No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
Molecular Mechanisms Activating the Nrf2-Keap1 Pathway of Antioxidant Gene Regulation
Abstract
Several years have passed since NF-E2-related factor 2 (Nrf2) was demonstrated to regulate the induction of genes encoding antioxidant proteins and phase 2 detoxifying enzymes. Following a number of studies, it was realized that Nrf2 is a key factor for cytoprotection in various aspects, such as anticarcinogenicity, neuroprotection, antiinflammatory response, and so forth. These widespread functions of Nrf2 spring from the coordinated actions of various categories of target genes. The activation mechanism of Nrf2 has been studied extensively. Under normal conditions, Nrf2 localizes in the cytoplasm where it interacts with the actin binding protein, Kelch-like ECH associating protein 1 (Keap1), and is rapidly degraded by the ubiquitin-proteasome pathway. Signals from reactive oxygen species or electrophilic insults target the Nrf2-Keap1 complex, dissociating Nrf2 from Keap1. Stabilized Nrf2 then translocates to the nuclei and transactivates its target genes. Interestingly, Keap1 is now assumed to be a substrate-specific adaptor of Cul3-based E3 ubiquitin ligase. Direct participation of Keap1 in the ubiquitination and degradation of Nrf2 is plausible. The Nrf2-Keap1 system is present not only in mammals, but in fish, suggesting that its roles in cellular defense are conserved throughout evolution among vertebrates. This review article recounts recent knowledge of the Nrf2-Keap1 system, focusing especially on the molecular mechanism of Nrf2 regulation.
... Nuclear factor-erythroid 2-related factor 2 (NRF2), a transcription factor, is crucial for defending cells against oxidative stress by regulating the expression of antioxidant genes (Itoh et al., 1999;Kobayashi and Yamamoto, 2005). Previous studies have highlighted NRF2 as a downstream target of estrogen signaling (Ishii and Warabi, 2019;Wu et al., 2014;Zhu et al., 2015). ...
... This suggests a protective role for E 2 in oxidative stress-induced Müller cell death across mammalian species. Nrf2 has been identified as a downstream target of estrogen signaling; moreover, Nrf2-regulating antioxidative molecules contribute to the inhibition of oxidative stress-induced tissue damage (Itoh et al., 1999;Kobayashi and Yamamoto, 2005). Estrogen-mediated activation of the PI3K pathway results in NRF2 activation, preventing light-induced retinal degeneration via Nrf2 upregulation and suppression of retinal reactive oxygen species (ROS) production (Wu et al., 2014;Zhu et al., 2015). ...
... Additionally, E 2 administration upregulates the expression of Esr2 and phase II detoxifying enzyme-encoding genes, such as hemo oxygenase 1 (HMOX-1) and superoxide dismutase (SOD) (Giddabasappa et al., 2010). NRF2 counteracts oxidative stress by upregulating the expression of HMOX-1 and SOD (Itoh et al., 1999;Kobayashi and Yamamoto, 2005). The expression of phase II detoxifying enzyme-encoding genes is regulated by both NRF2-dependent and independent pathways (Piao et al., 2012). ...
In this study, we aimed to investigate the effects of the deficient antioxidative gene, nuclear factor-erythroid 2-related factor 2 (Nrf2), on 17β-estradiol (E2)-mediated oxidative stress response, with a specific focus on growth factor production and cell death in Müller cells and retinal tissue. Administration of hydrogen peroxide (H2O2) reduced the viability of Müller cells derived from Nrf2 wild-type (WT) and knockout (KO) mice. However, this effect was more significant in the KO cells than in the WT cells. Pretreatment with E2 inhibited H2O2-induced cell death in both Nrf2 WT and KO Müller cell genotypes. Small interfering RNA-mediated gene silencing of estrogen receptor 2 (Esr2) attenuated the cell survival-promoting activity of E2 in Nrf2 KO Müller cells, while other identified estrogen receptors, Esr1 or G protein-coupled estrogen receptor 1 (Gper1), had no effect. Western blotting revealed higher ESR2 expression levels in Nrf2 KO cells than in WT Müller cells. Conditioned media from E2-and H2O2-treated Nrf2 WT or KO Müller cells enhanced the dissociated retinal cell viability compared with H2O2-treated cells. Both quantitative reverse-transcription polymerase chain reaction assay (qRT-PCR) and enzyme-linked immunosorbent assay exhibited a significant increase in fibroblast growth factor 2 (FGF2) expression levels in E2-and H2O2-treated Nrf2 WT and KO Müller cells compared to those in E2-treated cells. In vivo, administration of N-methyl-N-nitrosourea (MNU) reduced the thickness and cell density of the outer nuclear layer (ONL) in Nrf2 KO mice and enhanced the number of terminal deoxynucleotidyl transferase dUTP nick-end labeling-positive cells in the ONL. However, E2 administration attenuated these defects in MNU-treated mice. Concomitant administration of MNU and E2 enhanced FGF2 protein levels in retinal lysates of Nrf2 KO mice. In conclusion, E2 demonstrated a significant role in preventing oxidative stress-induced retinal cell death by stimulating FGF2 production in Müller cells, independent of the Nrf2 gene. Based on these findings, we anticipate that exogenous administration of estrogens or ESR2-selective agonists could aid in treating patients with oxidative stress-related retinal degenerative diseases such as age-related macular degeneration and retinitis pigmentosa.
... The nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor, belonging to a family of basic leucine zipper (bZIP) proteins, a member of the cap'n'collar (CNC) subfamily that in humans is encoded by the NFE2L2 gene [13,14]. Nrf2 has seven functional domains (Nrf2-ECH homology, Neh1-7), which are either implicated in the regulation of Nrf2 stability or necessary for its transcriptional activity. ...
... Inflammation, as a response against tissue homeostasis impairment, is involved in a plethora of pathologic processes [13]. After signal recognition, Toll-like receptors (TLRs) mediate the activation of specific immune cascades leading to NF-κB dissociation from IκB. ...
... Similar to NF-κB, CBP acts as a transcriptional co-activator of Nrf2, binding its Neh4 and Neh5 domains, nevertheless with a lower affinity than to p65. Thus, both transcription factors compete for CBP accessibility: when p65 is overexpressed the amount of free CBP for Nrf2 is reduced and, hence, Nrf2-mediated gene transcription decreases, as depicted in Figure 3 [13]. ...
Neuroblastoma is the most common solid extracranial tumor during childhood; it displays extraordinary heterogeneous clinical courses, from spontaneous regression to poor outcome in high-risk patients due to aggressive growth, metastasizing, and treatment resistance. Therefore, the identification and detailed analysis of promising tumorigenic molecular mechanisms are inevitable. This review highlights the abnormal regulation of NF-κB, Nrf2, and Phox2B as well as their interactions among each other in neuroblastoma. NF-κB and Nrf2 play a key role in antioxidant responses, anti-inflammatory regulation and tumor chemoresistance. Recent studies revealed a regulation of NF-κB by means of the Nrf2/antioxidant response element (ARE) system. On the other hand, Phox2B contributes to the differentiation of immature sympathetic nervous system stem cells: this transcription factor regulates the expression of RET, thereby facilitating cell survival and proliferation. As observed in other tumors, we presume striking interactions between NF-κB, Nrf2, and Phox2B, which might constitute an important crosstalk triangle, whose decompensation may trigger a more aggressive phenotype. Consequently, these transcription factors could be a promising target for novel therapeutic approaches and hence, further investigation on their regulation in neuroblastoma shall be reinforced.
