts a protective role against the development of DN, and the underlying mechanism may involve effective suppression of the generation of oxidants, protection of mitochondrial function, and up-regulating of VDAC expression.
PMCID: PMC3560498  PMID: 23386911
Diabetic nephropathy; reactive oxygen species; mitochondria; voltage-dependent anion channel; α-lipoic acid
12.  The Keap1–Nrf2 system in cancers: stress response and anabolic metabolism 
Frontiers in Oncology  2012;2:200.
The Keap1–Nrf2 [Kelch-like ECH-associated protein 1–nuclear factor (erythroid-derived 2)-like 2] pathway plays a central role in the protection of cells against oxidative and xenobiotic stresses. Nrf2 is a potent transcription activator that recognizes a unique DNA sequence known as the antioxidant response element (ARE). Under normal conditions, Nrf2 binds to Keap1 in the cytoplasm, resulting in proteasomal degradation. Following exposure to electrophiles or reactive oxygen species, Nrf2 becomes stabilized, translocates into the nucleus, and activates the transcription of various cytoprotective genes. Increasing attention has been paid to the role of Nrf2 in cancer cells because the constitutive stabilization of Nrf2 has been observed in many human cancers with poor prognosis. Recent studies have shown that the antioxidant and detoxification activities of Nrf2 confer chemo- and radio-resistance to cancer cells. In this review, we provide an overview of the Keap1–Nrf2 system and discuss its role under physiological and pathological conditions, including cancers. We also introduce the results of our recent study describing Nrf2 function in the metabolism of cancer cells. Nrf2 likely confers a growth advantage to cancer cells through enhancing cytoprotection and anabolism. Finally, we discuss the possible impact of Nrf2 inhibitors on cancer therapy.
doi:10.3389/fonc.2012.00200
PMCID: PMC3530133  PMID: 23272301
stress response; redox homeostasis; transcription; purine nucleotide; glutathione
13.  Pseudomonas aeruginosa Pyocyanin Activates NRF2-ARE-Mediated Transcriptional Response via the ROS-EGFR-PI3K-AKT/MEK-ERK MAP Kinase Signaling in Pulmonary Epithelial Cells 
PLoS ONE  2013;8(8):e72528.
The redox-active pyocyanin (PCN) secreted by the respiratory pathogen Pseudomonas aeruginosa generates reactive oxygen species (ROS) and causes oxidative stress to pulmonary epithelial cells. Nuclear factor (erythroid-derived 2)-like 2 (NRF2) confers protection against ROS-mediated cell death by inducing the expression of detoxifying enzymes and proteins via its binding to the cis-acting antioxidant response element (ARE). However, a clear relationship between NRF2 and PCN-mediated oxidative stress has not been established experimentally. In this study, we investigated the induction of NRF2-ARE response by PCN in the pulmonary epithelial cells. We analyzed the effect of PCN on NRF2 expression and nuclear translocation in cultured human airway epithelial cells, and in a mouse model of chronic PCN exposure. NRF2-dependent transcription of antioxidative enzymes was also assessed. Furthermore, we used inhibitors to examine the involvement of EGFR and its downstream signaling components that mediate NRF2-ARE-activation in response to PCN. PCN enhances the nuclear NRF2 accumulation and activates the transcription of ARE-mediated antioxidant genes. Furthermore, PCN activates NRF2 by inducing the EGFR-phosphoinositide-3-kinase (PI3K) signaling pathway and its main downstream effectors, AKT and MEK1/2-ERK1/2 MAP kinases. Inhibition of the EGFR-PI3K signaling markedly attenuates PCN-stimulated NRF2 accumulation in the nucleus. We demonstrate for the first time that PCN-mediated oxidative stress activates the EGFR-PI3K-AKT/MEK1/2-ERK1/2 MAP kinase signaling pathway, leading to nuclear NRF2 translocation and ARE responsiveness in pulmonary epithelial cells.
doi:10.1371/journal.pone.0072528
PMCID: PMC3755003  PMID: 24015256
14.  Transforming Growth Factor-β and Nuclear Factor E2–related Factor 2 Regulate Antioxidant Responses in Airway Smooth Muscle Cells 
Rationale: Aberrant airway smooth muscle cell (ASMC) function and overexpression of transforming growth factor (TGF)-β, which modulates ASMC proliferative and inflammatory function and induces oxidant release, are features of asthma. Nuclear factor E2-related factor 2 (Nrf2) activates antioxidant genes conferring protection against oxidative stress.
