Pharmacological Activation Of Nrf2 Research Articles

Transcription factor Nrf2 activation regulates NETosis, endothelial injury, and kidney disease in myeloperoxidase-positive antineutrophil cytoplasmic antibody-associated vasculitis

Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is a systemic autoimmune disease pathologically characterized by vascular necrosis with inflammation. During AAV development, activated neutrophils produce reactive oxygen species (ROS), leading to the aberrant formation of neutrophil extracellular traps (NETs) via NETosis and subsequent fibrinoid vascular necrosis. Nuclear factor-erythroid 2-related factor 2 (Nrf2) functions as an intracellular defense system to counteract oxidative stress by providing antioxidant properties. Herein, we explored the role of Nrf2 in the pathogenesis of AAV. The role and mechanism of Nrf2 in ANCA-stimulated neutrophils and subsequent endothelial injury were evaluated invitro using Nrf2 genetic deletion and Nrf2 activator treatment. In corresponding invivo studies, the role of Nrf2 in ANCA-transfer AAV and spontaneous AAV murine models was examined. Pharmacological activation of Nrf2 invitro suppressed ANCA-induced NET formation via the inhibition of ROS. In contrast, NET formation was enhanced in Nrf2-deficient neutrophils. Furthermore, Nrf2 activation protected endothelial cells from ANC-induced NETs-mediated injury. Invivo, Nrf2 activation ameliorated glomerulonephritis in two AAV models by upregulating antioxidants and inhibiting ROS-mediated NETs. Furthermore, Nrf2 activation restrained the expansion of splenic immune cells, including T lymphocytes and limited the infiltration of Th17 cells into the kidney. In contrast, Nrf2 genetic deficiency exacerbated vasculitis in a spontaneous AAV model. Thus, the pathophysiological process in AAV may be downregulated by Nrf2 activation, potentially leading to a new therapeutic strategy by regulating NETosis.

A Review of the Potential of Nuclear Factor [Erythroid-Derived 2]-like 2 Activation in Autoimmune Diseases.

An autoimmune disease is the consequence of the immune system attacking healthy cells, tissues, and organs by mistake instead of protecting them. Inflammation and oxidative stress (OS) are well-recognized processes occurring in association with acute or chronic impairment of cell homeostasis. The transcription factor Nrf2 (nuclear factor [erythroid-derived 2]-like 2) is of major importance as the defense instrument against OS and alters anti-inflammatory activities related to different pathological states. Researchers have described Nrf2 as a significant regulator of innate immunity. Growing indications suggest that the Nrf2 signaling pathway is deregulated in numerous diseases, including autoimmune disorders. The advantageous outcome of the pharmacological activation of Nrf2 is an essential part of Nrf2-based chemoprevention and intervention in other chronic illnesses, such as neurodegeneration, cardiovascular disease, autoimmune diseases, and chronic kidney and liver disease. Nevertheless, a growing number of investigations have indicated that Nrf2 is already elevated in specific cancer and disease steps, suggesting that the pharmacological agents developed to mitigate the potentially destructive or transformative results associated with the protracted activation of Nrf2 should also be evaluated. The activators of Nrf2 have revealed an improvement in the progress of OS-associated diseases, resulting in immunoregulatory and anti-inflammatory activities; by contrast, the depletion of Nrf2 worsens disease progression. These data strengthen the growing attention to the biological properties of Nrf2 and its possible healing power on diseases. The evidence supporting a correlation between Nrf2 signaling and the most common autoimmune diseases is reviewed here. We focus on the aspects related to the possible effect of Nrf2 activation in ameliorating pathologic conditions based on the role of this regulator of antioxidant genes in the control of inflammation and OS, which are processes related to the progression of autoimmune diseases. Finally, the possibility of Nrf2 activation as a new drug development strategy to target pathogenesis is proposed.

Nrf2 regulates iron-dependent hippocampal synapses and functional connectivity damage in depression

