Nuclear Factor-erythroid 2-related Factor-2 Pathways Research Articles

TAK1 inhibition mitigates intracerebral hemorrhage-induced brain injury through reduction of oxidative stress and neuronal pyroptosis via the NRF2 signaling pathway.

Intracerebral hemorrhage (ICH) often triggers oxidative stress through reactive oxygen species (ROS). Transforming growth factor-β-activated kinase 1 (TAK1) plays a pivotal role in regulating oxidative stress and inflammation across various diseases. 5Z-7-Oxozeaenol (OZ), a specific inhibitor of TAK1, has exhibited therapeutic effects in various conditions. However, the impact of OZ following ICH and its underlying molecular mechanisms remain elusive. This study aimed to explore the possible role of OZ in ICH and its underlying mechanisms by inhibiting oxidative stress-mediated pyroptosis. Adult male Sprague-Dawley rats were subjected to an ICH model, followed by treatment with OZ. Neurobehavioral function, blood-brain barrier integrity, neuronal pyroptosis, and oxidative stress markers were assessed using various techniques including behavioral tests, immunofluorescence staining, western blotting, transmission electron microscopy, and biochemical assays. Our study revealed that OZ administration significantly inhibited phosphorylated TAK1 expression post-ICH. Furthermore, TAK1 blockade by OZ attenuated blood-brain barrier (BBB) disruption, neuroinflammation, and oxidative damage while enhancing neurobehavioral function. Mechanistically, OZ administration markedly reduced ROS production and oxidative stress by facilitating nuclear factor-erythroid 2-related factor 2 (NRF2) nuclear translocation. This was accompanied by a subsequent suppression of the NOD-like receptor protein 3 (NLRP3) activation-mediated inflammatory cascade and neuronal pyroptosis. Our findings highlight that OZ alleviates brain injury and oxidative stress-mediated pyroptosis via the NRF2 pathway. Inhibition of TAK1 emerges as a promising approach for managing ICH.

Dextran sodium sulfate (DSS)-induced colitis is alleviated in mice after administration of flavone-derived NRF2-activating molecules

Inflammatory Bowel Disease (IBD) is a chronic and relapsing inflammatory condition characterized by severe symptoms such as diarrhea, fatigue, and weight loss. Growing evidence underscores the direct involvement of the nuclear factor-erythroid 2-related factor 2 (NRF2) in the development and progression of IBD, along with its associated complications, including colorectal cancer. The NRF2 pathway plays a crucial role in cellular responses to oxidative stress, and dysregulation of this pathway has been implicated in IBD. Flavones, a significant subclass of flavonoids, have shown pharmacological impacts in various diseases including IBD, through the NRF2 signaling pathway. In this study, we conducted a screening of compounds with a flavone structure and identified NJK15003 as a promising NRF2 activator. NJK15003 demonstrated potent NRF2 activation, as evidenced by the upregulation of downstream proteins, promoter activation, and NRF2 nuclear translocation in IBD cellular models. Treatment with NJK15003 effectively restored the protein levels of tight junctions in cells treated with dextran sodium sulfate (DSS) and in DSS-treated mice, suggesting its potential to protect cells from barrier integrity disruption in IBD. In DSS-treated mice, the administration of NJK15003 resulted in the prevention of body weight loss, a reduction in colon length shortening, and a decrease in the disease activity index. Furthermore, NJK15003 treatment substantially alleviated inflammatory responses and apoptotic cell death in the colon of DSS-treated mice. Taken together, this study proposes the potential utility of NRF2-activating flavone compounds, exemplified by NJK15003, for the treatment of IBD.

HP1 induces ferroptosis of renal tubular epithelial cells through NRF2 pathway in diabetic nephropathy.

