Expression Of Nrf2-dependent Genes Research Articles

Yinhuang granule alleviates carbon tetrachloride-induced liver fibrosis in mice and its mechanism.

Liver fibrosis is a formidable global medical challenge, with no effective clinical treatment currently available. Yinhuang granule (YHG) is a proprietary Chinese medicine comprising Scutellariae Radix and Lonicerae Japonicae Flos. It is frequently used for upper respiratory tract infections, pharyngitis, as well as acute and chronic tonsillitis. To investigate the potential of YHG in alleviating carbon tetrachloride (CCl4)-induced liver fibrosis in mice. To induce a hepatic fibrosis model in mice, this study involved intraperitoneal injections of 2 mL/kg of CCl4 twice a week for 4 wk. Meanwhile, liver fibrosis mice in the low dose of YHG (0.4 g/kg) and high dose of YHG (0.8 g/kg) groups were orally administered YHG once a day for 4 wk. Serum alanine/aspartate aminotransferase (ALT/AST) activity and liver hydroxyproline content were detected. Sirius red and Masson's trichrome staining assay were conducted. Real-time polymerase chain reaction, western-blot and enzyme-linked immunosorbent assay were conducted. Liver glutathione content, superoxide dismutase activity level, reactive oxygen species and protein carbonylation amount were detected. The administration of YHG ameliorated hepatocellular injury in CCl4-treated mice, as reflected by decreased serum ALT/AST activity and improved liver histological evaluation. YHG also attenuated liver fibrosis, evident through reduced liver hydroxyproline content, improvements in Sirius red and Masson's trichrome staining, and lowered serum hyaluronic acid levels. Furthermore, YHG hindered the activation of hepatic stellate cells (HSCs) and ameliorated oxidative stress injury and inflammation in liver from CCl4-treated mice. YHG prompted the nuclear accumulation of nuclear factor erythroid 2-related factor 2 (Nrf2) and upregulated the expression of Nrf2-dependent downstream antioxidant genes. In addition, YHG promoted mitochondrial biogenesis in liver from CCl4-treated mice, as demonstrated by increased liver adenosine triphosphate content, mitochondrial DNA levels, and the expression of peroxisome proliferator-activated receptor gamma coactivator 1 alpha and nuclear respiratory factor 1. YHG effectively attenuates CCl4-induced liver fibrosis in mice by inhibiting the activation of HSCs, reducing inflammation, alleviating liver oxidative stress damage through Nrf2 activation, and promoting liver mitochondrial biogenesis.

Thymidylate synthase promotes esophageal squamous cell carcinoma growth by relieving oxidative stress through activating nuclear factor erythroid 2-related factor 2 expression.

Thymidylate synthase (TYMS) is involved in the malignant process of multiple cancers, and has gained much attention as a cancer treatment target. However, the mechanism in carcinogenesis of esophageal squamous cell cancer (ESCC) is little reported. The present study was to clear the biological roles and carcinogenic mechanism of TYMS in ESCC, and explored the possibility to use TYMS as a tumor marker in diagnosis and a drug target for the treatment of ESCC. Stably TYMS-overexpression cells established by lentivirus transduction were used for the analysis of cell proliferation. RNA sequencing was performed to explore the possible carcinogenic mechanisms. GEPIA databases analysis showed that TYMS expression in esophageal cancer tissues was higher than that in normal tissues. The MTT assay, colony formation assay, and nude mouse subcutaneous tumor model found that the overexpression of TYMS increased cell proliferation. Transcriptome sequencing analysis revealed that the promoted cell proliferation in TYMS-overexpression ESCC cells were mediated through activating genes expression of nuclear factor erythroid 2-related factor 2 (Nrf2) and Nrf2 dependent antioxidant enzymes to relieve oxidative stress, which was confirmed by increased glutathione (GSH), glutathione peroxidase (GPX) activities, and reduced reactive oxygen species. Nrf2 active inhibitors (ML385) used in TYMS-overexpression cells inhibited the expression of Nrf2-dependent antioxidant enzyme genes, thereby increasing oxidative stress and blocking cell proliferation. Our study indicated a novel and effective regulatory capacity of TYMS in the cell proliferation of ESCC by relieving oxidative stress through activating expression of Nrf2 and Nrf2-dependent antioxidant enzymes genes. These properties make TYMS and Nrf2 as appealing targets for ESCC clinical chemotherapy.