... Nuclear factor erythroid 2-related factor 2 (Nrf 2 ) is a stress-responsive transcription factor, which is the downstream of ROS (Ma, 2013). During oxidative stress, Nrf 2 is translocated to the nucleus to activate the transcription of genes reliant on antioxidant response elements (AREs) for ROS elimination and the maintenance of the cellular redox balance (Kobayashi and Yamamoto, 2005). Heme oxygenase-1 (HO-1), a pivotal antioxidant enzyme, is regulated by Nrf 2 (Loboda et al., 2016). ...
Background
Diabetic nephropathy (DN) is a serious complication of diabetes and one of the leading causes of end-stage renal disease. Huangkui capsule (HKC), a traditional Chinese patent medicine, is widely used in clinical practice for the treatment of chronic glomerulonephritis. However, the therapeutic effects and underlying mechanisms of HKC in DN remain poorly understood.
Methods
DN was induced in db/db mice, which were randomly divided into the DN, HKC-L, HKC-H and IRB groups, and db/m mice served as the Control group. Biochemical indices of blood and urine samples from the mice were measured, and HE staining, Masson staining and PAS staining were used to verify the anti-DN effect of HKC. The levels of ROS and the expression of Nrf2 pathway-related proteins and mRNAs were detected. Metabonomic analysis was used to investigate the role of tryptophan metabolism in the regulation of DN by HKC. HK-2 cells were used to establish a model of high-glucose (HG) injury in vitro, and HKC treatment was given for supplementary verification. Sarpogrelate hydrochloride (SH) combined with HKC, a 5-HT2AR inhibitor, was used to verify the effect of the 5-HT pathway in an in vitro model.
Results
Treatment with HKC significantly inhibited the increase in blood glucose and Urinary albumin/creatinine ratio (UACR), improved kidney injury signs in mice, reduced the level of ROS and improved oxidative stress injury through the Keap1/ Nrf2/HO-1 pathway. Metabonomic analysis revealed that tryptophan metabolism is involved in the process by which HKC improves DN, and HKC can regulate the 5-HT pathway to improve the renal injury by oxidative stress regulation. HKC treatment also significantly improved the renal and oxidative stress injuries in HG HK-2 cell model through the Nrf2 pathway in vitro. SH administration revealed that inhibiting 5-HT2AR could significantly inhibit the synthesis of 5-HT and improve the renal injury induced by HG.
Conclusion
Our study demonstrate that HKC can inhibit kidney injury and oxidative stress injury in db/db mice and HK-2 cells by regulating tryptophan metabolism and the Keap1/Nrf2/HO-1 pathway, which provides new insight for the clinical use of HKC for treatment of DN.
... The activation of Nrf2 signaling is regulated by the actinassociated kelch-domain protein 1 (Keap1), which binds to and sequesters Nrf2 in the cytoplasm. Upon exposure to oxidative stress, Nrf2 dissociates from Keap1-facilitated degradation and ubiquitination, permitting its accumulation and translocation to the nucleus, where it attaches itself to target genes' antioxidant response elements (AREs) and starts the transcription of those genes (Itoh et al. 1997;Kensler et al. 2007;Kobayashi and Yamamoto 2005). ...
Cholestasis, a condition characterized by impaired bile flow, can lead to severe liver damage if left untreated. Current therapeutic options are limited, necessitating the development of novel treatment strategies. This study investigated the synergistic action of cilnidipine, a calcium channel blocker, and bexarotene, a retinoid X receptor (RXR) agonist, in mitigating cholestatic liver damage induced by alpha-naphthyl isothiocyanate (ANIT) in rats. The study aimed to elucidate the role of the farnesoid X receptor (FXR) signaling cascade in the protective effects of the combined treatment. Rats were divided into three groups: a negative control group, an ANIT-treated group, and a group pretreated with cilnidipine and bexarotene before ANIT administration. Biochemical markers of liver function, oxidative stress, and inflammation were assessed, along with histological examination of liver tissue. The expression of genes related to the FXR signaling pathway was also evaluated using quantitative polymerase chain reaction (qPCR). The results demonstrated that pretreatment with cilnidipine and bexarotene significantly attenuated ANIT-induced cholestatic liver damage, as evidenced by improved liver function markers, reduced oxidative stress and inflammation, and ameliorated histological changes. Furthermore, the combined treatment upregulated the expression of FXR and its target genes, suggesting that the protective effects may be mediated through the activation of the FXR signaling cascade. These findings highlight the potential of cilnidipine and bexarotene as a novel therapeutic approach for the management of cholestatic liver disorders and provide insights into the underlying molecular mechanisms involving the FXR signaling pathway.
... The downstream target proteins of the pathway include superoxide dismutase (SOD), glutathione (GSH), NAD(P) H dehydrogenase, quinone oxidoreductase 1 (NQO-1) and catalase (CAT). These antioxidant enzymes have cytoprotective effects and maintain cellular homeostasis in pancreatic acinar cells under conditions of stress and inflammation [23]. Furthermore, it has been shown that the induction of Nrf2 prevents ROS damage in pancreatic β-cells. ...
Acute pancreatitis is one of the serious diseases of the gastrointestinal tract with possible systemic complications. The goal of AP therapy is thorough supportive treatment after obtaining hemodynamic status, prevention of necrosis, infection of the pancreas and organ failure. At present, however, there is no suitable pharmacological treatment that would act in the basic process of the pathology of acute pancreatitis. According to available scientific information, suitable candidates could be natural antioxidants that act on processes affecting oxidative stress in pancreatic acinar cells and thus have anti-inflammatory, antioxidant and anti-tumour effects.
... The Nrf2/HO-1 signaling pathway, as an important regulator in oxidative stress, is involved in the occurrence and development of OA (Li et al. 2023). Under the stimulation of the external or internal oxidants, the Nrf2/HO-1 signaling pathway is activated, then Nrf2 in the cytoplasm is transferred to the nucleus, where it upregulates the expression of antioxidant genes, such as HO-1 and GPX4 (Kobayashi and Yamamoto 2005). Our previous study demonstrated that activating Nrf2/HO-1 signaling pathway could significantly inhibit the expression of inflammatory factors (Zhou et al. 2020). ...
Osteoarthritis (OA) is a degenerative musculoskeletal disease, featured by the destruction of articular cartilage. Oxidative stress, one of the drivers of the extracellular matrix degradation in cartilage, plays a vital role in OA pathogenesis. Senkyunolide I (SEI) is a natural compound with a prominent anti-oxidative stress property against multiple diseases. However, the protective effect of SEI on OA has not been explored. Here, we aimed to elucidate the effect of SEI on OA in vitro. Our results showed that SEI suppressed the expression of senescence-related markers such as P16 and P21 in IL-1β-induced chondrocytes. Besides, SEI alleviated IL-1β-induced the degradation of extracellular matrix (ECM) by suppressing the matrix proteinase like MMP13 and ATAMDS5 while promoting matrix synthesis regulated biomarkers like COL2A1 and ACAN in chondrocytes. Mechanically, the mitochondrial dysfunction and overproduction of intracellular reactive oxygen species (ROS) in chondrocytes induced by IL-1β were reversed by SEI. Additionally, the ROS inhibitor N-acetylcysteine (NAC) synergistically enhanced the biological effect of SEI in IL-1β-induced chondrocytes. Moreover, it was also found that the expression of Nrf2 and HO-1 was increased by the treatment of SEI in IL-1β-stimulated chondrocytes, while the Nrf2 inhibitor ML385 reversed the protective effect of SEI on OA chondrocytes. In conclusion, SEI could inhibit senescence, the degradation of ECM, and the production of ROS through activating Nrf2/ HO-1 signaling pathway, which provide a novel candidate for OA treatment.
