Activation Of Antioxidant Genes Research Articles

Melatonin ameliorates multiorgan injuries induced by severe acute pancreatitis in mice by regulating the Nrf2 signaling pathway

Severe acute pancreatitis (SAP) is a complicated inflammatory reaction that impacts the pancreas, often resulting in damage to numerous organs. This disorder encompasses a range of processes such as inflammation, oxidative stress, and pancreatitis. The hormone melatonin (MT) is primarily secreted by the pineal gland and plays a crucial role in mitigating inflammation, countering the harmful effects of free radicals, and regulating oxidative stress. The aim of this research was to investigate the potential protective impact and the underlying mechanism of melatonin in mice afflicted with SAP. The biochemical and histological assessments unequivocally demonstrated that melatonin effectively inhibited necrosis, infiltration, edema and cell death in pancreatic tissues, thereby suppressing acute pancreatitis. Notably, melatonin also alleviated the consequent harm to distant organs, notably the lungs, liver, and kidneys. Furthermore, both preventive and therapeutic administration of melatonin prompted nuclear factor E2-related factor 2 (Nrf2) activation followed by Nrf2 target gene expression. Nrf2 initiates the activation of antioxidant genes, thereby providing defense against oxidative stress. Conversely, Nrf2 reduction may contribute to impaired antioxidant protection in SAP. The beneficial impact of Nrf2 on antioxidants was absent in Nrf2-knockout mice, leading to the accumulation of LDH and exacerbation of cell death. This deterioration in both pancreatitis and injuries in distant organs intensified significantly. The results indicate that melatonin has an enhanced ability to protect against multiorgan damage caused by SAP, which is accomplished through the increase in Nrf2 expression. Additionally, Nrf2 initiates the activation of antioxidant genes that offer defense against cell death.

MLL1 histone methyltransferase and UTX histone demethylase functionally cooperate to regulate the expression of NRF2 in response to ROS-induced oxidative stress

The transcription factor NRF2 plays a pivotal role in maintaining redox and metabolic homeostasis by orchestrating oxidative stress-dependent transcription programs. Despite growing evidence implicating various cellular components in the regulation of NRF2 activity at the posttranslational stage, relatively less is known about the factors dictating the transcriptional activation of NRF2 in response to oxidative stress. In this study, we report the crucial roles of MLL1, an H3K4-specific methyltransferase, and UTX, an H3K27-specific histone demethylase, in the NRF2-dependent transcription program under oxidative stress. We find that the depletion of MLL1 or UTX results in increased susceptibility to oxidative stress, accompanied by higher intracellular ROS and the failed activation of antioxidant genes, including NRF2. In addition, MLL1 and UTX selectively target the NRF2 promoter, and exogenous FLAG-NRF2 expression increases the viability of MLL1-or UTX-depleted cells upon exposure to hydrogen peroxide. RNA-seq analysis demonstrates that depletion of MLL1 or UTX affects the changes in NRF2-dependent transcriptome in response to oxidative stress. Furthermore, ChIP and ChIP-seq analyses find that MLL1 and UTX functionally cooperate to establish a chromatin environment that favors active transcription at the H3K4me3/H3K27me3 bivalent NRF2 promoter in response to ROS-induced oxidative stress. Collectively, these findings provide a molecular mechanism underlying the cellular response to oxidative stress and highlight the importance of the chromatin structure and function in maintaining redox homeostasis.

A Transcriptional Analysis of Cattle Immune Cells Reveals a Central Role of Type 1 Interferon in the In Vitro Innate Immune Response against Mycobacterium bovis.

Bovine tuberculosis is a chronic infectious disease primarily caused by Mycobacterium bovis, a bacterium that affects cattle and other mammals, including humans. Despite the availability of vast research about the immune response mechanisms of human tuberculosis caused by Mycobacterium tuberculosis, the knowledge of bovine tuberculosis's immunology, particularly regarding the innate immune response, still remains scarce. In this study, we compared the transcriptome of cell cultures containing lymphocytes and M. bovis infected-macrophages with two strains of variable virulence, the virulent Mb04-303 strain and the attenuated Mb534. To that end, we infected bovine macrophages at a multiplicity of infection of one, and co-cultured the infections with autologous lymphocytes. RNA obtained from the co-cultures was sequenced to identify differentially expressed gene pathways by using the database Reactome. The RNA-seq analysis showed that the Mb04-303 infection upregulated the type 1 interferon signalling pathway, while it downregulated the KEAP1-NFE2L2 pathway. According to the literature, this last pathway is involved in the activation of antioxidant genes and inflammasome. In addition, the macrophages infected with Mb04-303 recruited more Galectin 8 than those infected with Mb534. This result indicates that Mb04-303 induced higher phagosome membrane damage, with the possible concomitant release of bacterial compounds into the cytoplasm that activates the type I signalling pathway. Altogether, Mb04-303 repressed the antioxidant and anti-inflammatory responses, likely impairing interleukin-1β activation, and trigged the canonical type 1 interferon signalling. Although these responses led to the control of bacterial replication during early infection, the virulent strain eventually managed to establish a successful infection.