Neuronal iron overload contributes to synaptic damage and neuropsychiatric disorders. However, the molecular mechanisms underlying iron deposition in depression remain largely unexplored. Our study aims to investigate how nuclear factor-erythroid 2 (NF-E2)-related factor 2 (Nrf2) ameliorates hippocampal synaptic dysfunction and reduces brain functional connectivity (FC) associated with excessive iron in depression. We treated mice with chronic unpredictable mild stress (CUMS) with the iron chelator deferoxamine mesylate (DFOM) and a high-iron diet (2.5% carbonyl iron) to examine the role of iron overload in synaptic plasticity. The involvement of Nrf2 in iron metabolism and brain function was assessed using molecular biological techniques and in vivo resting-state functional magnetic resonance imaging (rs-fMRI) through genetic deletion or pharmacologic activation of Nrf2. The results demonstrated a significant correlation between elevated serum iron levels and impaired hippocampal functional connectivity (FC), which contributed to the development of depression-induced CUMS. Iron overload plays a crucial role in CUMS-induced depression and synaptic dysfunction, as evidenced by the therapeutic effects of a high-iron diet and DFOM. The observed iron overload in this study was associated with decreased Nrf2 levels and increased expression of transferrin receptors (TfR). Notably, inhibition of iron accumulation effectively attenuated CUMS-induced synaptic damage mediated by downregulation of brain-derived neurotrophic factor (BDNF). Nrf2−/− mice exhibited compromised FC within the limbic system and the basal ganglia, particularly in the hippocampus, and inhibition of iron accumulation effectively attenuated CUMS-induced synaptic damage mediated by downregulation of brain-derived neurotrophic factor (BDNF). Activation of Nrf2 restored iron homeostasis and reversed vulnerability to depression. Mechanistically, we further identified that Nrf2 deletion promoted iron overload via upregulation of TfR and downregulation of ferritin light chain (FtL), leading to BDNF-mediated synapse damage in the hippocampus. Therefore, our findings unveil a novel role for Nrf2 in regulating iron homeostasis while providing mechanistic insights into poststress susceptibility to depression. Targeting Nrf2-mediated iron metabolism may offer promising strategies for developing more effective antidepressant therapies.

A Complex Interplay of DJ-1, LRRK2, and Nrf2 in the Regulation of Mitochondrial Function in Cypermethrin-Induced Parkinsonism.

Cypermethrin impairs mitochondrial function, induces redox imbalance, and leads to Parkinsonism in experimental animals. Knockdown of deglycase-1 (DJ-1) gene, which encodes a redox-sensitive antioxidant protein, aggravates cypermethrin-mediated α-synuclein overexpression and oxidative alteration of proteins. DJ-1 is also reported to be essential for maintaining stability of nuclear factor erythroid 2-related factor 2 (Nrf2), shielding cells against oxidative insult. Leucine-rich repeat kinase 2 (LRRK2), another protein associated with Parkinson's disease, is also involved in regulating mitochondrial function. However, underlying molecular mechanisms remain elusive. The study intended to explore an interaction of DJ-1, LRRK2, and Nrf2 in the regulation of mitochondrial function in cypermethrin-induced Parkinsonism. Small interfering RNA-mediated knockdown of DJ-1 and LRRK2 gene and pharmacological activation of Nrf2 were performed in rats and/or human neuroblastoma cells with or without cypermethrin. Indexes of oxidative stress, mitochondrial impairment, and Parkinsonism along with α-synuclein expression, post-translational modification, and aggregation were measured. DJ-1 gene knockdown exacerbated cypermethrin-induced increase in oxidative stress and intrinsic apoptosis and reduction in expression of mitochondrial antioxidant proteins via inhibiting nuclear translocation of Nrf2. Additionally, cypermethrin-induced oxidative stress, mitochondrial impairment, and α-synuclein expression and aggregation were found to be suppressed by LRRK2 gene knockdown, by promoting Nrf2 nuclear translocation and expression of mitochondrial antioxidant proteins. Furthermore, Nrf2 activator, sulforaphane, ameliorated cypermethrin-induced mitochondrial impairment and oxidative stress and provided protection against dopaminergic neuronal death. The findings indicate that DJ-1 and LRRK2 independently alter Nrf2-mediated changes and a complex interplay among DJ-1, LRRK2, and Nrf2 exists in the regulation of mitochondrial function in cypermethrin-induced Parkinsonism.

Astragaloside IV attenuates podocyte apoptosis through ameliorating mitochondrial dysfunction by up-regulated Nrf2-ARE/TFAM signaling in diabetic kidney disease.