... The cytoplasmic negative regulator Keap1 normally suppresses Nrf2. All vertebrates including zebrafish have the Nrf2-Keap1 system which protects cells from oxidative stress [18,17]. Reactive species from 4-nonylphenol influence signal transduction, gene expression, and DNA damage [7]. ...
... To date, some new therapies (tiny molecule) reused traditional medications have been recorded to have superior pro-oxidant features and marked effects (38,39). Elesclomol (STA-4783) is a tiny synthetic compound that has an anti-cancer function in several cancers, such as breast cancer and melanoma (40). ...
... Nrf2 is negatively regulated by the cytoplasmic protein, Kelch-like ECHassociated protein 1 (Keap1), which acts as a substrate adaptor protein for cul3-mediated proteasomal degradation and degrades Nrf2 under normal cellular conditions [2]. However, under conditions of oxidative stress or electrophilic insult, Keap1 undergoes conformational changes that disrupt Nrf2 degrading activity, leading to Nrf2 stabilization, nuclear accumulation, and binding to AREs [3,4]. Nrf2 activation has beneficial effects on various pathological conditions, including insulin resistance, obesity [5] and cognitive function [6]. ...
Objective
Various plants have been reported to contain compounds that promote the transcriptional activity of Nuclear factor erythroid 2-related factor 2 (Nrf2) to induce a set of xenobiotic detoxifying enzymes, such as NAD(P)H-quinone acceptor oxidoreductase 1 (NQO1), via the antioxidant response element (ARE). While conventional methods for evaluating Nrf2 induction potency include measurement of NQO1 activity, an ARE luciferase reporter assay was recently developed to specifically assess Nrf2 induction potency of compounds of interest. In this study, we compared the abilities of these two assays to evaluate and determine Nrf2 induction potency of plant-derived compounds.
Results
Although the compounds exhibited a high degree of consistency between assays, several compounds did not. These results suggest that although the NQO1 assay can be used as an evaluation method to estimate Nrf2 induction potency of a compound, an ARE luciferase reporter approach may offer greater precision. In summary, inconsistency in Nrf2 induction potency evaluated by the reporter and NQO1 assays of plant-derived compounds, including resveratrol, may be due to a variety of factors that may regulate NQO1 gene expression other than Nrf2 and/or directly modulate NQO1 enzymatic activity or protein levels, with each compound having a different degree of effect on these factors.
... This is consistent with the finding that the mRNA levels of SOD and CAT were decreased, though Nrf2 expression itself did not show significant changes. This might be because Nrf2 protein levels are tightly regulated by Keap1, and rapidly degrade under normal conditions, but stabilize under stress conditions [48,49]. Furthermore, CGA supplementation resulted in the decreased activity of antioxidant enzymes (e.g., GPx, CAT, and SOD) in the liver, probably because CGA reduces oxidative and inflammatory stress by regulating the expression of antioxidant and cytoprotective genes. ...
Simple Summary
This study examined the effects of chlorogenic acid (CGA) on largemouth bass’s growth, immunity, and antioxidant status. Over eight weeks, fish were fed diets with varying CGA levels. In this study, CGA improved the growth performance and lowered the FCR. In addition, CGA enhanced the antioxidant capacity and regulated the expression of genes related to immune responses, thereby strengthening immunity. Overall, the addition of CGA positively impacted the health and growth of largemouth bass.
Abstract
This experiment evaluated the function of chlorogenic acid (CGA) in the growth, health status, and inflammation of largemouth bass (Micropterus salmoides). Over eight weeks, CGA supplementation was designed at five levels: 0, 60, 120, 180, and 240 mg/kg. The 180 and 240 mg/kg CGA-supplemented groups showed significant improvements in the FBW, SGR, and WGR compared to the control group (0 mg/kg) (p < 0.05). All the CGA-supplemented groups exhibited a significant reduction in the FCR (p < 0.05), with the 180 mg/kg CGA group showing the lowest FCR. Nonetheless, there were no appreciable differences in the plasma concentrations of TP, ALT, or AST among the treatments (p > 0.05). Compared to the control group, the 180 mg/kg CGA group exhibited significantly lower TC and TG levels (p < 0.05). The ALP levels showed no significant differences from the control group (p > 0.05). In terms of antioxidant parameters, CGA supplementation considerably reduced the MDA content (p < 0.05) and increased the GSH levels, while decreasing the CAT, SOD, and GPx activity levels Meanwhile, CGA supplementation resulted in reduced mRNA levels of SOD, CAT, Nrf2, Keap1, and NF-κB compared to the control group. In contrast, the mRNA levels of GPx, IL-8, TLR2, and RelA were elevated in the liver. Our findings indicated that CGA supplementation improved the growth performance and antioxidant status and weakened the inflammatory response of largemouth bass. These findings suggest that CGA could be a valuable dietary supplement for enhancing the health and growth of this species.
... It is worth noting that the antioxidant function of RuPc-BSA may also have a synergistic effect on the regulation of Keap1/Nrf2 redox signaling [55][56][57]. It has been confirmed that exposure to high levels of ROS can trigger the Keap1/Nrf2 signaling pathway, hence facilitating the separation of Nrf2 and Keap1 [58,59]. Subsequently, the Nrf2 transcription factor activates the transcription of some cell protection genes, such as CAT and SOD [60,61]. ...
The prevention and treatment of hypobaric hypoxia brain injury (HHBI) remains an unprecedented challenge due to the complex oxidative stress response at the damage site. In this study, RuCO phthalocyanine compound (RuPc) and bovine serum albumin (BSA) were self-assembled to obtain RuPc-BSA nanoparticles for HHBI therapy. As a nanoprobe carrying and storing carbon monoxide (CO), RuPc-BSA delivers CO to pathologically damaged areas of the brain. CO specifically attaches itself to the heme functional groups on mitochondria and restricts the source of reactive oxygen species (ROS) generation. RuPc-BSA nanoparticles have been demonstrated in vitro to exhibit amazing stability as well as remarkable scavenging activity on hydroxyl radical, superoxide anion, and hydrogen peroxide. In vivo experiments showed that ROS levels in the brain of HHBI rats pretreated with RuPc-BSA decreased significantly, and neuronal function and oxidative stress levels were alleviated. Western blot and qRT-RCR results indicated that RuPc-BSA restricted the protein levels of Keap1, whereas enhanced the gene and protein levels of Nrf2. This study demonstrated that RuPc-BSA can ameliorate HHBI of mice by scavenging ROS partly via activating Keap1/Nrf2 signaling pathway.