Intracellular Iron Overload Induces Metabolic Switching in Active Hematopoietic Stem Cells in Beta-Thalassemia

Beta-thalassemia (BThal) is a complex monogenic disorder, causing severe anemia, ineffective erythropoiesis and multi-organ secondary defects. Iron overload (IO), associated with ineffective erythropoiesis and therapeutic blood transfusions, is one of the main complications of the disease, leading to morbidities in various organs. Despite the improvement in chelation therapies, IO toxicity is still a relevant issue. Correction of BThal is achieved by transplantation of hematopoietic stem cells (HSC) from healthy donors or autologous HSC from patients upon gene therapy. The quality and the engraftment of HSC depend on the bone marrow (BM) microenvironment, thus niche-HSC crosstalk plays a crucial role for transplantation outcome. We provided the first demonstration of impaired function of HSC caused by an altered BM niche in BThal (Aprile et al., Blood 2020). HSC from Hbbth3/+ (th3) BThal mice showed more active cycling profile and response to stress. We reported that IO reduces the hematopoietic supportive capacity of BM mesenchymal stromal cells from patients and recent evidence highlighted that both IO and iron deficiency are sources of cellular stress detrimental to HSC maintenance. Thus, we hypothesized that accumulation of iron might directly affect HSC function in BThal, as a model of chronic IO. Transcriptome analysis of sorted HSC from th3 mice showed a positive enrichment of genes involved in iron homeostasis, such as TfR1, Steap3, Fth1 and Ftl1, suggesting iron uptake and storage. Consistently, BThal HSC displayed higher levels of intracellular free reactive iron, as compared to wild-type (wt) controls (1.7±0.2 calcein MFI fold to wt, p<0.0001). We found that iron specifically accumulates in the mitochondria causing mitochondrial defects, as reduced size and lower membrane potential (0.4±0.05 TMRE/MTG fold to wt, p<0.001). These data, along with the downregulation of mitochondrial biogenesis and mitophagy genes, indicate an accumulation of damaged mitochondria in BThal HSC. In vivo administration of iron dextran to wt mice generated intracellular IO in HSC and was sufficient to decrease the mitochondrial membrane potential at levels comparable to those of th3 HSC, thus suggesting a direct effect of iron on mitochondrial activity. Since mitochondria are the powerhouse of the cell, we wondered if the mitochondrial dysfunction of BThal HSC could affect their production of metabolic energy. Metabolism indeed control HSC function: active HSC are more cycling and, because of their higher energy request, change the metabolism from glycolysis to mitochondrial oxidative phosphorylation (OXPHOS). Interestingly, we found that ATP levels in th3 HSC are lower compared to controls (wt 9.8±2.6 vs. th3 3.7±0.3 ATP nM, p<0.01). In line with mitochondrial dysfunction, in vitro inhibition of OXPHOS did not affect ATP content of BThal HSC since they switch to glycolysis for their energy demand. Consistently, th3 HSC showed positive enrichment of glycolytic genes and higher glucose uptake (wt 1639±163 vs. th3 2401±85 MFI 2NBDG, p<0.05). To unravel the basis of mitochondrial damage caused by IO, we investigated the role of reactive oxygen species (ROS). Iron is one of the sources of ROS and consistently, th3 HSC showed increased activation of antioxidant genes and higher levels of mitochondrial ROS (wt 2507±756 vs. th3 5205±459 MFI MitoSOX, p<0.01). In vivo reduction of mitochondrial ROS by MitoQ rescued mitochondrial activity (th3 0.3±0.07 vs. th3+MitoQ 0.9±0.2 TMRE/MTG fold to wt, p<0.05), thus demonstrating that mitochondrial dysfunction is reversible in BThal. Ongoing experiments will assess the metabolite profile, the restoration of HSC metabolism and in vivo function upon MitoQ and will also test HSC from BThal patients for mitochondrial defects. Overall, our study revealed that the multisystemic complications of IO in BThal also affect HSC. Iron has a direct impact on HSC metabolism by inducing oxidative stress and mitochondrial dysfunction. Despite the active cycling state, BThal HSC switch their metabolism to glycolysis because of the mitochondrial defect. Targeting mitochondrial iron or ROS might provide new tools to preserve HSC function in the setting of autologous transplantation upon gene therapy/editing for BThal. Moreover, this research will add novel insight about the regulation of HSC biology linking iron to mitochondrial metabolism.

Loss-of-function in GIGANTEA confers resistance to PPO-inhibiting herbicide tiafenacil through transcriptional activation of antioxidant genes in Arabidopsis

Herbicides play a crucial role in maintaining crop productivity by reducing competition between weeds and crops. Protoporphyrinogen oxidase (PPO)-inhibiting herbicides trigger the photooxidative damage that destroys cell membranes. Tiafenacil is a recently developed pyrimidinedione-type PPO-inhibiting herbicide that has low IC50 values in plants and is less toxic in humans compared to other PPO inhibitors. Previous reports confirmed that mutations in Arabidopsis circadian clock-controlled gene GIGANTEA (GI) were insensitive to phytooxidants, including chloroplast biogenesis inhibitors and herbicides. Here, we examined whether GI regulates the resistance to tiafenacil. Both gi mutant alleles, gi-1 and gi-2, were resistant to tiafenacil with survival rates of 97% and 83%, respectively, under 1 µM tiafenacil treatments, while 56% of wild-type and GI-overexpressing plants (GI-OX) survived. Both gi mutants were insensitive to tiafenacil-induced inhibition of photosystem efficiency and alleviated photooxidative damage. The gi mutants showed significant increases in transcriptional expressions and enzyme activities of antioxidants compared to wild-type and GI-OX. Moreover, loss-of-function in GI enhanced resistance to tiafenacil-containing commercial herbicide Terrad’or Plus®. Collectively, based on our results together with previous reports, mutations in GI confer resistance to herbicides with different MoAs and would be a crucial molecular target for non-target-site resistance strategies to develop herbicide-resistant crops.