Defective antioxidant system as well as mitochondrial dysfunction contributes to the pathogenesis and progression of diabetic kidney disease (DKD). Nuclear factor erythroid 2-related factor 2 (Nrf2)-mediated signaling is the central defensive mechanism against oxidative stress and therefore pharmacological activation of Nrf2 is a promising therapeutic strategy. In this study, using molecular docking we found that Astragaloside IV (AS-IV), an active ingredient from traditional formula of Huangqi decoction (HQD), exerted a higher potential to promote Nrf2 escape from Keap1-Nrf2 interaction via competitively bind to amino acid sites in Keap1. When podocyte exposed to high glucose (HG) stimulation, mitochondrial morphological alterations and podocyte apoptosis were presented and accompanied by Nrf2 and mitochondrial transcription factor A (TFAM) downregulation. Mechanistically, HG promoted a decrease in mitochondria-specific electron transport chain (ETC) complexes, ATP synthesis and mtDNA content as well as increased ROS production. Conversely, all these mitochondrial defects were dramatically alleviated by AS-IV, but suppression of Nrf2 with inhibitor or siRNA and TFAM siRNA simultaneously alleviated the AS-IV efficacy. Moreover, experimental diabetic mice exhibited significant renal injury as well as mitochondrial disorder, corresponding with the decreased expression of Nrf2 and TFAM. On the contrary, AS-IV reversed the abnormality and the Nrf2 and TFAM expression were also restored. Taken together, the present findings demonstrate the improvement of AS-IV on mitochondrial function, thereby resistance to oxidative stress-induced diabetic kidney injury and podocyte apoptosis, and the process is closely associated with activation of Nrf2-ARE/TFAM signaling.

Cytokine storm in individuals with severe COVID-19 decreases endothelial cell antioxidant defense via Downregulation of the Nrf2 transcriptional factor.

The cytokine storm in SARS-CoV-2 infection contributes to the onset of inflammation and target organ damage. The endothelium is a key player in COVID-19 pathophysiology and it is an important target for cytokines. Since cytokines trigger oxidative stress and negatively impact endothelial cell function, we sought to determine whether serum derived from severe COVID-19 individuals decreases endothelial cells' main antioxidant defense, i.e. the antioxidant transcriptional factor Nrf2. Human umbilical vein endothelial cells (HUVEC) were incubated with severe COVID-19 serum at different time points and the effects on redox balance and Nrf2 activity were determined. Serum from COVID-19 individuals increased oxidant species, as indicated by higher DHE oxidation, increased protein carbonylation and augmented mitochondrial reactive oxygen species (ROS) generation and dysfunction. COVID-19 serum, in comparison with serum from healthy individuals, induced cell death and diminished nitric oxide (NO) bioavailability. In parallel, Nrf2 nuclear accumulation and the expression of Nrf2-targeted genes were reduced in endothelial cells exposed to serum from COVID-19 individuals. Additionally, these cells exhibited higher expression of Bach-1, a negative regulator of Nrf2 that competes for DNA binding. All events were prevented by Tocilizumab, an IL-6 receptor blocker, indicating that IL-6 is key to the impairment of endothelial antioxidant defense. In conclusion, endothelial dysfunction related to SARS-Cov-2 infection is linked to an IL-6-dependent decrease in the endothelial antioxidant defense. Pharmacological activation of Nrf2 may decrease endothelial cell damage in individuals with severe COVID-19.

#2709 THE INVOLVEMENT OF NRF2-MEDIATED ANTIOXIDANT SYSTEM IN MYELOPEROXIDASE-POSITIVE ANTINEUTROPHIL CYTOPLASMIC ANTIBODY-ASSOCIATED VASCULITIS