The aim of this study was to investigate the role of ferroptosis in diabetic nephropathy (DN) and the mechanism of its regulatory genes. HK-2 cells were cultured with high glucose and mice were intraperitoneally injected with streptozotocin to establish DN models. GSE111154 was analyzed to identify the abnormal expression of genes associated with DN. Cell injury was evaluated through CCK-8 assay and 4',6-diamidino-2-phenylindole/phenylindole double staining. The levels of iron, glutathione, malondialdehyde, urinary albumin, and urinary creatinine were determined by ELISA. Furthermore, western blot and RT-qPCR were used to detect protein and mRNA levels, respectively. Our data showed that heterochromatin protein 1 is an abnormally elevated gene related to DN and is further elevated by ferroptosis activators. Inhibition of HP1 significantly inhibited ferroptosis but promoted cell viability. In addition, nuclear factor erythroid2-related factor2 (NRF2) was decreased in DN cell model, but increased under the action of ferroptosis activators. NRF2 silencing reversed the protective effects of HP1 inhibition on HK-2 cells. Additionally, HP1 silencing also alleviated kidney damage in DN mice. Collectively, these findings suggest that inhibiting HP1 inhibits ferroptosis via NRF2 pathway, thereby protecting renal tubular epithelial cells from damage.

Iron-mediated epigenetic activation of NRF2 targets

The toxic effects of excess dietary iron within the colonic lumen are well documented, particularly in the context of Inflammatory Bowel Disease (IBD) and Colorectal Cancer (CRC). Proposed mechanisms that underpin iron-associated intestinal disease include: (1) the pro-inflammatory and ROS-promoting nature of iron, (2) gene-expression alterations, and (3) intestinal microbial dysbiosis. However, to date no studies have examined the effect of iron on the colonic epigenome. Here we demonstrate that chronic iron exposure of colonocytes leads to significant hypomethylation of the epigenome. Bioinformatic analysis highlights a significant epigenetic effect on NRF2 (nuclear factor erythroid 2-related factor 2) pathway targets (including NAD(P)H Quinone Dehydrogenase 1 [NQO1] and Glutathione peroxidase 2 [GPX2]); this demethylating effect was validated and subsequent gene and protein expression quantified. These epigenetic modifications were not observed upon the diminishment of cellular lipid peroxidation with endogenous glutathione and the subsequent removal of iron. Additionally, the induction of TET1 expression was found post-iron treatment, highlighting the possibility of an oxidative-stress induction of TET1 and subsequent hypomethylation of NRF2 targets. In addition, a strong time dependence on the establishment of iron-orchestrated hypomethylation was found which was concurrent with the increase in the intracellular labile iron pool (LIP) and lipid peroxidation levels. These epigenetic changes were further validated in murine intestinal mucosa in models administered a chronic iron diet, providing evidence for the likelihood of dietary-iron mediated epigenetic alterations in vivo. Furthermore, significant correlations were found between NQO1 and GPX2 demethylation and human intestinal tissue iron-status, thus suggesting that these iron-mediated epigenetic modifications are likely in iron-replete enterocytes. Together, these data describe a novel mechanism by which excess dietary iron is able to alter the intestinal phenotype, which could have implications in iron-mediated intestinal disease and the regulation of ferroptosis.

Role of NRF2 cascade in determining the differential response of cervical cancer cells to anticancer drugs: an in vitro study.

Cervical cancers are usually treatable if detected in early stages by a combination of therapies. However, the prognosis of cervical cancer patients with metastasis remains unfavorable due to the fact that most of the cervical carcinomas are either resistant to anticancer drugs or show signs of relapse after initial treatment. Therefore, it is important to control the chemoresistance as it is the key to develop effective treatment options for cervical cancer. The current study aimed at evaluating the differential responses of cervical cancer cells to anti-cancer drugs and assessed whether the differences in the expression profiles of antioxidant genes regulated by nuclear factor erythroid-2-related factor 2 (NRF2), led to the variations in the sensitivities of the cancer cells to treatment. Three cervical cancer cell lines were investigated for their differences in NRF2 pathway by measuring the gene expression and enzyme activity. The differences in the sensitivity to anti-cancer drugs and variation in ROS profile was also evaluated. The addition of exogenous drugs to manipulate the intracellular ROS and its effect on NRF2 pathway genes was also investigated. HeLa and SiHa cells were more sensitive to cisplatin and oxaliplatin treatment than C33A cells. HeLa and SiHa cells had significantly lower NRF2 gene levels, NQO1 enzyme activity and basal GSH levels than C33A cells. Levels of ROS induced were higher in HeLa than C33A cells. Overall, the differences in the cellular levels of antioxidant regulatory genes led to the differential response of cervical cancer cells to anti-cancer drugs.