NRF2 Activation by Nitrogen Heterocycles: A Review.

Several nitrogen heterocyclic analogues have been applied to clinical practice, and about 75% of drugs approved by the FDA contain at least a heterocyclic moiety. Thus, nitrogen heterocycles are beneficial scaffolds that occupy a central position in the development of new drugs. The fact that certain nitrogen heterocyclic compounds significantly activate the NRF2/ARE signaling pathway and upregulate the expression of NRF2-dependent genes, especially HO-1 and NQO1, underscores the need to study the roles and pharmacological effects of N-based heterocyclic moieties in NRF2 activation. Furthermore, nitrogen heterocycles exhibit significant antioxidant and anti-inflammatory activities. NRF2-activating molecules have been of tremendous research interest in recent times due to their therapeutic roles in neuroinflammation and oxidative stress-mediated diseases. A comprehensive review of the NRF2-inducing activities of N-based heterocycles and their derivatives will broaden their therapeutic prospects in a wide range of diseases. Thus, the present review, as the first of its kind, provides an overview of the roles and effects of nitrogen heterocyclic moieties in the activation of the NRF2 signaling pathway underpinning their antioxidant and anti-inflammatory actions in several diseases, their pharmacological properties and structural-activity relationship are also discussed with the aim of making new discoveries that will stimulate innovative research in this area.

Indian traditional rice variety "Gathuwan" suppresses T-cell-mediated immune responses via activation of ERK/Nrf2/HO-1 signalling pathway.

The impact of food on immune functions has been recognized for centuries and is now being increasingly explored for therapeutic applications. Rice, in addition to being the staple food in most developing countries, exhibits diverse complexities of phytochemicals among its wide germplasm repertoire, which supports its development as a functional food. In the present study, we have explored the immunomodulatory properties of Gathuwan rice, a local rice variety grown in Chhattisgarh, India, and traditionally used for the treatment of rheumatism. Methanolic Gathuwan Brown Rice Extract (BRE) inhibits T-cell activation and proliferation and cytokine secretion (IL-2, IL-4, IL-6 and IFN-γ) without inducing cell death. BRE exhibits radical scavenging activity in a cell-free system and decreases intracellular reactive oxygen species (ROS) and glutathione levels in lymphocytes. BRE induces nuclear translocation of the immune-regulatory transcription factor Nrf2 via activation of ERK and p-38 MAP kinase and up-regulates the expression of Nrf2-dependent genes (SOD, CAT, HO-1, GPx and TrxR) in lymphocytes. BRE treatment had no effect on cytokine secretion by lymphocytes from Nrf2 knockout mice, confirming the role of Nrf2 in the immunosuppressive effects of BRE. Feeding of Gathuwan brown rice to mice had no effect on the basal haematological parameters, but lymphocytes isolated from these mice were hypo-responsive to mitogenic stimuli. Treatment of allografts with BRE significantly prevented graft-versus-host disease (GVHD)-associated mortality and morbidity in mice. Metabolic pathway enrichment analysis of ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) data revealed a high enrichment ratio of amino acid and vitamin B metabolism pathways, and among metabolite sets, pyridoxamines, phytosphingosines, hydroxybenzaldehydes, hydroxycinnamic acids and indoles were highly enriched bioactive components. In conclusion, Gathuwan BRE suppresses T-cell-mediated immune responses by altering the cellular redox balance and activating the Nrf2 signalling pathway.

Activation of NRF2 blocks HIV replication and apoptosis in macrophages

Abnormal oxidative stress caused by human immunodeficiency virus (HIV) infection affects viral replication and causes non-acquired immune deficiency syndrome-related complications in infected individuals. The transcription factor NFE2-related factor 2 (NRF2), a key regulator of oxidative stress, responds to abnormal oxidative stress by regulating the expression of NRF2-dependent cytoprotective genes. The present study aimed to determine whether inhibition of oxidative stress could control HIV replication and improve cell survival. In this study, the NRF2 activator, methyl bardoxolone, was used to treat cells for HIV infection. The effects on HIV replication and apoptosis pathways were confirmed by NRF2 activation or knockdown. The results showed that NRF2 activation could block HIV replication in macrophages before the integration phase and inhibited the expression of apoptotic pathways in virus-exposed macrophages. The study presents an unconventional anti-viral strategy of activation antioxidant response for HIV infection blocking.