Abstract Background and Aims Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is a systemic autoimmune disease pathologically characterized by vascular necrosis with inflammation. The necrotic lesions consist of ANCA-mediated neutrophil extracellular traps (NETs). During AAV, activated neutrophils form NETs via reactive oxygen spices (ROS)s-mediated signaling pathway. The excessive NETs lead to endothelial vascular damage and serve as autoantigens against ANCAs in a vicious cycle. Although the role of NETs in AAV has been revealed, its treatment for targeting NETs remains unestablished. Since nuclear factor-erythroid 2-related factor 2 (Nrf2) regulates antioxidant proteins against oxidative stress and functions as a defense system, we investigated the role of Nrf2 in the development of AAV. Method In vitro, human neutrophils treated with the Nrf2 activator bardoxolone methyl (Bard) were stimulated with ANCA, and the effects on NETs formation and signaling pathways including intracellular ROS were evaluated by cell imaging and biochemical approaches. The effects of Nrf2 activators on NETs-induced cytotoxicity were examined using human renal glomerular endothelial cells (HRGECs). Nrf2 knockout or WT mouse-derived neutrophils were stimulated with anti-MPO antibody to induce NETs. In vivo studies, Bard was administered to ANCA-transfer AAV model or spontaneous AAV developing mice models (SCG/Kj). Renal function was assessed by biochemical analysis, and the expression of ROS and antioxidant protein NAD(P)H Quinone Dehydrogenase 1 (NQO1) in tissues was evaluated by fluorescent immunostaining and immunoblotting. The formation of NETs in the glomeruli was evaluated by immunostaining and glomerular vascular endothelial damage was assessed by the expression of CD31 and TUNEL positivity. To assess the role of Nrf2 in immunological abnormality during AAV, murine splenocytes were collected and flow cytometry were conducted using CD4, CD8, B220, MCHCII, CD11c, F4/80, Ly 6G, Ly6b, and CD11b antibodies. Moreover, to assess the genetic role of Nrf2 in AAV, we established SCG/Kj mice carrying a germ-line deletion of the Nrf2-genes. A spontaneous mouse model of vasculitis genetically deficient in Nrf2 was analyzed by the same parameters to evaluate the effect of Nrf2. N1 offspring mice were generated by crossing Nrf2-/- mice with SCG/Kj mice. These backcrossed mice were obtained by crossing Nrf2-/- mice to background SCG/Kj mice for three generations. The experimental group was mated to N4 heterozygous mice, and female mice in the experimental group were analyzed at 18 weeks. Results In vitro, pharmacological activation of Nrf2 suppressed ANCA-induced NETs via the upregulation of NQO1 and inhibition of ROS. While, Nrf2-deficienet neutrophils showed the massive NETs in response to ANCA, compared to WT neutrophils. Furthermore, Nrf2 activation directly protected endothelium from cellular injury caused by the exposure of ANCA-mediated NETs. In vivo, the pharmacological activation of Nrf2 ameliorated glomerulonephritis in two kinds of AAV models through the upregulation of antioxidant and inhibition of ROS-mediated NETs. In contrast, Nrf2 genetic deficiency exacerbated vasculitis in spontaneous AAV model. The activation of Nrf2 regulated the expansion of splenocytes in AAV mice. In particular, pan-T cells in spleen and the infiltration of Th17 cells in kidney were suppressed by Nrf2 activation. Conclusion During ANCA-stimulation in neutrophils, the activation of Nrf2 initiates antioxidant system and inhibits intracellular ROS, leading to the suppression of NETs formation. Nrf2 may be a novel therapeutic target for AAV.

Nrf2 sensitizes ferroptosis through l-2-hydroxyglutarate–mediated chromatin modifications in sickle cell disease

AbstractSickle cell disease (SCD) is a chronic hemolytic and systemic hypoxia condition with constant oxidative stress and significant metabolic alterations. However, little is known about the correlation between metabolic alterations and the pathophysiological symptoms. Here, we report that Nrf2, a master regulator of cellular antioxidant responses, regulates the production of the metabolite l-2-hydroxyglutarate (L2HG) to mediate epigenetic histone hypermethylation for gene expression involved in metabolic, oxidative, and ferroptotic stress responses in SCD. Mechanistically, Nrf2 was found to regulate the expression of L2HG dehydrogenase (L2hgdh) to mediate L2HG production under hypoxia. Gene expression profile analysis indicated that reactive oxygen species (ROS) and ferroptosis responses were the most significantly affected signaling pathways after Nrf2 ablation in SCD. Nrf2 silencing and L2HG supplementation sensitize human sickle erythroid cells to ROS and ferroptosis stress. The absence of Nrf2 and accumulation of L2HG significantly affect histone methylation for chromatin structure modification and reduce the assembly of transcription complexes on downstream target genes to regulate ROS and ferroptosis responses. Furthermore, pharmacological activation of Nrf2 was found to have protective effects against ROS and ferroptosis stress in SCD mice. Our data suggest a novel mechanism by which Nrf2 regulates L2HG levels to mediate SCD severity through ROS and ferroptosis stress responses, suggesting that targeting Nrf2 is a viable therapeutic strategy for ameliorating SCD symptoms.

P235 Pharmacological activation of NRF2 has protective effects during Mycobacterium abscessus infection by promoting host defences and reducing inflammatory damage in the context of cystic fibrosis

Lung disease is the leading cause of mortality in cystic fibrosis (CF), a disorder caused by mutations in the CFTR gene, and is characterised by a repetitive circle of infection and inflammation. Among the pathogenic bacteria found in CF airway, the multidrug-resistant Mycobacterium abscessus (Mabs) is associated with accelerated inflammatory lung damage and is often refractory to antibiotic therapies. Therefore, we examined whether curcumin, an activator of the transcription factor NRF2, might provide a beneficial effect against Mabs infection in the context of CF.