Exercise mimicry: Characterization of nutraceutical agents that may contribute to mitochondrial homeostasis in skeletal muscle

While it is well established that endurance exercise improves mitochondrial health in skeletal muscle, optimizing nutraceutical agents to mimic these metabolic achievements in the cell remains a challenge. Nonetheless, the application of exercise mimetics is a fruitful direction to pursue, as they may target and activate the same mechanisms that are upregulated with exercise administration alone. This is particularly useful under conditions where contractile activity is compromised due to muscle disuse, disease, or aging. The agents Sulforaphane (SFN) and Urolithin A (UroA) represent our preliminary candidates for antioxidation and mitophagy, respectively, for maintaining mitochondrial turnover and homeostasis. SFN is a powerful inducer of the Nrf‐2‐ARE pathway, a mechanism that upregulates cellular defences against oxidative stress. On the other hand, the metabolite, UroA, has developed a reputable role in regulating mitochondrial turnover via mitophagy. The purpose of this ongoing study is to characterize these nutraceutical agents both in their time‐ and dose‐dependent capacities to induce changes in protein content relative to the antioxidant and mitophagy pathways, in C2C12 myotubes. Differentiated muscle cells were treated with two concentrations of each nutraceutical for 4 h, 24 h, or 48 h. Immunoblot analysis was conducted to measure changes in protein content. SFN treatment after 4 h rendered a marked increase in the master regulator for antioxidation, and transcription factor, Nrf‐2, with no apparent changes in its negative regulator Keap‐1 at any given time point. Nuclear‐cytoplasmic fractions confirmed Nrf‐2 translocation to the nucleus following 4 h of treatment, likely representing the potent activation of antioxidant genes. This aligns with the upregulation of the downstream antioxidant markers HO‐1 and NQO1, showing 4‐5‐fold increases as early as 4 h and 24 h, respectively, and maintained after 48 h. Despite modest effects with Urolithin A, some significant changes took place under basal conditions. The upstream kinase phospho‐AMPK was activated by 2‐fold as early as 4 hr of treatment with UroA, which subsided by 24 and 48 h. Previous reports observed changes with the agent on autophagy markers, which includes the modest 1.3‐fold increases in p62 and LC3‐II after 48 h of treatment. However, no observable changes took place with respect to the mitophagy markers phospho‐ULK1, Parkin, or PINK1. Nonetheless, our preliminary results suggest that these agents may be suitable candidates as exercise mimetics. These data also set the stage for an examination of the synergistic effect of these nutraceuticals, in combination with contractile activity, on mitochondrial turnover and function.

CAPE and its synthetic derivative VP961 restore BACH1/NRF2 axis in Down Syndrome

The cells possess several mechanisms to counteract the over-production of reactive oxygen species (ROS) and reactive nitrogen species (RNS), including enzymes such as superoxide dismutase, catalase and glutathione peroxidase. Moreover, an important sensor involved in the anti-oxidant response is KEAP1-NRF2-ARE signaling complex. Under oxidative stress (OS), the transcription factor NRF2 can dissociate from the KEAP1-complex in the cytosol and translocate into the nucleus to promote the transcriptional activation of anti-oxidant genes, such as heme oxygenase 1 and NADPH quinone oxidoreductase. Within this context, the activation of NRF2 response is further regulated by BACH1, a transcription repressor, that compete with the KEAP1-NRF2-ARE complex. In this work, we focused on the role of BACH1/NRF2 ratio in the regulation of the anti-oxidant response, proposing their antithetical relation as a valuable target for a therapeutic strategy to test drugs able to exert neuroprotective effects, notably in aging and neurodegenerative diseases. Among these, Down syndrome (DS) is a complex genetic disorder characterized by BACH1 gene triplication that likely results in the impairment of NRF2 causing increased OS. Our results revealed that BACH1 overexpression alters the BACH1/NRF2 ratio in the nucleus and disturbs the induction of antioxidant response genes ultimately resulting in the accumulation of oxidative damage both in Ts2Cje mice (a mouse model of DS) and human DS lymphoblastoid cell lines (LCLs).Based on this evidence, we tested Caffeic Acid Phenethyl Ester (CAPE) and the synthetic analogue VP961, which have been proven to modulate NRF2 activity. We showed that CAPE and VP961 administration to DS LCLs was able to promote NRF2 nuclear translocation, which resulted in the amelioration of antioxidant response. Overall, our study supports the hypothesis that BACH1 triplication in DS subjects is implicated in the alteration of redox homeostasis and therapeutic strategies to overcome this effect are under investigation in our laboratory.