Neonatal Selenium Deficiency Decreases Selenoproteins in the Lung and Impairs Pulmonary Alveolar Development.

Decreased selenium (Se) levels during childhood and infancy are associated with worse respiratory health. Se is biologically active after incorporation into Se-containing antioxidant enzymes (AOE) and proteins. It is unknown how decreased maternal Se during pregnancy and lactation impacts neonatal pulmonary selenoproteins, growth, and lung development. Using a model of neonatal Se deficiency that limits Se intake to the dam during pregnancy and lactation, we evaluated which neonatal pulmonary selenoproteins are decreased in both the saccular (postnatal day 0, P0) and early alveolar (postnatal day 7, P7) stages of lung development. We found that Se deficient (SeD) pups weigh less and exhibit impaired alveolar development compared to Se sufficient (SeS) pups at P7. The activity levels of glutathione peroxidase (GPx) and thioredoxin reductase (Txnrd) were decreased at P0 and P7 in SeD lungs compared to SeS lungs. Protein content of GPx1, GPx3 and Txnrd1 were decreased in SeD lungs at P0 and P7, whereas Txnrd2 content was unaltered compared to SeS controls. The expression of NRF-2 dependent genes and several non-Se containing AOE were similar between SeS and SeD lungs. SeD lungs exhibited a decrease in selenoprotein N, an endoplasmic reticulum protein implicated in alveolar development, at both time points. We conclude that exposure to Se deficiency during pregnancy and lactation impairs weight gain and lung growth in offspring. Our data identify multiple selenoproteins in the neonatal lung that are vulnerable to decreased Se intake, which may impact oxidative stress and cell signaling under physiologic conditions as well as after oxidative stressors.

Pharmacological characterization of a novel peptide inhibitor of the Keap1-Nrf2 protein-protein interaction

LAS200813 is a novel bicyclic lipopeptide that activates Nrf2 by binding to Keap1, thereby antagonising the Keap1-Nrf2 protein–protein interaction. In this work we report the pharmacological characterization of LAS200813 in Nrf2-dependent translational preclinical models. LAS200813 binds to Keap1 with high affinity (IC50: 0.73 nM) and is able to induce the translocation of Nrf2 to the nucleus. Furthermore, LAS200813 increases the expression of Nrf2 target genes in human bronchial epithelial cells (EC50 of 96 and 70 nM for srxn1 and nqo1, respectively). Similarly, the intratracheal administration of LAS200813 to rats increases the expression of Nrf2-dependent genes in lung tissue, an effect that lasts for a few hours. Moreover, in cells exposed to cigarette smoke, LAS200813 shows an antioxidant effect by increasing the production of glutathione and prevents cellular apoptosis. In conclusion, the results described herein demonstrate that LAS200813 is a potent non-electrophilic Nrf2-activating peptide designed to be administered by inhaled route which may be a potential therapeutic strategy for respiratory diseases driven by oxidative stress.

Compensatory expression of NRF2-dependent antioxidant genes is required to overcome the lethal effects of Kv11.1 activation in breast cancer cells and Patient-derived tumors

Compensatory expression of NRF2-dependent antioxidant genes is required to overcome the lethal effects of Kv11.1 activation in breast cancer cells and Patient-derived tumors

Four-Octyl Itaconate Protects Chondrocytes against H2O2-Induced Oxidative Injury and Attenuates Osteoarthritis Progression by Activating Nrf2 Signaling.

Nrf2 is a critical regulator of the antioxidant defense systems in cellular protection. Emerging evidence has shown that four-octyl itaconate (OI) activates Nrf2 cascade. In this study, the chondroprotective effects of OI on H2O2-stimulated chondrocytes and DMM-induced osteoarthritis (OA) progression were investigated. In primary murine chondrocytes, OI interrupted the binding of Keap1 and Nrf2, leading to accumulation and nuclear translocation of Nrf2 protein, as well as transcription and expression of Nrf2-dependent genes, such as HO-1, NQO1, and GCLC. Furthermore, OI inhibited cell death and apoptosis, as well as H2O2-stimulated ROS generation, lipid peroxidation, superoxide accumulation, and mitochondrial depolarization in chondrocytes, which were abolished by the silence or depletion of Nrf2. In addition, in vivo experiments revealed the therapeutic effects of OI on OA progression in a DMM mouse model. Collectively, these results suggested that OI might serve as a potential treatment for OA progression.