AGE-DEPENDENT CARTILAGE DEGENERATION THROUGH NRF2/ARE MODULATION OF SOX9 EXPRESSION

Nuclear factor erythroid 2–related factor 2 (Nrf2)/antioxidant response element (ARE) pathway is key in maintaining redox homeostasis and the pathogenesis of osteoarthritis (OA) involves oxidative distress. We thus investigated whether Nrf2/ARE signaling may control expression of key chondrogenic differentiation and hyaline cartilage maintenance factor SOX9.In human C-28/I2 chondrocytes SOX9 expression was measured by RT–qPCR after shRNA-mediated knockdown of Nrf2 or its antagonist the Kelch-like erythroid cell-derived protein with cap “n” collar homology-associated protein 1 (Keap1). Putative ARE-binding sites in the proximal SOX9 promoter region were inactivated, cloned into pGL3, and co-transfected with phRL–TK for dual-luciferase assays to verify whether Nrf2 transcriptionally regulates SOX9. SOX9 promoter activity without and with Nrf2-inducer methysticin were analyzed. Sox9 expression in articular chondrocytes was correlated to cartilage thickness and degeneration in wild-type (WT) and Nrf2-knockout mice. Data were analyzed by one-way ANOVA, a Student's t-test, or Wilcoxon rank-sum test, according to the normal distribution. Statistical significance was set to p < 0.05.While Keap1-specific RNAi increased SOX9 expression, Nrf2-specific RNAi significantly decreased it. Putative ARE sites (ARE1, ARE2) were identified in the SOX9 promoter region. ARE2 mutagenesis significantly reduced SOX9 promoter activity, while truncation of ARE1 did not. A functional ARE2 site was thus essential for methysticin-mediated induction of SOX9 promoter activity. Knee cartilage of young Nrf2-knockout mice further revealed significantly fewer Sox9-positive chondrocytes as compared to old Nrf2-knockout animals, which further showed thinner cartilage and more severe cartilage erosion.Our data suggest that SOX9 expression in articular cartilage is directly Nrf2-dependent and that pharmacological Nrf2 activation may hold potential to diminish age-dependent osteoarthritic changes in knee cartilage through improving protective SOX9 expression.

Nrf2 Activation Does Not Protect from Aldosterone-Induced Kidney Damage in Mice.

Nuclear factor erythroid 2-related factor 2 (Nrf2) is downregulated in chronic kidney disease (CKD). Activation of Nrf2 might be a therapeutic option in CKD. Here we investigate the effect of Nrf2 activation on aldosterone (Aldo)-induced renal injury. Wild-type (WT) mice, transgenic Keap1 hypomorphic (Nrf2ꜛ, genotype results in upregulation of Nrf2 expression) mice and WT mice treated with the Nrf2 activator sulforaphane (Sulf) received Aldo for 4 weeks. In Aldo-treated mice, kidneys were significantly heavier and pathologically altered, reflected by increased urinary albumin levels and tissue damage. In Nrf2ꜛ-Aldo mice the tubule damage marker NGAL was significantly decreased. Increased oxidative damage markers (8-OHdG, 15-isoprostane F2t) were measured in all Aldo-treated groups. Aldo-increased Nrf2 amounts were mainly found in the late tubule system. The amount of phosphorylated and thus putatively active Nrf2 was significantly increased by Aldo only in WT mice. However, expression of Nrf2 target genes NQO1 and HO1 was decreased in all Aldo-infused mice. GSK3β, which promotes Nrf2 degradation, was significantly increased in the kidneys of Aldo-treated WT mice. Neither genetic nor pharmacological Nrf2 activation was able to prevent oxidative injury induced by Aldo, probably due to induction of negative regulators of Nrf2.

The Role of the Transcription Factor Nrf2 in Alzheimer’s Disease: Therapeutic Opportunities

Alzheimer's disease (AD) is a common neurodegenerative disorder that affects the elderly. One of the key features of AD is the accumulation of reactive oxygen species (ROS), which leads to an overall increase in oxidative damage. The nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is a master regulator of the antioxidant response in cells. Under low ROS levels, Nrf2 is kept in the cytoplasm. However, an increase in ROS production leads to a translocation of Nrf2 into the nucleus, where it activates the transcription of several genes involved in the cells' antioxidant response. Additionally, Nrf2 activation increases autophagy function. However, in AD, the accumulation of Aβ and tau reduces Nrf2 levels, decreasing the antioxidant response. The reduced Nrf2 levels contribute to the further accumulation of Aβ and tau by impairing their autophagy-mediated turnover. In this review, we discuss the overwhelming evidence indicating that genetic or pharmacological activation of Nrf2 is as a potential approach to mitigate AD pathology.

A Nrf2-OSGIN1&2-HSP70 axis mediates cigarette smoke-induced endothelial detachment: implications for plaque erosion.