... This binding induces endogenous enzymes such as heme oxygenase-1 (HO-1), superoxide dismutase (SOD), and NAD (P) H quinone oxidoreductase 1 (NQO1). 39 HO-1 is an important antioxidant that is regulated by NRF-2 and affects intracellular ROS levels. The degree of HO-1/Nrf2 induction assists in the overall prognosis following a SCI. ...
Background
In parallel with population aging, the prevalence of neurological and neurodegenerative diseases has been dramatically increasing over the past few decades. Neurodegenerative diseases reduce the quality of life of patients and impose a high cost on the health system. These slowly progressive diseases can cause functional, perceptual, and behavioral deficits in patients. Therefore, neurodegenerative impairments have always been an interesting subject for scientists and clinicians. One of these diseases is spinal cord injury (SCI). SCI can lead to irreversible damage and is classified into two main subtypes: traumatic and non‐traumatic, each with very different pathophysiological features.
Aims
This review aims to gather relevant information about the beneficial effects of curcumin (Cur), with specific emphasis on its anti‐inflammatory properties towards spinal cord injury (SCI) patients.
Materials & Methods
The review collates data from extensive in‐vitro, in‐vivo, and clinical trials documenting the effects of CUR on SCI. It examines the modulation of pathophysiological pathways and regulation of the inflammatory cascades after CUR administration.
Results
Various pathophysiological processes involving the nuclear factor erythroid 2‐related factor 2 (Nrf2), nuclear factor kappa B (NF‐kB), and transforming growth factor beta (TGF‐β) signaling pathways have been suggested to exacerbate damages resulting from SCI. CUR administration showed to modulate these signaling pathways which lead to attenuation of SCI complications.
Discussion
Anti‐inflammatory compounds, particularly CUR, can modulate these pathophysiological pathways and regulate the inflammatory cascades. CUR, a well‐known natural product with significant anti‐inflammatory effects, has been extensively documented in experimental and clinical trials.
Conclusion
Curcumin's potential to alter key steps in the Nrf2, NF‐kB, and TGF‐β signaling pathways suggests that it may play a role in attenuating SCI complications.
... The nrf2-keap1 pathway, as a key antioxidant response element recognizer, holds potential as a crucial target for preventing liver disease development [39]. Nrf2 activation induces the transcription of numerous antioxidative enzymes, thereby increasing the antioxidative capacity [40], aligning with our results. Its activation could also indirectly induce the up-regulation of keap1 via the negative feedback regulation [41]. ...
A multi-strain yeast-based paraprobiotic (MsYbP) comprising inactive cells and polysaccharides (β-glucan, mannan oligosaccharides, and oligosaccharides) derived from Saccharomyces cerevisiae and Cyberlindnera jadinii could ensure optimal growth and health in farmed fish. This study assessed the impact of an MsYbP on the growth, immune responses, antioxidant capacities, and liver health of largemouth bass (Micropterus salmoides) through lab-scale (65 days) and pilot-scale (15 weeks) experiments. Two groups of fish were monitored: one fed a control diet without the MsYbP and another fed 0.08% and 0.1% MsYbP in the lab-scale and pilot-scale studies, respectively (referred to as YANG). In the lab-scale study, four replicates were conducted, with 20 fish per replicate (average initial body weight = 31.0 ± 0.8 g), while the pilot-scale study involved three replicates with approximately 1500 fish per replicate (average initial body weight = 80.0 ± 2.2 g). The results indicate that the MsYbP-fed fish exhibited a significant increase in growth in both studies (p < 0.05). Additionally, the dietary MsYbP led to a noteworthy reduction in the liver function parameters (p < 0.05), such as alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (AKP), and hepatic nuclear density, indicating improved liver health. Furthermore, the dietary MsYbP elevated the antioxidative capacity of the fish by reducing their malondialdehyde levels and increasing their levels and gene expressions related to antioxidative markers, such as total antioxidant ca-pacity (T-AOC), total superoxide dismutase (T-SOD), glutathione peroxidase (GSH-Px), catalase (CAT), nuclear factor erythroid 2-related factor 2 (nrf2) and kelch-1ike ech-associated protein (keap1) in both studies (p < 0.05). In terms of hepatic immune responses, the lab-scale study showed an increase in inflammation-related gene expressions, such as interleukin-1β (il-1β) and transforming growth factor β1 (tgf-β1), while the pilot-scale study significantly suppressed the expressions of genes related to inflammatory responses, such as tumor necrosis factor α (tnfα) and interleukin-10 (il-10) (p < 0.05). In summary, our findings underscore the role of dietary multi-strain yeast-based paraprobiotics in enhancing the growth and liver health of largemouth bass, potentially through increased antioxidative capacity and the modulation of immune responses, emphasizing the significance of employing yeast-based paraprobiotics in commercial conditions.
... The Nrf2-Keap1 system has evolved in all vertebrates, including zebrafish and mammals. It plays a significant role in cellular defense against oxidative stress (Kobayashi et al., 2002;Kobayashi and Yamamoto, 2005). Numerous reactive species produced by 4-NP impact gene expression, signal transduction and cause DNA damage (Duan et al., 2016). ...
... To combat free radicals, cells engage with the antioxidant response element (ARE) and several redox-sensitive transcription factors including nuclear factor (NF)-ϏB in response to ROS or RNS. Nuclear factor erythroid 2-related factor 2 (NRF2) is one of the responsible elements for controlling the expression of protective genes that maintain redox homeostasis within the cell (Kobayashi & Yamamoto, 2005). Nrf2 is found in the cytosol sequestered by Kelch-like ECHassociated protein 1 (Keap1) under physiological conditions and is released and translocates into the nucleus upon exposure to ROS or electrophilic chemicals. ...
... The outstanding role of Nrf2 in the cellular response to oxidative stress and inflammation has been mentioned before. Nrf2 is phosphorylated and activated via PKC, PI3K, MAPK, and Erk1/2 [98], signaling molecules associated with GPR55 [50,99]. Furthermore, activation of GPR55 using O-1602 increased levels of Nrf2-regulated anti-oxidative enzymes, such as SOD, GSH, and catalase, while it decreased levels of malondialdehyde, another lipid peroxidation marker, in β-amyloid [84] and streptozotocin [85] induced AD mice model. ...
Pharmacological treatment of psychiatric disorders remains challenging in clinical, pharmacological, and scientific practice. Even if many different substances are established for treating different psychiatric conditions, subgroups of patients show only small or no response to the treatment. The neuroinflammatory hypothesis of the genesis of psychiatric disorders might explain underlying mechanisms in these non-responders. For that reason, recent research focus on neuroinflammatory processes and oxidative stress as possible causes of psychiatric disorders. G-protein coupled receptors (GPCRs) form the biggest superfamily of membrane-bound receptors and are already well known as pharmacological targets in various diseases. The G-protein coupled receptor 55 (GPR55), a receptor considered part of the endocannabinoid system, reveals promising modulation of neuroinflammatory and oxidative processes. Different agonists and antagonists reduce pro-inflammatory cytokine release, enhance the synthesis of anti-inflammatory mediators, and protect cells from oxidative damage. For this reason, GPR55 ligands might be promising compounds in treating subgroups of patients suffering from psychiatric disorders related to neuroinflammation or oxidative stress. New approaches in drug design might lead to new compounds targeting different pathomechanisms of those disorders in just one molecule.