IMR-261, a Novel Oral Nrf2 Activator, Induces Fetal Hemoglobin in Human Erythroblasts, Reduces VOCs, and Ameliorates Ineffective Erythropoiesis in Experimental Mouse Models of Sickle Cell Disease and Beta-Thalassemia

BackgroundSickle cell disease (SCD) is an autosomal recessive disorder where mutated hemoglobin (HbS) polymerizes and can lead to irreversible red blood cell (RBC) sickling and painful vaso-occlusive crisis (VOC). The RBC sickling is amplified by inflammation, resulting in tissue and organ damage. The transcription factor Nuclear factor erythroid 2-related factor 2 (Nrf2) coordinates the expression of antioxidant genes in response to oxidative stress, regulates inflammation, inhibits the NFkB pathway, and induces fetal hemoglobin (HbF), making it an attractive target in SCD and beta-thalassemia. IMR-261 is a novel oral activator of Nrf2 and has been tested in Phase 2 clinical trials (previously as CXA-10).Methods & ResultsCD14+ human monocytes were exposed to IMR-261 at 3µM and 10µM for 3 hours, to determine via quantitative PCR (qPCR) its ability to induce expression of antioxidant genes. IMR-261 at 10 µM significantly increased (p<0.05) the expression of Nrf2-dependent genes (p<0.05), including HMOX1, HSPA1A, HSP90, GCLM, SOD1 and TXNRD1. Human monocytes were treated with lipopolysaccharide (LPS) to test the ability of IMR-261 to block inflammatory genes with a NFkB target dataset. IMR-261 significantly inhibited (p<0.05) LPS-induced expression of IL-1-beta, TNF-alpha and IL-6 in human monocytes.To test the effects of IMR-261 on HbF induction, human erythroblasts were derived from CD34+ blood marrow progenitor cells sourced from healthy or SCD subjects. IMR-261 induced expression of the gamma-globin gene (4.0-fold change at 3µM and 7.18-fold change at 6 µM). This was accompanied by increased %F-cells (2.8-fold change at 3µM and 3.0-fold change at 6 µM).IMR-261 was also tested in the Townes HbSS mouse model of SCD to assess the potential for HbF induction. Mice were dosed with IMR-261 at 12.5 mg/kg or 37.5 mg/kg BID for 4 weeks (N=4-8/group). After 4 weeks of treatment, IMR-261 at 12.5 mg/kg and 37.5 mg/kg resulted in a significant increase in HbF relative to control, and 37.5 mg/kg resulted in a significant increase in %F-cells relative to control (Table 1, p<0.05). In addition, both doses of IMR-261 led to significant increases in RBC counts and total hemoglobin (Hb) (Table 1, p<0.05). IMR-261 at 37.5 mg/kg also significantly decreased (p<0.05) both reticulocyte counts and spleen cellularity.The ability of IMR-261 to reduce VOCs was assessed in separate Townes HbSS mice after the administration of TNF-alpha (0.5 µg/mice i.p.). IMR-261 was dosed at 37.5 mg/kg BID for 5 days before triggering VOCs. RBCs were stained with Ter-119 antibodies on spleen and liver of mice. Compared to controls, IMR-261 significantly reduced the presence of RBC on occluded vessels. This was coupled with a reduction of P-selectin (3109±97 Mean Fluorescence Units [MFI] in vehicle-treated vs. 1974±379 MFI in IMR-261 group, p<0.05) and L-selectin (375±20 MFI in vehicle-treated vs. 242±60 MFI in IMR-261 group, p<0.05). IMR-261 also reduced select hemolysis biomarkers: bilirubin (11.2±0.3 mg/dL in vehicle-treated vs. 8.4±0.7 mg/dL in IMR-261 group, (p<0.05) and free-heme (325±52 µM in vehicle-treated vs. 203±51 µM in IMR-261 group, p<0.05).A beta-thalassemia experimental model Hbb th1/th1 was tested to evaluate whether IMR-261 could improve ineffective erythropoiesis seen in beta-thalassemia. IMR-261 treatment at 37.5 mg/kg BID significantly increased hemoglobin levels, RBC counts and hematocrit (p<0.05), with significant reductions observed in reticulocytes (p<0.05). flow cytometry analysis (CD71/Ter119) showed that IMR-261 significantly decreased late basophilic and polychromatic erythroblasts (Ery.B) and increased orthochromatic erythroblasts and reticulocytes (Ery.C) cell numbers in the spleen (p<0.05).ConclusionsIMR-261 activates Nrf2-dependent antioxidant genes and inhibits NFkB-induced pro-inflammatory genes in human monocytes. In human erythroblasts, IMR-261 significantly increased HbF and %F-cells. In vivo SCD models show that IMR-261 significantly induced HbF and %F-cells, improved hemolytic markers, and decreased VOCs. IMR-261 also increased Hb and improved ineffective erythropoiesis in a beta-thalassemia in-vivo model. Together these data suggest that IMR-261 is a promising, novel, oral therapy that warrants clinical testing in SCD and beta-thalassemia. [Display omitted] DisclosuresMaciel: Imara Inc.: Research Funding. Carvalho: Imara Inc.: Research Funding. Rignault: Imara Inc.: Research Funding. Pace: Imara Inc.: Consultancy. OCain: Imara Inc.: Current Employment, Current equity holder in publicly-traded company. Ballal: Imara Inc.: Current Employment, Current equity holder in publicly-traded company.