Effect of Kaempferol and Its Glycoside Derivatives on Antioxidant Status of HL-60 Cells Treated with Etoposide.

Kaempferol is a well-known antioxidant found in many plants and plant-based foods. In plants, kaempferol is present mainly in the form of glycoside derivatives. In this work, we focused on determining the effect of kaempferol and its glycoside derivatives on the expression level of genes related to the reduction of oxidative stress—NFE2L2, NQO1, SOD1, SOD2, and HO-1; the enzymatic activity of superoxide dismutases; and the level of glutathione. We used HL-60 acute promyelocytic leukemia cells, which were incubated with the anticancer drug etoposide and kaempferol or one of its three glycoside derivatives isolated from the aerial parts of Lens culinaris Medik.—kaempferol 3-O-[(6-O-E-caffeoyl)-β-d-glucopyranosyl-(1→2)]-β-d-galactopyranoside-7-O-β-d-glucuropyranoside (P2), kaempferol 3-O-[(6-O-E-p-coumaroyl)-β-d-glucopyranosyl-(1→2)]-β-d-galactopyranoside-7-O-β-d-glucuropyranoside (P5), and kaempferol 3-O-[(6-O-E-feruloyl)-β-d-glucopyranosyl-(1→2)]-β-d-galactopyranoside-7-O-β-d-glucuropyranoside (P7). We showed that none of the tested compounds affected NFE2L2 gene expression. Co-incubation with etoposide (1 µM) and kaempferol (10 and 50 µg/mL) leads to an increase in the expression of the HO-1 (9.49 and 9.33-fold at 10 µg/mL and 50 µg/mL, respectively), SOD1 (1.68-fold at 10 µg/mL), SOD2 (1.72-fold at 10–50 µg/mL), and NQO1 (1.84-fold at 50 µg/mL) genes in comparison to cells treated only with etoposide. The effect of kaempferol derivatives on gene expression differs depending on the derivative. All tested polyphenols increased the SOD activity in cells co-incubated with etoposide. We observed that the co-incubation of HL-60 cells with etoposide and kaempferol or derivative P7 increases the level of total glutathione in these cells. Taken together, our observations suggest that the antioxidant activity of kaempferol is related to the activation of antioxidant genes and proteins. Moreover, we observed that glycoside derivatives can have a different effect on the antioxidant cellular systems than kaempferol.

SPOP mutations promote p62/SQSTM1-dependent autophagy and Nrf2 activation in prostate cancer.

p62/SQSTM1 is a selective autophagy receptor that drives ubiquitinated cargos towards autophagic degradation. This receptor is also a stress-induced scaffold protein that helps cells to cope with oxidative stress through activation of the Nrf2 pathway. Functional disorders of p62 are closely associated with multiple neurodegenerative diseases and cancers. The gene encoding the E3 ubiquitin ligase substrate-binding adapter SPOP is frequently mutated in prostate cancer (PCa), but the molecular mechanisms underlying how SPOP mutations contribute to PCa tumorigenesis remain poorly understood. Here, we report that cytoplasmic SPOP binds and induces the non-degradative ubiquitination of p62 at residue K420 within the UBA domain. This protein modification decreases p62 puncta formation, liquid phase condensation, dimerization, and ubiquitin-binding capacity, thereby suppressing p62-dependent autophagy. Moreover, we show that SPOP relieves p62-mediated Keap1 sequestration, which ultimately decreases Nrf2-mediated transcriptional activation of antioxidant genes. We further show that PCa-associated SPOP mutants lose the capacity to ubiquitinate p62 and instead promote autophagy and the redox response in a dominant-negative manner. Thus, our findings indicate oncogenic roles of autophagy and Nrf2 activation in the tumorigenesis of SPOP-mutated PCa.

IN SILICO DOCKING STUDIES OF PHYTOSTEROL COMPOUNDS SELECTED FROM Ficus religiosa AS POTENTIAL CHEMOPREVENTIVE AGENT

Traditional medicine has employed Ficus reliosa (Moraceae) to treat a wide range of diseases such as central nervous system disorder, infectious disease, endocrine system, respiratory system, etc. This study aimed to evaluate phytosterol constituents that may serve as lead-drug such as 28-Isofucosterol, Stigmasterol, Sitosterol, and Campesterol towards their pharmacokinetic properties and chemopreventive activity to regulate nuclear receptor factor 2 (Nrf2) uptake bound by Kelch-like Activating Protein (Keap1). Structures were studied to evaluate their respective bioactivity using Milliprot followed by a docking study with Autodock Tools for macromolecule obtained from Protein Data Bank in 2FLU PDB file name. Chemopreventive activity from Sitosterol, Stigmasterol, Campesterol and 28-Isofucosterol in each respective bioactivity towards nuclear receptor target : 0.72; 0.74; 0.71; 0.91. A computational study performed by Autodock Tools against Keap1 targeted macromolecule covalent binding toward threonine 609 to obtain inhibition results. As respective Inhibition constant obtained (pM) : 522.97; 286.75; 620.16; 369.55. Free energy obtained (kcal/mol) : -8.57; -13.02; -12.56; -12.87. Hence the results revealed from the study showed that phytosterol compound from Ficus religiosa proven to be useful as a chemopreventive agent to promote antioxidant gene activity through regulation of Nrf2 factor by competing for receptor site with Keap1 during stressed conditions.