A strategy to prevent atherosclerosis via TNF receptor regulation

Atherosclerosis is a chronic inflammatory disease of the arterial wall. It has been known that development of atherosclerosis is closely related to activation of tumor necrosis factor α (TNF-α). The objective of this study was to elucidate the effects of TNF-α blockade with brusatol on endothelial activation under pro-atherosclerotic conditions. To this end, we examined the effects of brusatol on TNF-α-induced intracellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) expression in human aortic endothelial cells (HAECs) using western blot and THP-1 adhesion assays. Brusatol induced a decrease in TNF-α-induced ICAM-1 and VCAM-1 expression through inhibiting TNFR1 expression, leading to suppression of endothelial inflammation independently of the NRF2 (nuclear factor erythroid 2-related factor 2) pathway. The mechanism underlying brusatol-induced TNF receptor 1 (TNFR1) inhibition was investigated with the aid of protein synthesis, co-immunoprecipitation, and cytokine arrays. Notably, brusatol inhibited TNFR1 protein synthesis and suppressed both the canonical nuclear factor kappa-light-chain-enhancer of activated B cell (NF-κB) signaling pathway and TNF-α-induced cytokine secretion. We further tested the functional effect of brusatol on atherosclerosis development in vivo using two different atherosclerosis mouse models, specifically, acute partial carotid ligation and conventional chronic high-fat diet-fed mouse models. Administration of brusatol led to significant suppression of atherosclerosis development in both mouse models. Our finding that brusatol prevents atherosclerosis via inhibition of TNFR1 protein synthesis supports the potential of downregulation of cell surface TNFR1 as an effective therapeutic approach to inhibit development of atherosclerosis.

Liraglutide Ameliorates Lipotoxicity-Induced Oxidative Stress by Activating the NRF2 Pathway in HepG2 Cells.

Although glucagon-like peptide-1 (GLP-1) analogue has been reported to suppress oxidative stress in non-alcoholic fatty liver disease (NAFLD), an effective therapeutic agent for NAFLD is currently unavailable. Therefore, in this study, we aimed to investigate the protective effects of the GLP-1 analogue liraglutide against lipotoxicity-induced oxidative stress in HepG2 cells and to elucidate the underlying mechanisms. HepG2 cells were cultured for 48 hours and treated with a free fatty acid (FFA) mixture: FFA mixture and liraglutide or FFA mixture, liraglutide, and exendin (9-39). Lipid accumulation was examined by oil red O staining. Oxidative stress was assessed by measuring the levels of intracellular reactive oxygen species using 2',7'-dichlorofluorescein diacetate and thiobarbituric acid-reactive substances, whereas antioxidant capacity was assessed by measuring the activity of superoxide dismutase and catalase. Expression of the nuclear factor erythroid-2-related factor 2 (NRF2) gene and the genes encoding antioxidant enzymes was analyzed using quantitative RT-PCR. Cellular and nuclear NRF2 expression levels were assessed using immunofluorescence cell staining and western blotting. Liraglutide treatment reduced high fat-induced lipid formation and the levels of oxidative stress markers and increased antioxidant enzyme activity in HepG2 cells. Liraglutide treatment increased the mRNA expression of NRF2 target genes, induced NRF2 nuclear translocation, and increased nuclear NRF2 levels without altering NRF2 mRNA expression. Collectively, these results indicate that liraglutide exhibits a protective effect against lipotoxicity-induced oxidative stress, possibly via modulation of NRF2 and expression of antioxidant enzymes in liver cells.

Curcumin attenuates endothelial cell fibrosis through inhibiting endothelial-interstitial transformation.