... This heterodimer identifies the ARE as a cis-acting sequence in the promoters of a regulatory region genes, including phase II detoxification enzymes and antioxidant to induce their transcriptions, e.g., NAD(P)H:quinone oxidoreductase 1 (NQO1) and glutathione S-transferase (GST) [45]. More than 100 genes are regulated by the Nrf2-Keap1 in this regulation [46][47][48][49][50][51][52]. For instance, the reduction of ROS is achieved by direct regulation of the enzymatic formation by Nrf2. ...
Oxidative stress refers to a condition where there is an imbalance between the production of reactive oxygen species and their removal by antioxidants. While the function of reactive oxygen species as specific second messengers under physiological conditions is necessary, their overproduction can lead to numerous instances of cell and tissue damage. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a master regulator of many cytoprotective genes that respond to redox stresses. Nrf2 is regularly degraded by kelch-like ECH-associated protein 1 through the ubiquitin-proteasome pathway. The kelch-like ECH-associated protein 1 and Nrf2 complex have attracted attention in both basic and clinical infertility research fields. Oxidative stress is implicated in the pathogenesis of female infertility, including primary ovarian insufficiency, polycystic ovarian syndrome, and endometriosis, as well as male infertility, namely varicocele, cryptorchidism, spermatic cord torsion, and orchitis. Most scientists believe that Nrf2 is a potential therapeutic method in female and male infertility disorders due to its antioxidant effect. Here, the potential roles of oxidative stress and Nrf2 in female and male infertility disorders are reviewed. Moreover, the key role of Nrf2 in the inhibition or induction of these diseases is discussed.
... Interestingly, we predicted that NRF2, a master transcription factor in the antioxidant defense system, may interact with CARMN. NRF2 has been extensively studied in the context of inflammatory responses and oxidative stress through its regulation of the expression of genes encoding phase II detoxification enzymes and antioxidants [58,59]. NRF2 has also been reported to regulate PDGF-stimulated VSMC migration. ...
Phenotypic transformation of vascular smooth muscle cells (VSMCs) plays a crucial role in abdominal aortic aneurysm (AAA) formation. CARMN, a highly conserved, VSMC-enriched long noncoding RNA (lncRNA), is integral in orchestrating various vascular pathologies by modulating the phenotypic dynamics of VSMCs. The influence of CARMN on AAA formation, particularly its mechanisms, remains enigmatic. Our research, employing single-cell and bulk RNA sequencing, has uncovered a significant suppression of CARMN in AAA specimens, which correlates strongly with the contractile function of VSMCs. This reduced expression of CARMN was consistent in both 7- and 14-day porcine pancreatic elastase (PPE)-induced mouse models of AAA and in human clinical cases. Functional analyses disclosed that the diminution of CARMN exacerbated PPE-precipitated AAA formation, whereas its augmentation conferred protection against such formation. Mechanistically, we found CARMN's capacity to bind with SRF, thereby amplifying its role in driving the transcription of VSMC marker genes. In addition, our findings indicate an enhancement in CAMRN transcription, facilitated by the binding of NRF2 to its promoter region. Our study indicated that CARMN plays a protective role in preventing AAA formation and restrains the phenotypic transformation of VSMC through its interaction with SRF. Additionally, we observed that the expression of CARMN is augmented by NRF2 binding to its promoter region. These findings suggest the potential of CARMN as a viable therapeutic target in the treatment of AAA.
Graphical abstract
... Nrf2 is modi ed by the cytoplasmic protein Kelch-like ECH-associated protein 1 (Keap1) and localized in the cytoplasm under normal cellular conditions [2]. However, under conditions of oxidative stress or electrophilic insult, Keap1 undergoes conformational changes, leading Nrf2 to stabilization, nuclear accumulation, and binding to ARE, which activates the transcription of genes involved in antioxidant response and cellular detoxi cation [3,4]. ...
Objective
Various plants have been reported to contain compounds that promote the nuclear accumulation of Nrf2 to induce a set of xenobiotic detoxifying enzymes such as NAD(P)H-quinone acceptor oxidoreductase 1 (NQO1) via antioxidant response element (ARE). While conventional methods for evaluating the Nrf2 induction potency of compounds include NQO1 activity, recently, an ARE luciferase reporter was developed to directly assess the Nrf2 induction potency of compounds of interest. In this study, the ability of these two assays to evaluate and determine Nrf2 induction potency of plant-derived compounds was compared.
Results
Although the compounds overall showed a high degree of consistency between the assays, several compounds did not. The results suggest that although the NQO1 assay can be used as an evaluation method to estimate the Nrf2 induction potency of a compound, an ARE luciferase reporter may offer greater precision. In summary, the inconsistency in Nrf2 induction potency evaluated by the reporter and NQO1 assays for some of the plant-derived compounds evaluated herein, including resveratrol, may be due to a variety of factors that regulate NQO1 expression and activity other than Nrf2, with each compound having a different degree of effect on these factors.
... Nrf2/Keap1 signaling plays a vital role in resisting oxidative injury. Impaired oxidative redox status induces the dissociation of Nrf2 from Keap1 and translocation into the nucleus, which then binds to the antioxidant response element to regulate the expression of downstream antioxidant genes, such as CAT, SOD, and GPx, thus coping with cells from oxidative-stress-induced cellular injury [41,42]. In the present study, we found that Nrf2 protein expression significantly increased in response to silybin supplementation, and the mRNA expression of SOD1, GPX1, and GPX4 was also in parallel with the alteration trend of Nrf2 protein expression, suggesting that dietary silybin enhanced antioxidant capacity in the PQ-challenged piglets. ...
Silybin (Si) is the main element of silymarin isolated from the seeds of Silybum marianum L. Gaernt., which has superior antioxidant properties. However, the protective role of Si in maintaining liver health under oxidative stress remains ambiguous. This study aimed to investigate the underlying mechanism of the beneficial effect of dietary Si against hepatic oxidative injury induced by paraquat (PQ) in weaned piglets. A total of 24 piglets were randomly allocated to four treatments with six replicates per treatment and 1 piglet per replicate: the control group; Si group; PQ group; and Si + PQ group. Piglets in the control group and PQ group were given a basal diet, while piglets in the Si and Si + PQ groups were given a Si-supplemented diet. On the 18th day, the pigs in the PQ treatment group received an intraperitoneal injection of PQ, and the others were intraperitoneally injected with the same volume of saline. All piglets were sacrificed on day 21 for plasma and liver sample collection. The results showed that dietary Si supplementation mitigated PQ-induced liver damage, as proven by the reduction in liver pathological changes and plasma activity of alanine transaminase and aspartate transaminase. Si also improved superoxide dismutase and glutathione peroxidase activities and total antioxidant capacity, as well as decreased malondialdehyde and hydrogen peroxide concentration in the liver, which were closely related to the activation of the nuclear factor-erythroid 2-related factor 2 signaling pathway. Meanwhile, Si reduced tumor necrosis factor-α and interleukin-8 production and their transcript levels as well as abrogated the overactivation of nuclear factor-κB induced by PQ. Importantly, Si improved mitochondrial function by maintaining mitochondrial energetics and mitochondrial dynamics, which was indicated by the elevated activity of mitochondrial complexes I and V and adenosine triphosphate content, decreased expression of dynamin 1 protein, and increased expression of mitofusin 2 protein. Moreover, Si inhibited excessive hepatic apoptosis by regulating the B-cell lymphoma-2 (Bcl-2)/Bcl-2-associated-X-protein signaling pathway. Taken together, these results indicated that Si potentially mitigated PQ-induced hepatic oxidative insults by improving antioxidant capacity and mitochondrial function and inhibiting inflammation and cell apoptosis in weaned piglets.