Objectives: To determine the role of Nrf2 in ASMCs and its modulation by TGF-β, and compare Nrf2 activity in ASMCs from subjects with severe and nonsevere asthma and healthy subjects.
Methods: ASMCs were cultured from airways of subjects without asthma, and from airway biopsies from patients with severe and nonsevere asthma. We studied Nrf2 activation on antioxidant gene expression and proliferation, the effect of TGF-β on Nrf2 transcriptional activity, and the impact of Nrf2 activation on TGF-β–mediated proliferation and IL-6 release. Nrf2–antioxidant response elements binding and Nrf2-dependent antioxidant gene expression was determined in asthmatic ASMCs.
Measurements and Main Results: Activation of Nrf2 led to up-regulation of the antioxidant genes heme oxygenase (HO)-1, NAD(P)H:quinone oxidoreductase, and manganese superoxide dismutase, and a reduction in proliferation. TGF-β reduced Nrf2-mediated antioxidant gene transcription through induction of activating transcription factor-3 expression. Nrf2 activation attenuated TGF-β–mediated reduction in HO-1, ASMC proliferation, and IL-6 release. Nrf2–antioxidant response elements binding was reduced in ASMCs from patients with severe asthma compared with ASMCs from patients with nonsevere asthma and normal subjects. HO-1 expression was reduced in ASMCs from patients with both nonsevere and severe asthma compared with healthy subjects.
Conclusions: Nrf2 regulates antioxidant responses and proliferation in ASMCs and is inactivated by TGF-β. Nrf2 reduction may underlie compromised antioxidant protection and aberrant ASM function in asthma.
doi:10.1164/rccm.201011-1780OC
PMCID: PMC3402549  PMID: 21799075
asthma; airway smooth muscle; nuclear factor E2-related factor 2; transforming growth factor-β; antioxidant
15.  Targeted Deletion of Nrf2 Impairs Lung Development and Oxidant Injury in Neonatal Mice 
Antioxidants & Redox Signaling  2012;17(8):1066-1082.
Abstract
Aims: Nrf2 is an essential transcription factor for protection against oxidant disorders. However, its role in organ development and neonatal disease has received little attention. Therapeutically administered oxygen has been considered to contribute to bronchopulmonary dysplasia (BPD) in prematurity. The current study was performed to determine Nrf2-mediated molecular events during saccular-to-alveolar lung maturation, and the role of Nrf2 in the pathogenesis of hyperoxic lung injury using newborn Nrf2-deficient (Nrf2−/−) and wild-type (Nrf2+/+) mice. Results: Pulmonary basal expression of cell cycle, redox balance, and lipid/carbohydrate metabolism genes was lower while lymphocyte immunity genes were more highly expressed in Nrf2−/− neonates than in Nrf2+/+ neonates. Hyperoxia-induced phenotypes, including mortality, arrest of saccular-to-alveolar transition, and lung edema, and inflammation accompanying DNA damage and tissue oxidation were significantly more severe in Nrf2−/− neonates than in Nrf2+/+ neonates. During lung injury pathogenesis, Nrf2 orchestrated expression of lung genes involved in organ injury and morphology, cellular growth/proliferation, vasculature development, immune response, and cell–cell interaction. Bioinformatic identification of Nrf2 binding motifs and augmented hyperoxia-induced inflammation in genetically deficient neonates supported Gpx2 and Marco as Nrf2 effectors. Innovation: This investigation used lung transcriptomics and gene targeted mice to identify novel molecular events during saccular-to-alveolar stage transition and to elucidate Nrf2 downstream mechanisms in protection from hyperoxia-induced injury in neonate mouse lungs. Conclusion: Nrf2 deficiency augmented lung injury and arrest of alveolarization caused by hyperoxia during the newborn period. Results suggest a therapeutic potential of specific Nrf2 activators for oxidative stress-associated neonatal disorders including BPD. Antioxid. Redox Signal. 00, 000–000.
doi:10.1089/ars.2011.4288
PMCID: PMC3423869  PMID: 22400915
16.  An internal ribosomal entry site mediates redox-sensitive translation of Nrf2 
Nucleic Acids Research  2009;38(3):778-788.