Curcumin (Cur) has various pharmacological activities, including anti‐inflammatory, antiapoptotic and anticancer effects. However, there is no report on the effect of Cur on endothelial cell fibrosis. This study was designed to investigate the effect and mechanism of Cur on endothelial cell fibrosis. An endothelial cell fibrosis model was established by using transforming growth factor (TGF) induction. Proliferation assays, qRT‐PCR, western blotting and immunostaining were performed to investigate the effects and mechanism of Cur on endothelial cell fibrosis. We found that in human umbilical vein endothelial cells (HUVECs), TGF‐β1 treatment significantly decreased the expression of nuclear factor erythroid‐2‐related factor 2 (NRF‐2), dimethylarginine dimethylaminohydrolase‐1 (DDAH1), and VE‐cadherin, the secretion of cellular nitric oxide (NO) and the activity of nitrous oxide synthase (NOS), while asymmetric dimethylarginine (ADMA) and the release of inflammatory factors were elevated. Immunofluorescence showed decreased CD31 and increased α‐smooth muscle actin (α‐SMA). Overexpression of NRF‐2 significantly attenuated the effects of TGF‐β1, while downregulation of DDAH1 potently counteracted the effect of NRF‐2. In addition, ADMA treatment resulted in similar results to those of TGF‐β1, and Cur significantly attenuated the effect of TGF‐β1, accompanied by increased VE‐cadherin, DDAH1 and NRF‐2 and decreased matrix metalloproteinase‐9 (MMP‐9) and extracellular regulated protein kinases 1/2 (ERK1/2) phosphorylation. The NRF‐2 inhibitor ML385 had the opposite effect as that of Cur. These results demonstrated that Cur inhibits TGF‐β1‐induced endothelial‐to‐mesenchymal transition (EndMT) by stimulating DDAH1 expression via the NRF‐2 pathway, thus attenuating endothelial cell fibrosis.

Oleic acid and hydroxytyrosol present in olive oil promote ROS and inflammatory response in normal cultures of murine dermal fibroblasts through the NF-κB and NRF2 pathways.

Few studies have evaluated the effects of olive oil on normal tissues like skin and its components. Hence, we investigated whether olive oil could increase the production of ROS and oxidative damage in murine dermal fibroblast cultures in a short-term exposition. In addition, we evaluated the role of oleic acid and hydroxytyrosol, which are the two most important components of olive oil, in the associated mechanisms of action, and the metabolism of long-chain fatty acids from olive oil. To study this, neonatal murine dermal fibroblasts (NMDF) were incubated with olive oil, oleic acid, or hydroxytyrosol for 24 or 72h. The NMDF incubated with olive oil or oleic acid showed an increase in the production of ROS after 24h, lipid peroxidation, and protein carbonylation after 72h, as well as increased expression of nuclear factor-kappa B (NF-κB) p65 and cyclooxygenase-2 (COX-2) after 72h. However, NMDF treated with olive oil or hydroxytyrosol demonstrated an increase in the expression of nuclear factor-erythroid2-related factor 2 (NRF2) and heme oxygenase-1 (HO-1) after 72h. In addition, NMDF treated with olive oil also showed an increase in the protein expression of diacylglycerol acyltransferase1 (DGAT1), which promotes triacylglycerol synthesis, and in the levels of triacylglycerols. The microscopic analysis showed Nile red-positive lipid droplets inside olive oil-treated NMDF after 72h. Moreover, gas chromatography-mass spectrometry demonstrated high levels of oleic acid in the olive oil-treated NMDF after 72h. In conclusion, oleic acid present in the olive oil promotes the production of ROS and oxidative damage in murine dermal fibroblasts, which leads to NF-κB p65 and COX-2 expression, while hydroxytyrosol promotes NRF2 and HO-1 expression. In addition, NMDF area capable of absorbing long-chain fatty acids derived from olive oil, which promotes the synthesis and the accumulation of triacylglycerols into cytoplasm of NMDF through DGAT1 activation.

Regulation of Wound Healing by the NRF2 Transcription Factor-More Than Cytoprotection.

The nuclear factor-erythroid 2-related factor 2 (NRF2) transcription factor plays a central role in mediating the cellular stress response. Due to their antioxidant properties, compounds activating NRF2 have received much attention as potential medications for disease prevention, or even for therapy. Accumulating evidence suggests that activation of the NRF2 pathway also has a major impact on wound healing and may be beneficial in the treatment of chronic wounds, which remain a considerable health and economic burden. While NRF2 activation indeed shows promise, important considerations need to be made in light of corresponding evidence that also points towards pro-tumorigenic effects of NRF2. In this review, we discuss the evidence to date, highlighting recent advances using gain- and loss-of-function animal models and how these data fit with observations in humans.

Three novel genetic variants in NRF2 signaling pathway genes are associated with pancreatic cancer risk.