... 6,7 When intra-cellular redox imbalance exists, Nrf2 binding to Keap1 is disrupted, translocation of unbound Nrf2 to the nucleus is enabled, and binding of Nrf2 to the promoter region of the antioxidant response element is facilitated, resulting in the upregulation of numerous cytoprotective genes. 8,9 The multi-functional adaptor protein sequestosome 1 (SQSTM1) (p62) interacts with, and participates in the regulation of, various cell signalling pathways including protein and redox homoeostasis. 10 Evidence exists that p62 binds to Keap1 via its Keap1-interacting region (KIR), disrupts Nrf2-Keap1 binding, and allows nuclear translocation of unbound Nrf2 to initiate an antioxidant defence in non-cardiac cells. ...
Aims
Heart failure due to ischaemic heart disease (IHD) is a leading cause of mortality worldwide. A major contributing factor to IHD-induced cardiac damage is hypoxia. Sequestosome 1 (p62) is a multi-functional adaptor protein with pleiotropic roles in autophagy, proteostasis, inflammation, and cancer. Despite abundant expression in cardiomyocytes, the role of p62 in cardiac physiology is not well understood. We hypothesized that cardiomyocyte-specific p62 deletion evokes hypoxia-induced cardiac pathology by impairing hypoxia-inducible factor 1α (Hif-1α) and nuclear factor erythroid 2-related factor 2 (Nrf2) signalling.
Methods and results
Adult mice with germline deletion of cardiomyocyte p62 exhibited mild cardiac dysfunction under normoxic conditions. Transcriptomic analyses revealed a selective impairment in Nrf2 target genes in the hearts from these mice. Demonstrating the functional importance of this adaptor protein, adult mice with inducible depletion of cardiomyocyte p62 displayed hypoxia-induced contractile dysfunction, oxidative stress, and cell death. Mechanistically, p62-depleted hearts exhibit impaired Hif-1α and Nrf2 transcriptional activity. Because findings from these two murine models suggested a cardioprotective role for p62, mechanisms were evaluated using H9c2 cardiomyoblasts. Loss of p62 in H9c2 cells exposed to hypoxia reduced Hif-1α and Nrf2 protein levels. Further, the lack of p62 decreased Nrf2 protein expression, nuclear translocation, and transcriptional activity. Repressed Nrf2 activity associated with heightened Nrf2-Keap1 co-localization in p62-deficient cells, which was concurrent with increased Nrf2 ubiquitination facilitated by the E3 ligase Cullin 3, followed by proteasomal-mediated degradation. Substantiating our results, a gain of p62 in H9c2 cells stabilized Nrf2 and increased the transcriptional activity of Nrf2 downstream targets.
Conclusion
Cardiac p62 mitigates hypoxia-induced cardiac dysfunction by stabilizing Hif-1α and Nrf2.
... These PTMs disrupt the interaction between Nrf2 and Keap1, allowing Nrf2 to translocate into the nucleus and bind to the promoter region of antioxidant responsive genes (de Freitas Silva et al., 2018;Tu et al., 2019;Ulasov et al., 2022). The resulting antioxidant response proteins, which include antioxidant enzymes like superoxide dismutase, glutathione reductase, and catalase (Sajadimajd and Khazaei, 2018;Yuan et al., 2021), as well as phase II detoxifying enzymes like glutathione S-transferase (GST), NADPH quinone oxidoreductase, gammaglutamylcysteine synthetase (GGS) and heme oxygenase-1 (HO-1), and translation detoxification enzymes like glutathione synthetase (GSS) (Alam et al., 1999;Kobayashi and Yamamoto, 2005), possess the capability to safeguard cells against oxidative damage by scavenging intracellular free radicals and maintaining intracellular redox balance. ...
The Keap1-Nrf2 signaling pathway plays a crucial role in cellular defense against oxidative stress-induced damage. Its activation entails the expression and transcriptional regulation of several proteins involved in detoxification and antioxidation processes within the organism. Keap1, serving as a pivotal transcriptional regulator within this pathway, exerts control over the activity of Nrf2. Various post-translational modifications (PTMs) of Keap1, such as alkylation, glycosylation, glutathiylation, S-sulfhydration, and other modifications, impact the binding affinity between Keap1 and Nrf2. Consequently, this leads to the accumulation of Nrf2 and its translocation to the nucleus, and subsequent activation of downstream antioxidant genes. Given the association between the Keap1-Nrf2 signaling pathway and various diseases such as cancer, neurodegenerative disorders, and diabetes, comprehending the post-translational modification of Keap1 not only deepens our understanding of Nrf2 signaling regulation but also contributes to the identification of novel drug targets and biomarkers. Consequently, this knowledge holds immense importance in the prevention and treatment of diseases induced by oxidative stress.
... It has been suggested that Nrf2 signaling is impaired in an aging RPE, which increases the risk of developing AMD [32]. The activation of Nrf2 could induce the expression of antioxidative enzymes such as HO-1, NQO-1, GLC, glutathione peroxidase, and glutathione transferase, the activation of which would increase the production of glutathione as well as the total cellular antioxidant capacity [33]. Therefore, Nrf2 has been well-recognized as a therapeutic target for the treatment of AMD. ...
Nuclear factor E2-related factor 2 (Nrf2) is fundamental to the maintenance of redox homeostasis within cells via the regulation of a series of phase II antioxidant enzymes. The unique olive-derived phenolic compound hydroxytyrosol (HT) is recognized as an Nrf2 activator, but knowledge of the HT derivative hydroxytyrosol acetate (HTac) on Nrf2 activation remains limited. In this study, we observed that an HT pretreatment could protect the cell viability, mitochondrial membrane potential, and redox homeostasis of ARPE-19 cells against a t-butyl hydroperoxide challenge at 50 μM. HTac exhibited similar benefits at 10 μM, indicating a more effective antioxidative capacity compared with HT. HTac consistently and more efficiently activated the expression of Nrf2-regulated phase II enzymes than HT. PI3K/Akt was the key pathway accounting for the beneficial effects of HTac in ARPE-19 cells. A further RNA-Seq analysis revealed that in addition to the consistent upregulation of phase II enzymes, the cells presented distinct expression profiles after HTac and HT treatments. This indicated that HTac could trigger a diverse cellular response despite its similar molecular structure to HT. The evidence in this study suggests that Nrf2 activation is the major cellular activity shared by HTac and HT, and HTac is more efficient at activating the Nrf2 system. This supports its potential future employment in various disease management strategies.
Cardiovascular diseases (CVDs) are the leading cause (32%) of global morbidomortalities. Understanding how dietary interventions influence molecular pathways offers practical, preventive solutions for reducing this burden. Additionally, this topic targets a wide range of stakeholders, including researchers, healthcare professionals, and the public seeking strategies to ensure healthy hearts.