Nrf2 plays pivotal roles in coordinating the antioxidant response and maintaining redox homeostasis. Nrf2 expression is exquisitely regulated; Nrf2 expression is suppressed under unstressed conditions but strikingly induced under oxidative stress. Previous studies showed that stress-induced Nrf2 up-regulation results from both the inhibition of Nrf2 degradation and enhanced Nrf2 translation. In the present study, we elucidate the mechanism underlying translational control of Nrf2. An internal ribosomal entry site (IRES) was identified within the 5′ untranslated region of human Nrf2 mRNA. The IRESNrf2 contains a highly conserved 18S rRNA binding site (RBS) that is required for internal initiation. This IRESNrf2 also contains a hairpin structured inhibitory element (IE) located upstream of the RBS. Deletion of this IE remarkably enhanced translation. Significantly, treatment of cells with hydrogen peroxide (H2O2) and phyto-oxidant sulforaphane further stimulated IRESNrf2-mediated translation initiation despite the attenuation of global protein synthesis. Polyribosomal profile assay confirmed that endogenous Nrf2 mRNAs were recruited into polysomal fractions under oxidative stress conditions. Collectively, these data demonstrate that Nrf2 translation is suppressed under normal conditions and specifically enhanced upon oxidant exposure by internal initiation, and provide a mechanistic explanation for translational control of Nrf2 by oxidative stress.
doi:10.1093/nar/gkp1048
PMCID: PMC2817467  PMID: 19934254
17.  Preclinical Evaluation of Targeting the Nrf2 Pathway by Triterpenoids (CDDO-Im and CDDO-Me) for Protection from LPS-Induced Inflammatory Response and Reactive Oxygen Species in Human Peripheral Blood Mononuclear Cells and Neutrophils 
Antioxidants & redox signaling  2007;9(11):1963-1970.
Sepsis is characterized by an inappropriate host immune-inflammatory response and sustained oxidative damage. Nrf2, a bZIP oxidant-responsive transcription factor, regulates a battery of cytoprotective genes including antioxidants and maintains cellular redox homeostasis. Mouse studies have demonstrated a critical role of Nrf2 in improving survival during sepsis. This preclinical ex vivo study using neutrophils and peripheral blood mononuclear cells (PBMCs) as a surrogate cells evaluates the efficacy of CDDO-Im and CDDO-Me [imidazole and methyl ester derivative of 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid (CDDO)] to activate the Nrf2 pathway and protect from lipopolysaccharide (LPS)-induced inflammatory response in humans. CDDO-Im treatment significantly induced Nrf2–dependent antioxidative genes (HO-1, GCLC, GCLM, and NQO1) in PBMCs isolated from six normal subjects. CDDO-Im increased nuclear accumulation of Nrf2 protein. Pretreatment of PBMC by CDDO-Im significantly attenuated LPS-induced cytokine expression. Similar increases in levels of antioxidant genes and suppression of LPS-induced cytokine expression was observed after CDDO-Me pretreatment. CDDO-Im also greatly inhibited LPS, fMLP, TNF-α, and TPA-induced ROS generation in neutrophils. In conclusion, these results demonstrate that activation of the Nrf2-dependent antioxidative pathway by CDDO-Im or CDDO-Me protects against the LPS-induced inflammatory response and suggest that they can be potential therapeutic candidates for intervening sepsis syndrome.
doi:10.1089/ars.2007.1745
PMCID: PMC2396226  PMID: 17822364
18.  Coordinated Induction of Nrf2 Target Genes Protects Against Iron Nitrilotriacetate (FeNTA)-Induced Nephrotoxicity 
Toxicology and applied pharmacology  2008;231(3):364-373.
The iron chelate, ferric nitrilotriacetate (FeNTA), induces acute proximal tubular necrosis as a consequence of lipid peroxidation and oxidative tissue damage. Chronic exposure of FeNTA leads to a high incidence of renal adenocarcinomas in rodents. NF-e2-related factor 2 (Nrf2) is a transcription factor that is activated by oxidative stress and electrophiles, and regulates the basal and inducible expression of numerous detoxifying and antioxidant genes. To determine the roles of Nrf2 in regulating renal gene expression and protecting against oxidative stress-induced kidney damage, wild-type and Nrf2-null mice were administered FeNTA. Renal Nrf2 protein translocated to the nucleus at 6 h after FeNTA treatment. FeNTA increased mRNA levels of Nrf2 target genes, including NQO1, GCLC, GSTpi1/2, Mrp1, 2, and 4 in kidneys from wild-type mice, but not Nrf2-null mice. Protein expression of NQO1, a prototypical Nrf2 target gene, was increased in wild-type mice, with no change in Nrf2-null mice. FeNTA produced more nephrotoxicity in Nrf2-null mice than wild-type mice as indicated by higher serum urea nitrogen and creatinine levels, as more urinary NAG, stronger 4-hydroxynonenal protein adduct staining, and more extensive proximal tubule damage. Furthermore, pretreatment with CDDO-Im, a potent small molecule Nrf2 activator, protected mice against FeNTA-induced renal toxicity. Collectively, these results suggest that activation of Nrf2 protects mouse kidneys from FeNTA-induced oxidative stress damage by coordinately up-regulating the expression of cytoprotective genes.