Pancreatic cancer (PanC) is one of the most lethal solid malignancies, and metastatic PanC is often present at the time of diagnosis. Although several high‐ and low‐penetrance genes have been implicated in PanC, their roles in carcinogenesis remain only partially elucidated. Because the nuclear factor erythroid2‐related factor2 (NRF2) signaling pathway is involved in human cancers, we hypothesize that genetic variants in NRF2 pathway genes are associated with PanC risk. To test this hypothesis, we assessed associations between 31 583 common single nucleotide polymorphisms (SNP) in 164 NRF2‐related genes and PanC risk using three published genome‐wide association study (GWAS) datasets, which included 8474 cases and 6944 controls of European descent. We also carried out expression quantitative trait loci (eQTL) analysis to assess the genotype‐phenotype correlation of the identified significant SNP using publicly available data in the 1000 Genomes Project. We found that three novel SNP (ie, rs3124761, rs17458086 and rs1630747) were significantly associated with PanC risk (P = 5.17 × 10−7, 5.61 × 10−4 and 5.52 × 10−4, respectively). Combined analysis using the number of unfavorable genotypes (NUG) of these three SNP suggested that carriers of two to three NUG had an increased risk of PanC (P < 0.0001), compared with those carrying zero to one NUG. Furthermore, eQTL analysis showed that both rs3124761 T and rs17458086 C alleles were associated with increased mRNA expression levels of SLC2A6 and SLC2A13, respectively (P < 0.05). In conclusion, genetic variants in NRF2 pathway genes could play a role in susceptibility to PanC, and further functional exploration of the underlying molecular mechanisms is warranted.

Dimethyl fumarate is highly cytotoxic in KRAS mutated cancer cells but spares non-tumorigenic cells.

KRAS mutation, one of the most common molecular alterations observed in adult carcinomas, was reported to activate the anti-oxidant program driven by the transcription factor NRF2 (Nuclear factor-erythroid 2-related factor 2). We previously observed that the antitumoral effect of Dimethyl fumarate (DMF) is dependent of NRF2 pathway inhibition. We used in vitro methods to examine the effect of DMF on cell death and the activation of the NRF2/DJ-1 antioxidant pathway. We report here that DMF is preferentially cytotoxic against KRAS mutated cancer cells. This effect was observed in patient-derived cancer cell lines harbouring a G12V KRAS mutation, compared with cell lines without such a mutation. In addition, KRAS*G12V over-expression in the human Caco-2 colon cancer cell line significantly promoted DMF-induced cell death, as well as DMF-induced- reactive oxygen species (ROS) formation and -glutathione (GSH) depletion. Moreover, in contrast to malignant cells, our data confirms that the same concentration of DMF has no significant cytotoxic effects on non-tumorigenic human ARPE-19 retinal epithelial, murine 3T3 fibroblasts and primary mice bone marrow cells; but is rather associated with NRF2 activation, decreased ROS and increased GSH levels. Furthermore, DJ-1 down-regulation experiments showed that this protein does not play a protective role against NRF2 in non-tumorigenic cells, as it does in malignant ones. This, interestingly, could be at the root of the differential effect of DMF observed between malignant and non-tumorigenic cells. Our results suggest for the first time that the dependence on NRF2 observed in mutated KRAS malignant cells makes them more sensitive to the cytotoxic effect of DMF, which thus opens up new prospects for the therapeutic applications of DMF.

Valproic Acid Sensitizes Hepatocellular Carcinoma Cells to Proton Therapy by Suppressing NRF2 Activation

Although efficacy of combined histone deacetylase (HDAC) inhibitors and conventional photon radiotherapy is being tested in clinical trials, their combined effect with proton beam radiotherapy has yet to be determined. Here, we compared combined effect of valproic acid (VPA), a class I and II HDAC inhibitor and antiepileptic drug with proton and photon irradiation in hepatocellular carcinoma (HCC) cells in vitro and in vivo. We found that VPA sensitized more Hep3B cells to proton than to photon irradiation. VPA prolonged proton-induced DNA damage and augmented proton-induced apoptosis. In addition, VPA further increased proton-induced production of intracellular reactive oxygen species and suppressed expression of nuclear factor erythroid-2-related factor 2 (NRF2), a key transcription factor regulating antioxidant response. Downregulation of NRF2 by siRNA transfection increased proton-induced apoptotic cell death, supporting NRF2 as a target of VPA in radiosensitization. In Hep3B tumor xenograft models, VPA significantly enhanced proton-induced tumor growth delay with increased apoptosis and decreased NRF2 expression in vivo. Collectively, our study highlights a proton radiosensitizing effect of VPA in HCC cells. As NRF2 is an emerging prognostic marker contributing to radioresistance in HCC, targeting NRF2 pathway may impact clinical outcome of proton beam radiotherapy.