From 2023 to 2030, the market of functional foods and nutraceuticals is expected to see a growth of 7.5% CAGR that indicates increasing public and scientific interest in health-promoting diets. It has been estimated that balanced diets like the Mediterranean or traditional Brazilian diets can reduce the global CVD deaths by 20%.
Diet therapies are significant in mitigating cardiovascular risk factors and enhancing heart health. The bioactive compounds in various cardioprotective foods modulate molecular pathways associated with inflammation, oxidative stress, and lipid metabolism. These mechanisms mostly converge on key pathways like Nitric oxide, NF-κB, Nrf2/ARE, PI3K/Akt, mitochondria regulatory, and epigenetic pathways. We utilized molecular docking techniques and found significant interactions between the bioactive compounds like Epicatechin, Resveratrol, Quercetin, and Curcumin found in cardioprotective foods particularly with the key proteins involved in Nitric oxide signaling. Our study supports the cardioprotective implications of traditional dietary recommendations by highlighting how bioactive compounds act at the cellular and molecular levels.
Cancer is a significant cause of death worldwide. It has been suggested that the consumption of flavonoids decreases the risk for cancer by increasing phase II enzymes, such as Nicotinamide Adenine Dinucleotide Phosphate Hydrogen (NAD(P)H) quinone oxidoreductase 1 (NQO1), glutathione S-transferases, and Uridine 5'-diphospho- (UDP)-glucuronosyltransferases that assist in removing carcinogens from the human body. Flavonoids are bioactive compounds found in a variety of dietary sources, including fruits, vegetables, legumes, nuts, and teas. As such, it is important to investigate which flavonoids are involved in the metabolism of carcinogens to help reduce the risk of cancer. Therefore, the objective of this narrative review was to investigate the effects of commonly consumed flavonoids on NQO1 mRNA expression, protein, and activity in human cell and murine models. PubMed was used to search for peer-reviewed journal articles, which demonstrated that selected flavonoids (e.g., quercetin, apigenin, luteolin, genistein, and daidzein) increase NQO1, and therefore, increase the excretion of carcinogens. However, more research is needed regarding the mechanisms by which flavonoids induce NQO1. Furthermore, it is suggested that future efforts focus on providing precise flavonoid recommendations to decrease the risk factors for chronic diseases.
Bisphenol-A (BPA) is categorized as a major endocrine-disrupting chemical (EDC) used to manufacture many plastic products. BPA affects reproductive performance and promotes infertility by causing hormonal imbalance, mitochondrial dysfunction, and altered gene expression. The present investigation aimed to evaluate the effects of BPA exposure for 28 days on the activity or level of antioxidant response elements (AREs), mRNA expressions of antioxidant genes, and histomorphological changes in the ovary of adult zebrafish. The adult female zebrafish were randomly divided into four experimental groups, viz. control, vehicle (0.01% ethanol), low dose (BPA: 350 µg/L), and high dose (BPA: 700 µg/L) exposure groups. After BPA exposure in both groups, superoxide dismutase (SOD) activity and total antioxidant capacity (TAC) level were significantly (p < 0.05) decreased in the zebrafish ovary. Whereas, catalase (CAT) activity and malondialdehyde (MDA) level were significantly (p < 0.05) increased in both treatment groups. The sod mRNA expression was significantly (p < 0.05) down-regulated in the high-dose BPA-exposed group. Whereas, cat and nuclear factor erythroid 2-related factor 2 (nrf2) mRNA expressions were significantly (p < 0.05) up-regulated in both BPA-treated groups. Noticeable histomorphological alterations were recorded in the ovary of zebrafish exposed to low and high doses of BPA. The alterations in the activity of ARE, mRNA expressions of antioxidant genes, and histopathological changes suggest that exposure to BPA can cause endocrine disruption and damage to the ovary of adult zebrafish caused by oxidative stress.
Background
Among erectile dysfunction (ED) caused by metabolic abnormalities, diabetes mellitus‐induced ED (DMED) progresses rapidly, manifests with severe symptoms, and shows reduced responsiveness to conventional medications. Hyperglycemia in the corpus cavernosum has been linked to the induction of both ferroptosis and oxidative stress, which are mediated by nuclear factor E2 related factor 2 (Nrf2). Hesperidin (Hes), a flavonoid compound, has been revealed to activate Nrf2 in certain diabetic complications, yet the efficacy of Hes on DMED and the specific mechanism remain unclear.
Objectives
To elucidate the potential mechanism and efficacy of Hes in regulating Nrf2‐mediated ferroptosis and oxidative stress in DMED.
Materials and methods
DMED rats were constructed through the intraperitoneal injection of streptozotocin (STZ), partially supplemented with Hes. In parallel, in vitro research utilized human umbilical vein endothelial cells (HUVECs), with glucose addition to simulating a high glucose (HG) environment, and induced with Hes or ML385 (an Nrf2 inhibitor). Penile tissues and HUVECs were harvested for subsequent analyses.
Results
The results of this study indicate that Hes partially reversed the impaired erectile function. The expression of Nrf2, glutathione peroxidase 4 (GPX4), and heme oxygenase‐1 (HO‐1) in the corpus cavernosum elevated after supplementing with Hes, resulted in an inhibition in ferroptosis and oxidative stress. Moreover, the quantity and function of erectile effector cells were restored, and cavernous fibrosis was ameliorated. In HG‐induced HUVECs, Hes ameliorated Nrf2‐mediated ferroptosis and oxidative stress, effects which ML385 partially reversed.
Conclusions
Hes exerts a therapeutic effect on DMED rats and a regulatory mechanism on the Nrf2–HO‐1/GPX4 axis, concurrently revitalizing endothelial and smooth muscle cells, and diminishing fibrosis. Our study provides robust preclinical evidence for employing Hes in treating DMED.
Anthropogenic contamination of surface water by chemicals is a worldwide concern, which affects human health and aquatic organisms including fish. Acrylamide (ACR), a Neo-Formed Contaminants (NFC) leads to serious toxic effects on the respiratory system of marine inhabitants. The mechanisms underlying ACR-induced oxidative stress and altered mRNA expressions in zebrafish are unclear. The present investigation aimed to evaluate the effects of ACR exposure for 28 days on the activity or level of antioxidant response elements (ARE), mRNA expressions of antioxidant genes and histomorphological changes in the gills of adult zebrafish. The 270 adult zebrafish were randomly allocated into 03 experimental groups viz. control group, T1 group (ACR: 8.5 mg/L) and T2 group (ACR: 17 mg/L). After 28 days of ACR exposure, Superoxide Dismutase (SOD) activity was significantly (p<0.01) decreased in the gills of zebrafish of the T2 group; and no change in Catalase (CAT) activity was observed in both the treatment groups. The levels of reduced Glutathione (GSH) were significantly (p<0.001) decreased and Malondialdehyde (MDA) were significantly (p<0.001) increased in a concentration-dependent manner. The sod and nuclear factor erythroid 2-related factor 2 (nrf2) mRNA expressions were significantly (p<0.001) downregulated in the gills of zebrafish of both treatment groups. Noticeable histomorphological alterations were recorded in the gills of zebrafish of the T2 group. Alterations in ARE activity, mRNA expressions of antioxidant genes and histopathological findings suggest ACR exposure has been shown to produce oxidative stress-mediated damage in the gills of adult zebrafish.