Direct effects of phenolic compounds on the mammary gland: In vivo and ex vivo evidence

We assessed the potential of Pistacia lentiscus (lentisk) phenolic compounds to enhance production of milk composition in lactating goats and caprine primary mammary epithelial cells (MEC). Damascus goats were given a lentisk infusion (LI) or fresh water (FW) to drink, in a crossover design. Milk from LI vs. FW goats was 0.43% richer in fat and 30% in omega 3 fatty acids. Lentisk infusion enhanced antioxidant capacity of plasma and milk by 37% and 30% respectively, and induced transcriptional activation of antioxidant genes. To assess the direct effect of polyphenols on milk quality in terms of composition and antioxidant capacity, we used plasma collected from goats fed hay (HP) or browsed on phenolic compounds-rich pasture (primarily lentisk; PP) as a conditioning medium for primary culture of MEC. PP increased 2-fold cellular triglyceride content and 2.4-fold intracellular casein, and increased ATP production and non-mitochondrial oxygen consumption. Taken together, the results imply that lentisk phenolic compounds affect blood, MEC and milk oxidative status, which increase fat production by the mammary gland.

Moringa isothiocyanate-1 regulates Nrf2 and NF-κB pathway in response to LPS-driven sepsis and inflammation.

This study aims to document the dual mode of pharmacological action of moringa isothiocyanate-1 (MIC-1) derived from seeds of Moringa oleifera Lam. Oral administration of chemically stable MIC-1 (80 mg/kg) significantly reduced the expression of inflammatory markers (Tnf-α, Ifn-α, IL-1β, IL-6) in the liver, kidney, spleen, and colon and decreased spleen weight in the lipopolysaccharide (LPS)-induced sepsis / acute inflammation model in mice. Transcriptomic analysis of the effect of MIC-1 on the liver and in the LPS-induced RAW264.7 murine macrophage showed that MIC-1 decreases inflammation via inflammation, immunity, and oxidative stress pathways. These results are supported by the immunocytochemical observations that MIC-1 increased the nuclear accumulation of nuclear factor (erythroid-derived 2)-like 2 (Nrf2) transcription factor and decreased the nuclear accumulation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) in the LPS-induced macrophages. Transcriptional activation of antioxidant genes by MIC-1 translated into a reduction of reactive oxygen species (ROS) in the cytoplasm, decrease of mitochondrial superoxide content, and restoration of the mitochondrial membrane potential in the LPS-induced macrophages. Our data indicate that MIC-1 affects inflammation and oxidative stress, two key processes involved in the etiology of many chronic diseases. These effects involve upstream regulation of two key transcriptional factors regulating responses to these processes at a gene expression level.

Genetic Silencing of KEAP1 Induces NRF2 Mediated Oxidative Stress Pathway in Human Erythroid Cells

Sickle cell disease (SCD) is a monogenic hereditary disorder resulting from a mutation in the β-globin gene which renders the red blood cells (RBCs) prone to sickling and hemolysis. SCD is characterized by several severe pathophysiologies including anemia, chest pain, stroke and vaso-occlusive crisis (VOC). Hemolysis has been attributed as one of the major contributing factors to these pathologies and has been linked to oxidative stress in RBCs that carry high levels of pro-oxidant heme. Additionally, sickle RBCs have an increased rate of auto-oxidation cycle that results in production of superoxide. Other factors including deformation and subsequent physical stress on the membrane during transit through narrow capillaries aggravate lipid peroxidation mediated damage. Therapies that can improve the antioxidant status and overall RBC health may offer a protective effect in this disease. NRF2 is a transcription factor that orchestrates activation of antioxidant genes in response to oxidative stress. We previously showed that genetic activation of NRF2 by KEAP1 knockout in HUDEP-2 and primary CD34+ cells induces fetal hemoglobin (HbF) levels and elicits a protective effect against oxidative stress in-vitro (Gupta et al. 2019). In this study, we employed the KEAP1 knockout HUDEP-2 and primary CD34+ cells to induce NRF2 pathway and subjected them to a systemic multi-omics analysis to identify key genes that are implicated in oxidative stress defense mechanisms. KEAP1 KO HUDEP-2 cells were subjected to RNA sequencing and proteomics profiling and the results were analyzed using gene set enrichment analysis (GSEA) and ingenuity pathway analysis (IPA). IPA analysis in RNA seq results showed association with NRF2 mediated oxidative stress and heme Biosynthesis II pathways. Global proteomic profiling was carried in HUDEP KEAP1 KO populations using TMT-label based peptide quantitation. Top hallmark gene sets by GSEA in proteomics analysis included reactive oxygen species pathway, xenobiotic metabolism and oxidative phosphorylation. Some of the top genes included NQO1, GSR, TXNRD1 and GCLM. In order to confirm the findings in primary CD34+ cells, a CRISPR Cas9 RNP mediated approach was used to knockout KEAP1. Two guide RNA targeting KEAP1, in two different healthy donors were employed for RNA seq and proteomics analysis. KEAP1 KO CD34+ cells were subjected to erythroid differentiation and samples were harvested for RNA seq on day 5 and proteomics on day 7 of differentiation. IPA analysis of RNA seq results showed positive enrichment of NRF2 mediated oxidative stress responses, aryl hydrocarbon receptor signaling and xenobiotic general signaling pathway. Some of the top genes regulated in CD34+ cells included NQO1, OSGIN1, GCLM, GSR, HMOX-1 and TXNRD1. Global proteomic profiling followed by IPA analysis showed association with pathways including NRF2 mediated oxidative stress pathway and xenobiotic metabolism. Furthermore, genes including NQO1, GCLM, GSR, HMOX-1 and TXNRD1 were confirmed to be upregulated by proteomics. Both, RNA seq and proteomics data confirmed the upregulation of HBG1/2 and downregulation of KEAP1, and moreover, IPA analysis of both the data sets confirmed NRF2 as the upstream regulator. In order to evaluate the functional effects of multi-omics data, KEAP1 KO CD34+ cells were subjected to hydrogen peroxide treatment and reactive oxygen species (ROS) levels were measured. Relative to control populations, KEAP1 KO cells showed attenuated response in ROS generation indicative of a protective effect against oxidative stress. Overall, these results provide a mechanistic insight towards the improvement of antioxidant status of erythroid populations with KEAP1 silencing that can be helpful in bolstering the red blood cell antioxidant responses. Improvement in antioxidant status can reduce the propensity to hemolyze and further, provides strong evidence towards NRF2 as a therapeutic target in SCD. Disclosures Gupta: Sanofi: Current Employment. Lessard:Sanofi: Current Employment. Dai:Sanofi: Current Employment. Hicks:Sanofi: Current Employment. Krishnamoorthy:Sanofi: Current Employment.