doi:10.1016/j.taap.2008.05.022
PMCID: PMC2582522  PMID: 18617210
Nrf2; FeNTA; NQO1; oxidative stress; Mrp; kidney
19.  Oxidative Stress/Angiotensinogen/Renin-Angiotensin System Axis in Patients with Diabetic Nephropathy 
Although recent studies have proven that renin-angiotensin system (RAS) blockades retard the progression of diabetic nephropathy, the detailed mechanisms of their reno-protective effects on the development of diabetic nephropathy remain uncertain. In rodent models, it has been reported that reactive oxygen species (ROS) are important for intrarenal angiotensinogen (AGT) augmentation in the progression of diabetic nephropathy. However, no direct evidence is available to demonstrate that AGT expression is enhanced in the kidneys of patients with diabetes. To examine whether the expression levels of ROS- and RAS-related factors in kidneys are increased with the progression of diabetic nephropathy, biopsied samples from 8 controls and 27 patients with type 2 diabetes were used. After the biopsy, these patients were diagnosed with minor glomerular abnormality or diabetes mellitus by clinical and pathological findings. The intensities of AGT, angiotensin II (Ang II), 4-hydroxy-2-nonenal (4-HNE), and heme oxygenase-1 (HO-1) were examined by fluorescence in situ hybridization and/or immunohistochemistry. Expression levels were greater in patients with diabetes than in control subjects. Moreover, the augmented intrarenal AGT mRNA expression paralleled renal dysfunction in patients with diabetes. These data suggest the importance of the activated oxidative stress/AGT/RAS axis in the pathogenesis of diabetic nephropathy.
doi:10.3390/ijms141123045
PMCID: PMC3856105  PMID: 24284398
angiotensinogen; clinical study; diabetic nephropathy; oxidative stress; renin-angiotensin system
20.  NF-E2–Related Factor 2 Promotes Atherosclerosis by Effects on Plasma Lipoproteins and Cholesterol Transport That Overshadow Antioxidant Protection 
Objective
To test the hypothesis that NF-E2–related factor 2 (Nrf2) expression plays an antiatherogenic role by its vascular antioxidant and anti-inflammatory properties.
Methods and Results
Nrf2 is an important transcription factor that regulates the expression of phase 2 detoxifying enzymes and antioxidant genes. Its expression in vascular cells appears to be an important factor in the protection against vascular oxidative stress and inflammation. We developed Nrf2 heterozygous (HET) and homozygous knockout (KO) mice on an apolipoprotein (apo) E–null background by sequential breeding, resulting in Nrf2−/−, apoE−/− (KO), Nrf2−/+, apoE−/− (HET) and Nrf2+/+, and apoE−/− wild-type littermates. KO mice exhibited decreased levels of antioxidant genes with evidence of increased reactive oxygen species generation compared with wild-type controls. Surprisingly, KO males exhibited 47% and 53% reductions in the degree of aortic atherosclerosis compared with HET or wild-type littermates, respectively. Decreased atherosclerosis in KO mice correlated with lower plasma total cholesterol in a sex-dependent manner. KO mice also had a decreased hepatic cholesterol content and a lower expression of lipogenic genes, suggesting that hepatic lipogenesis could be reduced. In addition, KO mice exhibited atherosclerotic plaques characterized by a lesser macrophage component and decreased foam cell formation in an in vitro lipid-loading assay. This was associated with a lower rate of cholesterol influx, mediated in part by decreased expression of the scavenger receptor CD36.