Endothelial erosion of plaques is responsible for ∼30% of acute coronary syndromes (ACS). Smoking is a risk factor for plaque erosion, which most frequently occurs on the upstream surface of plaques where the endothelium experiences elevated shear stress. We sought to recreate these conditions in vitro to identify potential pathological mechanisms that might be of relevance to plaque erosion. Culturing human coronary artery endothelial cells (HCAECs) under elevated flow (shear stress of 7.5 Pa) and chronically exposing them to cigarette smoke extract (CSE) and tumour necrosis factor-alpha (TNFα) recapitulated a defect in HCAEC adhesion, which corresponded with augmented Nrf2-regulated gene expression. Pharmacological activation or adenoviral overexpression of Nrf2 triggered endothelial detachment, identifying Nrf2 as a mediator of endothelial detachment. Growth/Differentiation Factor-15 (GDF15) expression was elevated in this model, with protein expression elevated in the plasma of patients experiencing plaque erosion compared with plaque rupture. The expression of two Nrf2-regulated genes, OSGIN1 and OSGIN2, was increased by CSE and TNFα under elevated flow and was also elevated in the aortas of mice exposed to cigarette smoke in vivo. Knockdown of OSGIN1&2 inhibited Nrf2-induced cell detachment. Overexpression of OSGIN1&2 induced endothelial detachment and resulted in cell cycle arrest, induction of senescence, loss of focal adhesions and actin stress fibres, and disturbed proteostasis mediated in part by HSP70, restoration of which reduced HCAEC detachment. In ACS patients who smoked, blood concentrations of HSP70 were elevated in plaque erosion compared with plaque rupture. We identified a novel Nrf2-OSGIN1&2-HSP70 axis that regulates endothelial adhesion, elevated GDF15 and HSP70 as biomarkers for plaque erosion in patients who smoke, and two therapeutic targets that offer the potential for reducing the risk of plaque erosion.

Is Nrf2 Behind Endogenous Neuroprotection of the Hippocampal CA2-4,DG Region?

The transcription factor nuclear factor-erythroid 2-related factor 2 (Nrf2) is the master regulator of genes known to be involved in antioxidant, and anti-inflammatory processes, metabolic regulation, and other cellular functions. Here, we also hypothesize a core role for it in endogenous neuroprotection, i.e., the natural adaptive mechanisms protecting the brain from ischemia–reperfusion (I/R) episode. An example of endogenous neuroprotection is ischemia-resistance of the hippocampal regions comprising the CA2, CA3, CA4 and dentate gyrus subfields (here abbreviated to CA2-4,DG) which can be contrasted with the ischemia-vulnerable CA1 region. In the work detailed here, we used a gerbil model of transient cerebral ischemia to examined Nrf2 activation in CA1 and CA2-4,DG, in a control group, and post I/R episode. Data obtained indicate enhanced Nrf2 activity in CA2-4,DG as compared with CA1 in the control, with this difference seen to persist even after I/R. While I/R does indeed cause further activation of Nrf2 in CA2-4,DG, it is associated with slight and transient activation in CA1. Sub-regional differences in Nrf2 activity correlate with immunoreactivity of Keap1 (an Nrf2 suppressor) and Nrf2 target proteins, including heme oxygenase 1, the catalytic and modulatory sub-units of glutamate-cysteine ligase, and glutathione peroxidase 1. Pharmacological Nrf2 activation by sulforaphane results in protection of CA1 after I/R episode. Our results therefore suggest that high Nrf2 activity in CA2-4,DG may guarantee resistance of this region to I/R, potentially explaining the differential sensitivities of the hippocampal regions.

Activation of Nrf2 in fibroblasts promotes a skin aging phenotype via an Nrf2-miRNA-collagen axis

Aging is associated with progressive skin fragility and a tendency to tear, which can lead to severe clinical complications. The transcription factor NRF2 is a key regulator of the cellular antioxidant response, and pharmacological NRF2 activation is a promising strategy for the prevention of age-related diseases. Using a combination of molecular and cellular biology, histology, imaging and biomechanical studies we show, however, that constitutive genetic activation of Nrf2 in fibroblasts of mice suppresses collagen and elastin expression, resulting in reduced skin strength as seen in aged mice. Mechanistically, the “aging matrisome” results in part from direct Nrf2-mediated overexpression of a network of microRNAs that target mRNAs of major skin collagens and other matrix components. Bioinformatics and functional studies revealed high NRF2 activity in aged human fibroblasts in 3D skin equivalents and human skin biopsies, highlighting the translational relevance of the functional mouse data. Together, these results identify activated NRF2 as a promoter of age-related molecular and biomechanical skin features.