The Role of BRG1 in Antioxidant and Redox Signaling.

Redox homeostasis is regulated by critical molecules that modulate antioxidant and redox signaling (ARS) within the cell. Imbalances among these molecules can lead to oxidative stress and damage to cell functions, causing a variety of diseases. Brahma-related gene 1 (BRG1), also known as SMARCA4, is the central ATPase catalytic subunit of the switch/sucrose nonfermentable (SWI/SNF) chromatin remodeling complex, which plays a core role in DNA replication, repair, recombination, and transcriptional regulation. Numerous recent studies show that BRG1 is involved in the regulation of various cellular processes associated with ARS. BRG1, as a major factor in chromatin remodeling, is essential for the repair of oxidative stress-induced DNA damage and the activation of antioxidant genes under oxidative stress. Consequently, a comprehensive understanding of the roles of BRG1 in redox homeostasis is crucial to understand the normal functioning as well as pathological mechanisms. In this review, we summarized and discussed the role of BRG1 in the regulation of ARS.

Effect of Phytochrome Deficit on Activity of Ascorbate Peroxidase and Phenylalanine Ammonia-Lyase and Expression of Genes APX1, tAPX, sAPX, and PAL in the Leaves of Arabidopsis thaliana Plants Exposed to UV-A and Red Light

Effect of UV-A radiation (10 W/m2, 2 h) and modifying effect of brief exposure to red light (RL) (1.8 W/m2, 20 min) on photochemical activity of photosystem II (PSII) (Fv/Fm), as well as on activity of ascorbate peroxidase (AsP) and phenylalanine ammonia-lyase (PAL), were investigated in the leaves of 26–28-day-old Аrabidopsis thaliana (L.) Heynh. plants of wild type (WT) and mutants: hy3 deficient in phytochrome B (phyB) and hy2 defective in phytochromobilin synthesis. UV-A reduced photochemical activity of PSII and activities of AsP and PAL. Exposure to RL increased PAL activity in only WT plants and in mutant hy3. Elevation of expression of genes APX1, sAPX, tAPX, and PAL encoding enzymes AsP and PAL was detected upon the exposure to RL and UV-A. For instance, expression of gene APX1 in WT plants and in mutant hy3 rose upon exposure to RL and UV-A; in mutant hy2, it increased only upon UV-A treatment. Expression of gene tAPX in WT plants rose only under the effect of RL, whereas expression of gene sAPX did not rise in any type of treatment. In mutant hy2, expression of the above-named genes decreased upon the effect of RL. Upon joint action of RL and UV-A (RL followed by UV), the negative effect of UV-A on the activity of the total pool of AsP was nullified by RL in only WT plants and in mutant hy3. Partial removal of the negative effect of UV-A on activity of PSII as a result of pretreatment of plants with RL is probably related to a rise in the activity of antioxidant enzymes. This is evident in WT plants whose PSII is more sensitive to RL radiation than in mutants deficient in phytochromes. We believe that PSII resistance to UV-A radiation depends on the potential of the plant antioxidant system, which rises when the share of the active form of phytochrome in its total pool increases as a result of plants’ pretreatment with RL and is partially realized by way of induction of transcriptional activity of antioxidant genes.