Conclusion
Nrf2 expression unexpectedly promotes atherosclerotic lesion formation in a sex-dependent manner, most likely by a combination of systemic metabolic and local vascular effects.
doi:10.1161/ATVBAHA.110.210906
PMCID: PMC3037185  PMID: 20947826
atherosclerosis; cytokines; lipoproteins; reactive oxygen species; foam cell formation; lipogenesis; Nrf2
21.  Role of Bone Morphogenetic Proteins-7 (BMP-7) in the Renal Improvement Effect of DangGui (Angelica sinensis) in Type-1 Diabetic Rats 
Hyperglycemia induced reactive oxygen species (ROS) generation is believed as major factors leading to diabetic nephropathy (DN). DangGui (Angelica sinensis) is mentioned to show renal protective effect in combination with other herbs. Bone morphogenetic proteins-7 (BMP-7) is produced merit in protection of DN. The role of BMP-7 in DangGui-induced renal improvement is not clear. The present study investigated the effects of DangGui on renal functions, BMP-7 expression and the levels of ROS in streptozotocin (STZ)-induced diabetic rats and high glucose-exposed rat mesangial cells (RMCs). After 1- or 4-week treatment, DangGui improved renal functions and increased renal BMP-7 expression in diabetic rats. The BMP-7 expression in RMCs was reduced by high glucose treatment and this could be reversed by DangGui. Moreover, RMCs exposed to high glucose were expired by BMP-7 RNAi transfection but those cells remained alive by scramble transfection. Thus, we employed regular RMCs to knock down BMP-7 with RNAi and we found that DangGui increased BMP-7 expression in these RMCs. Direct activation of BMP-7 expression by DangGui could be considered. The results of DPPH assay, DHE stain and lucigenin assay indicated that DangGui could inhibit high glucose-induced ROS in RMCs. These results suggest that DangGui has an ability to improve renal functions in STZ-diabetic rats through increasing endogenous BMP-7 expression and decreasing oxidative stress in kidney. The present study suggest that DangGui could be applied to improve renal functions in diabetic disorders.
doi:10.1093/ecam/nep167
PMCID: PMC3163074  PMID: 21876712
22.  Protective Role of Cannabinoid Receptor Type 2 in a Mouse Model of Diabetic Nephropathy 
Diabetes  2011;60(9):2386-2396.
OBJECTIVE
The cannabinoid receptor type 2 (CB2) has protective effects in chronic degenerative diseases. Our aim was to assess the potential relevance of the CB2 receptor in both human and experimental diabetic nephropathy (DN).
RESEARCH DESIGN AND METHODS
CB2 expression was studied in kidney biopsies from patients with advanced DN, in early experimental diabetes, and in cultured podocytes. Levels of endocannabinoids and related enzymes were measured in the renal cortex from diabetic mice. To assess the functional role of CB2, streptozotocin-induced diabetic mice were treated for 14 weeks with AM1241, a selective CB2 agonist. In these animals, we studied albuminuria, renal function, expression of podocyte proteins (nephrin and zonula occludens-1), and markers of both fibrosis (fibronectin and transforming growth factor-β1) and inflammation (monocyte chemoattractant protein-1 [MCP-1], CC chemokine receptor 2 [CCR2], and monocyte markers). CB2 signaling was assessed in cultured podocytes.
RESULTS
Podocytes express the CB2 receptor both in vitro and in vivo. CB2 was downregulated in kidney biopsies from patients with advanced DN, and renal levels of the CB2 ligand 2-arachidonoylglycerol were reduced in diabetic mice, suggesting impaired CB2 regulation. In experimental diabetes, AM1241 ameliorated albuminuria, podocyte protein downregulation, and glomerular monocyte infiltration, without affecting early markers of fibrosis. In addition, AM1241 reduced CCR2 expression in both renal cortex and cultured podocytes, suggesting that CB2 activation may interfere with the deleterious effects of MCP-1 signaling.
CONCLUSIONS
The CB2 receptor is expressed by podocytes, and in experimental diabetes, CB2 activation ameliorates both albuminuria and podocyte protein loss, suggesting a protective effect of signaling through CB2 in DN.
doi:10.2337/db10-1809
PMCID: PMC3161308  PMID: 21810593
23.  Oridonin Confers Protection against Arsenic-Induced Toxicity through Activation of the Nrf2-Mediated Defensive Response 
Environmental Health Perspectives  2008;116(9):1154-1161.
Background
Groundwater contaminated with arsenic imposes a big challenge to human health worldwide. Using natural compounds to subvert the detrimental effects of arsenic represents an attractive strategy. The transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) is a critical regulator of the cellular antioxidant response and xenobiotic metabolism. Recently, activation of the Nrf2 signaling pathway has been reported to confer protection against arsenic-induced toxicity in a cell culture model.