Correction: Differential regulation of innate immune cytokine production through pharmacological activation of Nuclear Factor-Erythroid-2-Related Factor 2 (NRF2) in burn patient immune cells and monocytes.

[This corrects the article DOI: 10.1371/journal.pone.0184164.].

Differential regulation of innate immune cytokine production through pharmacological activation of Nuclear Factor-Erythroid-2-Related Factor 2 (NRF2) in burn patient immune cells and monocytes.

Burn patients suffer from immunological dysfunction for which there are currently no successful interventions. Similar to previous observations, we find that burn shock patients (≥15% Total Burn Surface Area (TBSA) injury) have elevated levels of the innate immune cytokines Interleukin-6 (IL-6) and Monocyte Chemoattractant Protein-1 (MCP-1)/CC-motif Chemokine Ligand 2(CCL2) early after hospital admission (0–48 Hours Post-hospital Admission (HPA). Functional immune assays with patient Peripheral Blood Mononuclear Cells (PBMCs) revealed that burn shock patients (≥15% TBSA) produced elevated levels of MCP-1/CCL2 after innate immune stimulation ex vivo relative to mild burn patients. Interestingly, treatment of patient PBMCs with the Nuclear Factor-Erythroid-2-Related Factor 2 (NRF2) agonist, CDDO-Me(bardoxolone methyl), reduced MCP-1 production but not IL-6 or Interleukin-10 (IL-10) secretion. In enriched monocytes from healthy donors, CDDO-Me(bardoxolone methyl) also reduced LPS-induced MCP1/CCL2 production but did not alter IL-6 or IL-10 secretion. Similar immunomodulatory effects were observed with Compound 7, which activates the NRF2 pathway through a different and non-covalent Mechanism Of Action (MOA). Hence, our findings with CDDO-Me(bardoxolone methyl) and Compound 7 are likely to reflect a generalizable aspect of NRF2 activation. These observed effects were not specific to LPS-induced immune responses, as NRF2 activation also reduced MCP-1/CCL2 production after stimulation with IL-6. Pharmacological NRF2 activation reduced Mcp-1/Ccl2 transcript accumulation without inhibiting either Il-6 or Il-10 transcript levels. Hence, we describe a novel aspect of NRF2 activation that may contribute to the beneficial effects of NRF2 agonists during disease. Our work also demonstrates that the NRF2 pathway is retained and can be modulated to regulate important immunomodulatory functions in burn patient immune cells.

Aldo-keto reductases are biomarkers of NRF2 activity and are co-ordinately overexpressed in non-small cell lung cancer.

Background:Although the nuclear factor-erythroid 2-related factor 2 (NRF2) pathway is one of the most frequently dysregulated in cancer, it is not clear whether mutational status is a good predictor of NRF2 activity. Here we utilise four members of the aldo-keto reductase (AKR) superfamily as biomarkers to address this question.Methods:Twenty-three cell lines of diverse origin and NRF2-pathway mutational status were used to determine the relationship between AKR expression and NRF2 activity. AKR expression was evaluated in lung cancer biopsies and Cancer Genome Atlas (TCGA) and Oncomine data sets.Results:AKRs were expressed at a high basal level in cell lines carrying mutations in the NRF2 pathway. In non-mutant cell lines, co-ordinate induction of AKRs was consistently observed following activation of NRF2. Immunohistochemical analysis of lung tumour biopsies and interrogation of TCGA data revealed that AKRs are enriched in both squamous cell carcinomas (SCCs) and adenocarcinomas that contain somatic alterations in the NRF2 pathway but, in the case of SCC, AKRs were also enriched in most other tumours.Conclusions:An AKR biomarker panel can be used to determine NRF2 status in tumours. Hyperactivation of the NRF2 pathway is far more prevalent in lung SCC than previously predicted by genomic analyses.