Role of Nrf2 in Disease: Novel Molecular Mechanisms and Therapeutic Approaches - Pulmonary Disease/Asthma.

Nuclear factor erythroid 2-related factor 2 (Nrf2) is a major transcription factor involved in redox homeostasis and in the response induced by oxidative injury. Nrf2 is present in an inactive state in the cytoplasm of cells. Its activation by internal or external stimuli, such as infections or pollution, leads to the transcription of more than 500 elements through its binding to the antioxidant response element. The lungs are particularly susceptible to factors that generate oxidative stress such as infections, allergens and hyperoxia. Nrf2 has a crucial protective role against these ROS. Oxidative stress and subsequent activation of Nrf2 have been demonstrated in many human respiratory diseases affecting the airways, including asthma and chronic obstructive pulmonary disease (COPD), or the pulmonary parenchyma such as acute respiratory distress syndrome (ARDS) and pulmonary fibrosis. Several compounds, both naturally occurring and synthetic, have been identified as Nrf2 inducers and enhance the activation of Nrf2 and expression of Nrf2-dependent genes. These inducers have proven particularly effective at reducing the severity of the oxidative stress-driven lung injury in various animal models. In humans, these compounds offer promise as potential therapeutic strategies for the management of respiratory pathologies associated with oxidative stress but there is thus far little evidence of efficacy through human trials. The purpose of this review is to summarize the involvement of Nrf2 and its inducers in ARDS, COPD, asthma and lung fibrosis in both human and in experimental models.

Increased expression of autophagy and Nrf2-dependent signaling pathway genes by new monophenolic antioxidants depends on their structure

Under certain conditions, both activators and inhibitors of Nrf2-dependent signaling and autophagy can serve as potential agents for the prevention, treatment, and maintenance therapy of cancer, as well as overcoming chemoresistance. We are developing a panel of structurally related synthetic monophenolic compounds, in addition to a direct antioxidant effect, having the ability to affect intracellular signaling pathways and processes, including the above mentioned. Aim of the study was to investigate the relationship between its structure and the ability to influence the expression of autophagy and Nrf2 pathway genes.Material and methods. Five original hydrophilic phenolic structurally related compounds were synthesized; the prototypical inductor of the Keap1/Nrf2/ARE system, monosubstituted phenol tert-butylhydro-quinone (tBHQ), served as a reference substance. We used cultures of murine Lewis lung carcinoma (LLC) and human breast adenocarcinoma MCF-7 cells, with the test compounds at a concentration of 20 gM cells were incubated for 24 hours. Changes in mRNA expression of Nrf2, Nqo1, LC3b, and Sqstm1 genes encoding Nrf2, NAD(P)H:quinone oxidoreductase-1, LC3B, p62/sequestosome-1, respectively, were determined by TaqMan real-time PCR.Results and discussion. Structurally related synthetic monophenols effectively induced the Keap1/Nrf2/ARE system in LLC and MCF-7 cells, increasing the expression of the Nrf2-driven Nqo1 gene, and ambiguously affected the transcription of genes of proteins mediating autophagy: the content of mRNA of the LC3B protein did not change, while the expression of mRNA of the p62 protein, which affects the operation of both systems (processes), is increased only by the action of ortho-monosubstituted phenol with an «active» sulfur atom in the para-propyl substituent TS-13 and its structural analogue TS-12 with a para-substituent shortened by one methylene. It can be assumed that the observed effect either indicates the ability of TS-13 and TS-12 both to induce the Keap1/Nrf2/ARE system and to stimulate autophagy, or reflects a compound-mediated mutually reinforcing cross-activation of these processes.