Nrf2 as a regulator of mitochondrial function: Energy metabolism and beyond

Mitochondria are unique and essential organelles that mediate many vital cellular processes including energy metabolism and cell death. The transcription factor Nrf2 (NF-E2 p45-related factor 2) has emerged in the last few years as an important modulator of multiple aspects of mitochondrial function. Well-known for controlling cellular redox homeostasis, the cytoprotective effects of Nrf2 extend beyond its ability to regulate a diverse network of antioxidant and detoxification enzymes. Here, we review the role of Nrf2 in the regulation of mitochondrial function and structure. We focus on Nrf2 involvement in promoting mitochondrial quality control and regulation of basic aspects of mitochondrial function, including energy production, reactive oxygen species generation, calcium signalling, and cell death induction. Given the importance of mitochondria in the development of multiple diseases, these findings reinforce the pharmacological activation of Nrf2 as an attractive strategy to counteract mitochondrial dysfunction.

P1560: IMR-261, A NOVEL, ORAL NRF2 ACTIVATOR, REDUCES IRON OVERLOAD IN AN EXPERIMENTAL MOUSE MODEL BY ACTIVATING BMP6-MEDIATED HEPCIDIN SYNTHESIS

Background: Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor that regulates several cellular defense mechanisms, including the oxidative stress response. Recent studies elucidated a protective role for Nrf2 under conditions of iron overload. Accumulation of toxic iron activates Nrf2, which activates Bmp6-mediated synthesis of hepcidin, thereby inhibiting ferroportin and improving iron homeostasis (Lim et al, Nat Metab 2019). IMR-261 is a novel, oral Nrf2 activator which has been tested in Phase 2 clinical trials (previously as CXA-10). Our previous work in mouse models of sickle cell disease and b-thalassemia showed that activation of Nrf2 by IMR-261 increases fetal hemoglobin (HbF) and hemoglobin (Hb), decreases markers of hemolysis and adhesion, reduces VOCs and improves ineffective erythropoiesis. Aims: The objective of this study was to evaluate the ability of IMR-261 to activate Bmp6-mediated synthesis of hepcidin (encoded by the Hamp gene), regulate iron stores and modulate transferrin saturation (TSAT) in a mouse model of iron overload. Methods: b-thalasssemic mice (Hbbth1/th1) were treated with IMR-261 at 37.5 or 50 mg/kg BID for 4 weeks. To assess activation of the Bmp6-hepcidin pathway, hepatic mRNA and plasma protein levels were measured. Iron concentrations in serum and tissue (liver, kidney and spleen) as well as markers of iron overload were also evaluated, and standard hematologic assessments were performed. Results: IMR-261 showed a significant, dose-dependent increase in hepatic mRNA levels of Bmp6 and Hamp (~3-fold increase; high dose vs vehicle) as well as Bmp6 target genes, Smad7 and Id1. A corresponding significant increase in Bmp6 and hepcidin protein levels in plasma was also observed. IMR-261 significantly decreased iron in serum by 32% (high dose vs vehicle) and in all tissues tested (34% in liver, 39% in spleen and 24% in kidney; high dose vs vehicle). Mice treated with IMR-261 also showed a significant decrease in TSAT and ferritin levels (up to 30% and 27%, respectively), as well as a reduction in GDF15 and erythropoietin (up to 72% and 48%, respectively) relative to vehicle. IMR-261 improved hematologic markers including a 30% decrease in reticulocytes and significant increases in RBCs, hemoglobin and hematocrit (consistent with our previous data). Image:Summary/Conclusion: Pharmacologic activation of Nrf2 by IMR-261 in a mouse model of iron overload activates hepcidin synthesis (likely via the Bmp6 signaling pathway) and decreases iron in serum, liver, spleen and kidney. IMR-261 also showed significant reductions in TSAT, ferritin, GDF15 and erythropoietin, which further support its role in iron homeostasis. Induction of hepcidin synthesis via Nrf2 activation is a novel therapeutic approach, and these results support further clinical development of IMR-261 as a treatment for conditions associated with iron dysregulation, including hereditary hemochromatosis.

Nrf2 Activation in Chronic Kidney Disease: Promises and Pitfalls.

The nuclear factor erythroid 2-related factor 2 (Nrf2) protects the cell against oxidative damage. The Nrf2 system comprises a complex network that functions to ensure adequate responses to redox perturbations, but also metabolic demands and cellular stresses. It must be kept within a physiologic activity range. Oxidative stress and alterations in Nrf2-system activity are central for chronic-kidney-disease (CKD) progression and CKD-related morbidity. Activation of the Nrf2 system in CKD is in multiple ways related to inflammation, kidney fibrosis, and mitochondrial and metabolic effects. In human CKD, both endogenous Nrf2 activation and repression exist. The state of the Nrf2 system varies with the cause of kidney disease, comorbidities, stage of CKD, and severity of uremic toxin accumulation and inflammation. An earlier CKD stage, rapid progression of kidney disease, and inflammatory processes are associated with more robust Nrf2-system activation. Advanced CKD is associated with stronger Nrf2-system repression. Nrf2 activation is related to oxidative stress and moderate uremic toxin and nuclear factor kappa B (NF-κB) elevations. Nrf2 repression relates to high uremic toxin and NF-κB concentrations, and may be related to Kelch-like ECH-associated protein 1 (Keap1)-independent Nrf2 degradation. Furthermore, we review the effects of pharmacological Nrf2 activation by bardoxolone methyl, curcumin, and resveratrol in human CKD and outline strategies for how to adapt future Nrf2-targeted therapies to the requirements of patients with CKD.