The genotoxic effects of mixture of aluminum, arsenic, cadmium, cobalt, and chromium on the gill tissue of adult zebrafish (Danio rerio, Hamilton 1822)

The aim of this study is to investigate the genotoxic effects of mixtures of five metals on zebrafish at two different concentrations; at the permissible maximum contamination levels in drinking water and irrigation waters. The drinking water limits are as follows: 300 µg/L for Aluminum (Al+3), 10 µg/L for Arsenic (As+3), 5 µg/L for Cadmium (Cd+2), 10 µg/L for Cobalt (Co+2), and 50 µg/L for Chromium (Cr+2). The irrigation water limits: 5000 µg/L for Al+3, 100 µg/L for As+3, 10 µg/L for Cd+2, 50 µg/L for Co+2, and 100 µg/L for Cr+2. The zebrafish underwent chronic exposure for periods of 5, 10, and 20 days. The gene expressions for mitochondrial superoxide dismutase (SOD2), stress-specific receptor protein NCCRP1, the heat shock proteins: Hsp9, Hsp14, Hsp60, Hsp70, DNA repair (XRCC1 and EXO1), and apoptosis (BOK and BAX) were evaluated. It was found that exposure to the low- and high-concentrations of the heavy metal mixtures caused cell stress, an increased expression of the antioxidant genes, and repair proteins. As the duration of exposure was increased, the cells progressed through the apoptotic pathway. This was more evident in the high-concentration exposure groups. The results demonstrated the necessity for a reevaluation of the maximum values of heavy metal and toxic element concentrations as prescribed by the Local Standing Rules of Water Pollution Control Regulation, as well as a reevaluation of the limitations of heavy metal mixture interactions with respect to ecological balance and environmental health. Highlights The purpose of this study was to investigate the genotoxic effects of a mixture of Aluminum, Arsenic, Cadmium, Cobalt, Chromium on zebrafish, within drinking water, and irrigation water limits determining the concentration. The zebrafish were exposed to two different concentrations of each metal mixture for 5-, 10-, and 20-day periods. Following exposure, gene expressions of the zebrafish’s gill tissues were examined. As a result of the exposure to the metal mixtures, the following occurred: cell stress, increased antioxidant gene activity, and attempts to protect cell viability. However, the cells progressed through the apoptotic pathway after prolonged exposure. The results demonstrated the necessity for a reevaluation of the maximum limits of metal and toxic element concentrations as stated in the Standing Rules of Water Pollution Control Regulation.

Traumatic brain injury-induced downregulation of Nrf2 activates inflammatory response and apoptotic cell death.

Recent studies from our group and others have demonstrated that oxidative stress, Ca2+ signaling, and neuroinflammation are major mechanisms contributing to post-traumatic neurodegeneration. The present study investigated the mechanisms of regulation of nuclear factor E2-related factor 2 (Nrf2) and its role in regulating antioxidant genes and oxidative stress-induced neuroinflammation and neurodegeneration following TBI. Nrf2 transcriptional system is the major regulator of endogenous defense mechanisms operating within the cells. Wild-type (Nrf2+/+) and Nrf2-deficient mice (Nrf2-/-) were subjected to 15 psi fluid percussion injury and demonstrated the regulatory role of Nrf2 in the expression antioxidant genes and oxidative stress, neuroinflammation, and cell death. Immunohistochemistry, q-RT-PCR, and western blotting techniques detected downregulation of Nrf2 and antioxidant proteins such as HO-1, GPx1, GSTm1, and NQO1 in mouse brain samples. Further, our study demonstrated that the downregulation of Nrf2 and antioxidant genes in TBI correlated with the induction of free radical-generating enzyme NADPH oxidase 1 and inducible nitric oxide synthase and their corresponding oxidative/nitrosative stress markers 4-hydroxynonenal and 3-nitrotyrosine. The decrease in Nrf2 with subsequent increase in oxidative stress markers led to the activation of MMP3/9, TGF-β1, and NF-kB that further led to neuroinflammation and apoptosis. The absence of Nrf2 function in mice resulted in exacerbated brain injury as shown by the increased oxidative stress markers, pro-inflammatory cytokines, and apoptosis markers at 24 h after TBI. In conclusion, this study could establish the significance of Nrf2 in transforming into a novel preventive approach against the pathophysiology of TBI. KEY MESSAGES: • Traumatic brain injury impairs Nrf2 signaling in mouse. • Nrf2-mediated activation of antioxidant genes are altered after TBI. • Impairment of Nrf2 signaling leads to oxidative stress. • TBI-induced downregulation of Nrf2 activates MMPs, TGF-β1, and NF-kB. • Nrf2 regulates neuroinflammation and apoptotic cell death in TB.

Bioenergetics and redox adaptations of astrocytes to neuronal activity.