Compensatory expression of NRF2-dependent antioxidant genes is required to overcome the lethal effects of Kv11.1 activation in breast cancer cells and PDOs

Potassium channels are important regulators of cellular homeostasis and targeting these proteins pharmacologically is unveiling important mechanisms in cancer cell biology. Here we demonstrate that pharmacological stimulation of the Kv11.1 potassium channel activity results in mitochondrial reactive oxygen species (ROS) production and fragmentation in breast cancer cell lines and patient-derived organoids independent of breast cancer subtype. mRNA expression profiling revealed that Kv11.1 activity significantly altered expression of genes controlling the production of ROS and endoplasmic-reticulum (ER) stress. Characterization of the transcriptional signature of breast cancer cells treated with Kv11.1 potassium channel activators strikingly revealed an adaptive response to the potentially lethal augmentation of ROS by increasing Nrf2-dependent transcription of antioxidant genes. Nrf2 in this context was shown to promote survival in breast cancer, whereas knockdown of Nrf2 lead to Kv11.1-induced cell death. In conclusion, we found that the Kv11.1 channel activity promotes oxidative stress in breast cancer cells and that suppression of the Nrf2-mediated anti-oxidant survival mechanism strongly sensitized breast cancer cells to a lethal effect of pharmacological activation of Kv11.1.

Four-octyl itaconate activates Nrf2 cascade to protect osteoblasts from hydrogen peroxide-induced oxidative injury

Four-octyl itaconate (4-OI) is the cell-permeable derivative of itaconate that can activate Nrf2 signaling by alkylating Keap1’s cysteine residues. Here, we tested the potential effect of 4-OI on hydrogen peroxide (H2O2)-induced oxidative injury in osteoblasts. In OB-6 cells and primary murine osteoblasts, 4-OI was able to activate Nrf2 signaling cascade and cause Keap1–Nrf2 disassociation, Nrf2 protein stabilization, cytosol accumulation, and nuclear translocation. 4-OI also augmented antioxidant-response element reporter activity and promoted expression of Nrf2-dependent genes (HO1, NQO1, and GCLC). Pretreatment with 4-OI inhibited H2O2-induced reactive oxygen species production, cell death, and apoptosis in osteoblasts. Furthermore, 4-OI inhibited H2O2-induced programmed necrosis by suppressing mitochondrial depolarization, mitochondrial cyclophilin D-ANT1 (adenine nucleotide translocase 1)-p53 association, and cytosol lactate dehydrogenase release in osteoblasts. Ectopic overexpression of immunoresponsive gene 1 (IRG1) increased endogenous itaconate production and activated Nrf2 signaling cascade, thereby inhibiting H2O2-induced oxidative injury and cell death. In OB-6 cells, Nrf2 silencing or CRISPR/Cas9-induced Nrf2 knockout blocked 4-OI-induced osteoblast cytoprotection against H2O2. Conversely, forced Nrf2 activation, by CRISPR/Cas9-induced Keap1 knockout, mimicked 4-OI-induced actions in OB-6 cells. Importantly, 4-OI was ineffective against H2O2 in Keap1-knockout cells. Collectively, 4-OI efficiently activates Nrf2 signaling to inhibit H2O2-induced oxidative injury and death of osteoblasts.

Activation of Nrf2 signaling by oltipraz inhibits death of human macrophages with mycobacterium tuberculosis infection

Mycobacterium tuberculosis (MTB) infection can induce cytotoxicity to the host macrophages, promoting bacterial spread. We here tested the potential effect of oltipraz, a synthetic dithiolethione, in MTB-infected human macrophages. We show that oltipraz significantly inhibited MTB-induced death and apoptosis in human macrophages. MTB-induced reactive oxygen species production, mitochondrial depolarization and programmed necrosis were attenuated by oltipraz in macrophages. Oltipraz activated Nrf2 signaling, causing Keap1-Nrf2 disassociation, Nrf2 protein stabilization and nuclear translocation, simultaneously promoting expression of Nrf2-dependent genes (HO1, NQO1 and GST) in human macrophages. Nrf2 shRNA or CRISPR/Cas9-induced Nrf2 knockout completely reversed oltipraz-induced macrophage protection against MTB infection. Furthermore, CRISPR/Cas9-mediated Keap1 knockout induced Nrf2 cascade activation and protected human macrophages from MTB. Importantly, oltipraz was unable to offer further cytoprotection against MTB in Keap1 knockout macrophages. Collectively we conclude that oltipraz activates Nrf2 signaling cascade to protect human macrophages from MTB-induced oxidative injury and cell death.