Objectives
The goal of the present work was to identify a potent Nrf2 activator from plants as a chemopreventive compound and to demonstrate the efficacy of the compound in battling arsenic-induced toxicity.
Results
Oridonin activated the Nrf2 signaling pathway at a low subtoxic dose and was able to stabilize Nrf2 by blocking Nrf2 ubiquitination and degradation, leading to accumulation of the Nrf2 protein and activation of the Nrf2-dependent cytoprotective response. Pretreatment of UROtsa cells with 1.4 μM oridonin significantly enhanced the cellular redox capacity, reduced formation of reactive oxygen species (ROS), and improved cell survival after arsenic challenge.
Conclusions
We identified oridonin as representing a novel class of Nrf2 activators and illustrated the mechanism by which the Nrf2 pathway is activated. Furthermore, we demonstrated the feasibility of using natural compounds targeting Nrf2 as a therapeutic approach to protect humans from various environmental insults that may occur daily.
doi:10.1289/ehp.11464
PMCID: PMC2535615  PMID: 18795156
antioxidant responsive element; antitumor; ARE; arsenic; chemoprevention; diterpenoid; Keap1; Nrf2; oridonin; oxidative stress; rubescensin
24.  Fufang Xue Shuan Tong capsules inhibit renal oxidative stress markers and indices of nephropathy in diabetic rats 
Fufang Xue Shuan Tong (FXST) capsules, a traditional Chinese medicine, have been used to treat diabetic nephropathy for many years. FXST has been shown to attenuate elevated levels of oxidative stress in the retina of diabetic rats. However, whether FXST protects kidneys through the same mechanism(s) remains unclear. In this study, diabetes was induced in rats by administration of a high-fat diet and low-dose streptozotocin. Rats were administered low (450 mg/kg/day), middle (900 mg/kg/day) or high (1800 mg/kg/day) doses of FXST orally for 3 months. Another group was administered 50 mg/kg/day orally for the same period. The results indicated that all doses of FXST reduced urinary protein excretion and creatinine clearance and ameliorated the diabetic nephropathy-related mesangial matrix expansion. However, only middle and high doses of FXST prevented glomerular hypertrophy in diabetic rats, and the high dose showed the greatest inhibitory effect with regard to mesangial matrix expansion. Furthermore, superoxide dismutase activities were significantly elevated, whereas malondialdehyde levels were significantly reduced in the renal cortex following FXST treatment. The kidney-protective role of FXST is not inferior to that of captopril, one of the most commonly used drugs for the treatment of diabetic nephropathy. In conclusion, FXST retards the progression of diabetic nephropathy, while high-dose FXST shows the most prominent effect in counteracting the pathological changes of diabetic nephropathy. The renoprotective action of FXST is induced by the reduction of oxidative stress in diabetic nephropathy.
doi:10.3892/etm.2012.680
PMCID: PMC3493747  PMID: 23226741
Fufang Xue Shuan Tong capsule; diabetic nephropathy; oxidative stress; malondialdehyde; superoxide dismutase
25.  Dual Roles of Nrf2 in Cancer 
In response to oxidative stress, the transcription factor NF-E2-related factor 2 (Nrf2) controls the fate of cells through transcriptional upregulation of antioxidant response element (ARE)-bearing genes, including those encoding endogenous antioxidants, phase II detoxifying enzymes, and transporters. Expression of the Nrf2-dependent proteins is critical for ameliorating or eliminating toxicants/carcinogens to maintain cellular redox homeostasis. As a result, activation of the Nrf2 pathway, by naturally-occurring compounds or synthetic chemicals at sub-toxic doses, confers protection against subsequent toxic/carcinogenic exposure. Thus, the use of dietary compounds or synthetic chemicals to boost the Nrf2-dependent adaptive response to counteract environmental insults has emerged to be a promising strategy for cancer prevention. Interestingly, recent emerging data has revealed the “dark” side of Nrf2. Nrf2 and its downstream genes are overexpressed in many cancer cell lines and human cancer tissues, giving cancer cells an advantage for survival and growth. Furthermore, Nrf2 is upregulated in resistant cancer cells and is thought to be responsible for acquired chemoresistance. Therefore, it may be necessary to inhibit the Nrf2 pathway during chemotherapy. This review is primarily focused on the role of Nrf2 in cancer, with emphasis on the recent findings indicating the cancer promoting function of Nrf2 and its role in acquired chemoresistance.
doi:10.1016/j.phrs.2008.09.003
PMCID: PMC2652397  PMID: 18838122

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