Neuronal activity is a high‐energy demanding process recruiting all neural cells that adapt their metabolism to sustain the energy and redox balance of neurons. During neurotransmission, synaptic cleft glutamate activates its receptors in neurons and in astrocytes, before being taken up by astrocytes through energy costly transporters. In astrocytes, the energy requirement for glutamate influx is likely to be met by glycolysis. To enable this, astrocytes are constitutively glycolytic, robustly expressing 6‐phosphofructo‐2‐kinase/fructose‐2,6‐bisphosphatase‐3 (PFKFB3), an enzyme that is negligibly present in neurons by continuous degradation because of the ubiquitin‐proteasome pathway via anaphase‐promoting complex/cyclosome (APC)‐Cdh1. Additional factors contributing to the glycolytic frame of astrocytes may include 5′‐AMP‐activated protein kinase (AMPK), hypoxia‐inducible factor‐1 (HIF‐1), pyruvate kinase muscle isoform‐2 (PKM2), pyruvate dehydrogenase kinase‐4 (PDK4), lactate dehydrogenase‐B, or monocarboxylate transporter‐4 (MCT4). Neurotransmission‐associated messengers, such as nitric oxide or ammonium, stimulate lactate release from astrocytes. Astrocyte‐derived glycolytic lactate thus sustains the energy needs of neurons, which in contrast to astrocytes mainly rely on oxidative phosphorylation. Neuronal activity unavoidably triggers reactive oxygen species, but the antioxidant defense of neurons is weak; hence, they use glucose for oxidation through the pentose‐phosphate pathway to preserve the redox status. Furthermore, neural activity is coupled with erythroid‐derived erythroid‐derived 2‐like 2 (Nrf2) mediated transcriptional activation of antioxidant genes in astrocytes, which boost the de novo glutathione biosynthesis in neighbor neurons. Thus, the bioenergetics and redox programs of astrocytes are adapted to sustain neuronal activity and survival. Developing therapeutic strategies to interfere with these pathways may be useful to combat neurological diseases. Our current knowledge on brain's management of bioenergetics and redox requirements associated with neural activity is herein revisited. The astrocyte‐neuronal lactate shuttle (ANLS) explains the energy needs of neurotransmission. Furthermore, neurotransmission unavoidably triggers increased mitochondrial reactive oxygen species in neurons. By coupling glutamatergic activity with transcriptional activation of antioxidant genes, astrocytes provide neurons with neuroprotective glutathione through an astrocyte‐neuronal glutathione shuttle (ANGS). This article is part of the 60th Anniversary special issue.

Restoration of Nrf2 Signaling Normalizes the Regenerative Niche.

Chronic hyperglycemia impairs intracellular redox homeostasis and contributes to impaired diabetic tissue regeneration. The Keap1/Nrf2 pathway is a critical regulator of the endogenous antioxidant response system, and its dysfunction has been implicated in numerous pathologies. Here we characterize the effect of chronic hyperglycemia on Nrf2 signaling within a diabetic cutaneous regeneration model. We characterized the effects of chronic hyperglycemia on the Keap1/Nrf2 pathway within models of diabetic cutaneous wound regeneration. We assessed reactive oxygen species (ROS) production and antioxidant gene expression following alterations in the Nrf2 suppressor Keap1 and the subsequent changes in Nrf2 signaling. We also developed a topical small interfering RNA (siRNA)–based therapy to restore redox homeostasis within diabetic wounds. Western blotting demonstrated that chronic hyperglycemia–associated oxidative stress inhibits nuclear translocation of Nrf2 and impairs activation of antioxidant genes, thus contributing to ROS accumulation. Keap1 inhibition increased Nrf2 nuclear translocation, increased antioxidant gene expression, and reduced ROS production to normoglycemic levels, both in vitro and in vivo. Topical siKeap1 therapy resulted in improved regenerative capacity of diabetic wounds and accelerated closure. We report that chronic hyperglycemia weakens the endogenous antioxidant response, and the consequences of this defect are manifested by intracellular redox dysregulation, which can be restored by Keap1 inhibition. Targeted siRNA-based therapy represents a novel, efficacious strategy to reestablish redox homeostasis and accelerate diabetic cutaneous tissue regeneration.

Sildenafil activates antioxidant and antiapoptotic genes and inhibits proinflammatory cytokine genes in a rat model of renal ischemia/reperfusion injury.

To study the possible renoprotective effect of sildenafil against renal ischemia/reperfusion (I/R) injury and its effect on the expression of some antioxidant, antiapoptotic gene and proinflammatory cytokine genes in rat model of renal I/R injury. One hundred and twenty male Sprague Dawley rats were subdivided into three equal groups: sham (underwent right nephrectomy without ischemia), control (underwent right nephrectomy and left ischemia for 45 min) and study [as control with 1 mg/kg sildenafil (per oral) 60 min before anesthesia]. Serum creatinine and BUN were measured at the baseline and the study endpoints (2, 24, 48 h and 7 days), and the left kidney was harvested at study endpoints for histopathological examination as well as for assessment of the expression of antioxidant genes (Nrf-2, HO-1 and NQO-1), antiapoptotic gene (Bcl-2) and inflammatory cytokines, e.g., TNF-a, IL-1β and ICAM-1. I/R caused significant increase in serum creatinine, BUN, histopathological damage score (p < 0.001) and significant reduction in antioxidant genes (nrf2, HO-1 and NQO-1) and antiapoptotic gene (Bcl2) with significant increase in TNF-a, IL-1β and ICAM-1 genes in kidney tissues. Pretreatment with sildenafil caused significant attenuation of serum creatinine and BUN as well as significant increase in the expression of antioxidant genes and Bcl-2 genes with significant reduction in the expression of proinflammatory cytokine genes (p value < 0.001). The renoprotective effect of sildenafil against renal I/R might be due to the activation of antioxidant genes (Nrf2, HO-1 and NQO-1) and antiapoptotic gene (Bcl2) and attenuation of proinflammatory cytokines (TNF-a, IL-1β and ICAM-1).