Effects of 2-MCPD on oxidative stress in different organs of male mice

2-Monochloropropane-1,3-diol (2-MCPD) and its isomer 3-monochloropropane-1,2-diol (3-MCPD) are widespread food contaminants. 3-MCPD has been classified as a non-genotoxic carcinogen, whereas very limited toxicological data are available for 2-MCPD. Animal studies indicate that heart and skeletal muscle are target organs of 2-MCPD. Oxidative stress may play a role in this process, and the potential of 3-MCPD to induce oxidative stress in vivo has already been demonstrated. To investigate the potential of 2-MCPD to induce oxidative stress in vivo, a 28-day oral feeding study in male HOTT reporter mice was conducted. This mouse model allows monitoring substance-induced oxidative stress in various target organs on the basis of Hmox1 promoter activation. Repeated daily doses of up to 100 mg 2-MCPD/kg body weight did not result in substantial toxicity. Furthermore, the highest dose of 2-MCPD had only minor effects on oxidative stress in kidney and testes, whereas brain, heart and skeletal muscle were not affected. Additionally, 2-MCPD caused only mild changes in the expression of Nrf2-dependent genes and only slightly affected the redox status of the redox-sensor protein DJ-1. Thus, the data indicate that 2-MCPD, in contrast to its isomer 3-MCPD, does not lead to a considerable induction of oxidative stress in male mice.

Discovery of Eicosapentaenoic Acid Esters of Hydroxy Fatty Acids as Potent Nrf2 Activators.

Branched fatty acid esters of hydroxy fatty acids (FAHFAs) are a recently discovered class of biologically active lipids with anti-inflammatory and anti-diabetic properties. Despite the possible link between endogenous FAHFA levels and nuclear factor erythroid 2-related factor 2 (Nrf2), their possible function as antioxidants and the mechanisms involved in this are unknown. Here, we investigate FAHFAs’ plausible antioxidant potential with reference to their effect on the Nrf2 levels, oxidative stress, and lipid droplet oxidation in human hepatocytes (C3A). Six authentic FAHFAs were chemically synthesized and performed activity-based screening by reporter gene assay. Among them, eicosapentaenoic acid (EPA) esterified 12-hydroxy stearic acid (12-HSA) and 12-hydroxy oleic acid (12-HOA) FAHFAs showed less cytotoxicity compared to their free fatty acids and potent activators of Nrf2. To define their mode of action, relative levels of nuclear Nrf2 were determined, which found a higher amount of Nrf2 in nucleus of cells treated with 12-EPAHSA compared to the control. Furthermore, 12-EPAHSA increased the expression of Nrf2-dependent antioxidant enzyme genes (NQO1, GCLM, GCLC, SOD-1, and HO-1). Fluorescence imaging analysis of linoleic-acid-induced lipid droplets (LDs) in C3A cells treated with 12-EPAHSA revealed the strong inhibition of small-size LD oxidation. These results suggest that EPA-derived FAHFAs as a new class of lipids with less cytotoxicity, and strong Nrf2 activators with plausible antioxidant effects via the induction of cytoprotective proteins against oxidative stress, induced cellular damage.

Anthocyanins reduce inflammation and improve glucose and lipid metabolism associated with inhibiting nuclear factor-kappaB activation and increasing PPAR-γ gene expression in metabolic syndrome subjects