12-OR: Preventing ß-Cell Death under Glucotoxic Conditions: The Role of ChREBPb

Glucotoxicity is a major mediator of β-cell dysfunction derived from prolonged hyperglycemia. A better understanding of glucotoxicity and preservation of β-cell mass are urgently needed. Carbohydrate response element binding protein (ChREBP) is a glucose-responsive, lipogenic transcription factor with two splice isoforms, ChREBPα and ChREBPβ. We previously showed ChREBPβ is induced by glucose and is required for glucose-stimulated β-cell proliferation in both rodent and human β-cells. In addition, we identified ChREBPβ as a marker for hyperglycemia in β-cells, with its nuclear expression reflecting the progression and severity of the diabetic state. Here, we demonstrate that β-cell death due to glucolipotoxicity (20 mM glucose + 1 mM palmitate) can be prevented by silencing ChREBPβ (6.5±3.1% TUNEL+/Insulin+ in ChREBPβ knock-out islets compared with 47.2±13.6% in control islets, N=7) , most likely via inhibition of lipogenesis and prevention of ceramide-mediated β-cell death. Furthermore, we generated mice with β-cell specific overexpression of ChREBPβ. These mice exhibited increased non-fasting blood glucose levels, impaired glucose tolerance and a marked decrease in β-cell mass, all in a gene dose-dependent manner (OE of one or two alleles of ChREBPβ, N=5) . ChREBPβ overexpression in human beta cells also mimicked glucotoxicity (93.6±3.1% TUNEL+/Insulin+, N=3) . Importantly, we show that ChREBPβ-mediated rodent and human β-cell death was rescued by co-expression with ChREBPα, or by pharmacological activation of Nrf2, a downstream effector of ChREBPα (6.6±1.9 and 2.8±0.9%, respectively, TUNEL+/Insulin+, N=3) . Altogether, our data demonstrate that ChREBPβ is a marker and a causative effector of glucotoxicity, and that downregulating ChREBPβ levels or upregulating ChREBPα levels or its downstream effector (NRF2) rescues β-cells from glucotoxicity. These results position ChREBPβ as a therapeutic target for the prevention of β-cell loss in T2D, an important unmet need in diabetes research. Disclosure L.S.Katz: None. G.Brill: None. M.A.Herman: Research Support; Eli Lilly and Company. A.Garcia-ocana: Consultant; Sun Pharmaceutical Industries Ltd. D.Scott: None. Funding NIH, R01DK130300NIH, R01DK114338NIH, R01DK108905

Thyroid Disorders in Patients Treated with Dimethyl Fumarate for Multiple Sclerosis: A Retrospective Observational Study.

Background: Dimethyl fumarate (DMF), a drug used for the treatment of multiple sclerosis (MS) and psoriasis, has been shown to activate the Keap1/Nrf2 antioxidant response. Nrf2 exerts pleiotropic roles in the thyroid gland; among others, single nucleotide polymorphisms (SNPs) in the gene encoding Nrf2 modulate the risk of Hashimoto’s thyroiditis (HT), suggesting that pharmacological activation of Nrf2 might also be protective. However, a patient with acute exacerbation of HT after starting DMF for MS was recently reported, raising questions about the thyroidal safety of Nrf2 activators. Methods: In a retrospective observational study, we investigated the prevalence and incidence of thyroid disorders (TD) among 163 patients with MS treated with DMF. Results: Only 7/163 patients (4.3%) were diagnosed with functional TD; most (5/163, 3.0%) were diagnosed before DMF treatment. Functional TD were diagnosed under or after DMF in only 2 patients (1.2%). Under DMF, one patient developed transient mild hypothyroidism with negative thyroid autoantibodies. After DMF discontinuation, another patient developed hyperthyroidism due to Graves’ disease. No patient developed thyroid structural disease under or after DMF. Conclusions: The very low incidence of functional TD indicates an overall very good thyroid tolerance of DMF, arguing against screening for TD in MS patients considered for or treated with DMF, and supporting the further study of Nrf2 activators for the prevention and treatment of TD.