Anthocyanins exhibit antioxidant and anti-inflammatory activities via a multitude of biochemical mechanisms. However, the signaling pathways involved in the actions of anthocyanins against chronic inflammation are not fully understood.The effects of berry-rich anthocyanin supplements (320 mg/day) for four weeks were examined on features of metabolic syndrome components and the expression of PPAR-γ, Nrf2, and NF-κB dependent genes in MetS and healthy subjects. Total RNA was isolated from whole blood with the PAXgene proprietary blood collection system.Four weeks anthocyanin consumption significantly decreased fasting blood glucose (15.7% vs 3.2%), TG (18.2% vs -1.39%), cholesterol (33.5% vs 1.56%) and LDL (28.4% vs -15.6%) in the MetS compared to Control group (P-value < 0.05, 95% CI). There was a significant up regulation in the expression PPAR-γ gene associated with the lipid and glucose metabolism in MetS subjects which negatively correlated (P-value < 0.01) with the change in the FBG (r = -0.488), Cholesterol (r = -0.496), TG (r = -0.513) and LDL (r = -0.519). Moreover, anthocyanin supplementation decreases serum hs-CRP (-36.3% vs 6.25%) in MetS in compared to Control group (P-value < 0.05). Anthocyanin supplementation also down-regulated the expression of NF-κB dependent genes including TNF-α (-28% and -15%), IL-6 (-16.1% and -13.6%), IL-1A (-21.5% and -12.9%), PCAM-1 (-15% and -17.5%), and COX-2(-26% and -27%) in both MetS and Control group respectively (P-value < 0.05).The study results suggested that berry supplements improved selected features of metabolic syndrome and related cardiovascular risk factors. These benefits may be due to the inhibition of NF-κB dependent gene expression and enhancement of PPAR-γ.

Compound 13 activates AMPK-Nrf2 signaling to protect neuronal cells from oxygen glucose deprivation-reoxygenation.

Oxygen glucose deprivation-reoxygenation (OGD-R) causes the production of reactive oxygen species (ROS) and oxidative injury in neuronal cells. We tested the potential neuroprotective function of compound 13 (C13), a novel AMP-activated protein kinase (AMPK) activator, against OGD-R. We show that C13 pretreatment protected SH-SY5Y neuronal cells and primary hippocampal neurons from OGD-R. C13 activated AMPK signaling in SH-SY5Y cells and primary neurons. It significantly inhibited OGD-R-induced apoptosis activation in neuronal cells. Conversely, AMPKα1 shRNA or knockout reversed C13-mediated neuroprotection against OGD-R. C13 potently inhibited OGD-R-induced ROS production and oxidative stress in SH-SY5Y cells and primary neurons. Furthermore, C13 induced Keap1 downregulation and Nrf2 activation, causing Nrf2 stabilization, nuclear accumulation, and expression of Nrf2-dependent genes. Nrf2 silencing or knockout in SH-SY5Y cells abolished C13-mediated neuroprotection against OGD-R. In conclusion, C13 activates AMPK-Nrf2 signaling to protect neuronal cells from OGD-R.

Nrf2 activation mediates tumor-specific hepatic stellate cells-induced DIgR2 expression in dendritic cells.

Our previous studies discovered that tumor-specific hepatic stellate cells (tHSCs) induced dendritic cell-derived immunoglobulin receptor 2 (DIgR2) expression in bone marrow-derived dendritic cells (mDCs), inhibiting splenic T cell activation. The current study aims to explore the underlying mechanism of DIgR2 expression by focusing on Nrf2 (nuclear-factor-E2-related factor 2) signaling. We show that tHSCs co-culture induced significant Nrf2 signaling activation in mDCs. The latter was evidenced by Nrf2-Keap1 disassociation, Nrf2 protein stabilization, accumulation and nuclear translocation. Expression of Nrf2-dependent genes, including heme oxygenase-1 (HO-1) and NAD(P)H:quinone oxidoreductase 1 (NQO1), were detected in tHSCs-co-cultured mDCs. Importantly tHSCs-induced DIgR2 expression was blocked by Nrf2 shRNA or knockout (KO, by CRISPR/Cas9 method). Conversely, forced activation of Nrf2, by Keap1 shRNA or the Nrf2 activators (3H-1,2-dithiole-3-thione and MIND4-17), induced significant DIgR2 expression. tHSCs stimulation induced reactive oxygen species (ROS) production in mDCs. Conversely, ROS scavengers inhibited tHSCs-induced ROS production, Nrf2 activation and DIgR2 expression in mDCs. Significantly, tHSCs inhibited production of multiple cytokines (CD80, CD86 and IL-12) in mDCs, reversed by Nrf2 depletion. Moreover, Nrf2 shRNA or KO attenuated splenic T cell inhibition by tHSCs-stimulated mDCs. Together, we conclude that Nrf2 activation mediates tHSCs-induced DIgR2 expression in mDCs.