Ferroptosis, a form of cell death characterized by lipid peroxidation, is involved in neurodegenerative diseases such as Alzheimer´s disease (AD). Recent studies have shown that a first-line antimalarial drug artemisinin is effective to counteract AD pathology. In this study, we investigated the protective effect of artemisinin against neuronal ferroptosis and the underlying mechanisms. In hippocampal HT22 cells, pretreatment with artemisinin dose-dependently protected against Erastin-induced cell death with an EC50 value of 5.032 µM, comparable to the ferroptosis inhibitor ferrostatin-1 (EC50 = 4.39 µM). We demonstrated that artemisinin (10 μM) significantly increased the nuclear translocation of Nrf2 and upregulated SLC7A11 and GPX4 in HT22 cells. Knockdown of Nrf2, SLC7A11 or GPX4 prevented the protective action of artemisinin, indicating that its anti-ferroptosis effect is mediated by the Nrf2-SLC7A11-GPX4 pathway. Molecular docking and Co-Immunoprecipitation (Co-IP) analysis revealed that artemisinin competitively binds with KEAP1, promoting the dissociation of KEAP1-Nrf2 complex and inhibiting the ubiquitination of Nrf2. Intrahippocampal injection of imidazole-ketone-Erastin (IKE) induced ferroptosis in mice accompanied by cognitive deficits evidenced by lower preference for exploration of new objects and new object locations in the NOR and NOL tests. Artemisinin (5, 10 mg/kg, i.p.) dose-dependently inhibited IKE-induced ferroptosis in hippocampal CA1 region and ameliorated learning and memory impairments. Moreover, we demonstrated that artemisinin reversed Aβ1-42-induced ferroptosis, lipid peroxidation and glutathione depletion in HT22 cells, primary hippocampal neurons, and 3×Tg mice via the KEAP1-Nrf2 pathway. Our results demonstrate that artemisinin is a novel neuronal ferroptosis inhibitor that targets KEAP1 to activate the Nrf2-SLC7A11-GPX4 pathway.
Redox imbalance is reported to play a pivotal role in tumorigenesis, cancer development, and drug resistance. Severe oxidative damage is a general consequence of cancer cell responses to treatment and may cause cancer cell death or severe adverse effects. To maintain their longevity, cancer cells can rescue redox balance and enter a state of resistance to anticancer drugs. Therefore, targeting redox signalling pathways has emerged as an attractive and prospective strategy for enhancing the efficacy of anticancer drugs and decreasing their adverse effects. Over the past few decades, natural products (NPs) have become an invaluable source for developing new anticancer drugs due to their high efficacy and low toxicity. Increasing evidence has demonstrated that many NPs exhibit remarkable antitumour effects, whether used alone or as adjuvants, and are emerging as effective approaches to enhance sensitivity and decrease the adverse effects of conventional cancer therapies by regulating redox balance. Among them are several novel anticancer drugs based on NPs that have entered clinical trials. In this review, we summarize the synergistic anticancer effects and related redox mechanisms of the combination of NPs with conventional anticancer drugs. We believe that NPs targeting redox regulation will represent promising novel candidates and provide prospects for cancer treatment in the future.
Graphical Abstract
Drug-induced liver injury (DILI) is the most common trigger for acute liver failure and the leading cause of attrition in drug development. In this study, we developed an in-silico framework to screen drug-induced hepatocellular toxicity (INSIGHT) by integrating the post-treatment transcriptomic data from both rodent models and primary human hepatocytes. We first built an early prediction model using logistic regression with elastic net regularization for 123 compounds and established the INSIGHT framework that can screen for drug-induced hepatotoxicity. The 235 signature genes identified by INSIGHT were involved in metabolism, bile acid synthesis, and stress response pathways. Applying the INSIGHT to an independent transcriptomic dataset treated by 185 compounds predicted that 27 compounds show a high DILI risk, including zoxazolamine and emetine. Further integration with cell image data revealed that predicted DILI compounds can induce abnormal morphological changes in the endoplasmic reticulum (ER) and mitochondrion. Clustering analysis of the treatment-induced transcriptomic changes delineated distinct DILI mechanisms induced by these compounds. Our study presents a computational framework for a mechanistic understanding of long-term liver injury and the prospective prediction of DILI.
Objectives: Long-term exposure to heat-not-burn cigarette smoke can serve as a risk factor for smoking-related diseases such as chronic obstructive pulmonary disease (COPD), similar to the exposure to conventional cigarettes. In this study, we evaluated the effects of heat-not-burn cigarettes on the alveoli and the cytokine expression in COPD muscle atrophy model mice treated with heat-not-burn cigarettes.
Methods: We used 17 wild-type mice (C57BL6, male, 12 weeks old) and divided them into heat-not-burn cigarette and control groups; next, we randomly divided them into three groups depending on whether they were subjected/not subjected to hind limb suspension. The COPD muscle atrophy mice model was developed through the intratracheal administration of a solution of heat-not-burn cigarettes along with hind limb suspension of mice. The mRNA expression levels were analyzed by real-time PCR, the morphological analysis of the histological image was obtained from general staining, the macrophage expression level was examined using immunostaining, and pulmonary function was measured using spirometry.
Results: The results indicated increases in the mean linear intercept, inflammatory cytokine expression, and macrophage count in the lungs in the COPD muscle atrophy model treated with heat-not-burn cigarettes. Besides, a decrease in the mRNA levels of optic atrophy 1 was observed during mitochondrial fusion in the muscle tissues.
Conclusions: These results indicate that the heat-not-burn model can be used as a COPD model, and that the use of heat-not-burn cigarettes affects pulmonary tissues, inflammatory cytokine expression, macrophage count, and muscle mitochondrial dynamics in the lung.
Methotrexate (MTX) is an anti-folate chemotherapeutic commonly used to treat cancer and autoimmune diseases. Despite its widespread clinical use, MTX has been linked to serious neurotoxicity side effects. Vinpocetine (VNP) has been widely used clinically to treat many neurological conditions. This study was conducted to study the potential neuroprotective effects of VNP against MTX hippocampal intoxication in rats. Thirty-two rats were randomly allocated into 4 groups: (I) control (Vehicle); (II) VNP-treated group (20 mg/kg/day, p.o); (III) MTX-control (20 mg/kg/once, i.p.) group; and (IV) the VNP + MTX group. VNP was administered orally for 10 days, during which MTX was given intraperitoneally once at the end of day 5. Our data indicated that VNP administration significantly improved MTX-induced neuronal cell death, odema, vacuolation and degeneration. VNP attenuated oxidative injury mediated by significant upregulation of the Nrf2, HO-1, and GCLC genes, while the Keap-1 mRNA expression downregulated. Moreover, VNP suppressed cytokines release mediated by increasing IκB expression level while it caused a marked downregulation in NF-κB and AP-1 (C-FOS and C-JUN) levels. Additionally, VNP attenuated apoptosis by reducing hippocampal Bax levels while increasing Bcl2 levels in MTX-intoxicated rats. In conclusion, our results suggested that VNP significantly attenuated MTX hippocampal intoxication by regulating Keap-1/Nrf2, NF-κB/AP-1, and apoptosis signaling in these effects.
Osteoarthritis (OA) is a chronic degenerative joint disease that affects worldwide. Oxidative stress plays a critical role in the chronic inflammation and OA progression. Scavenging overproduced reactive oxygen species (ROS) could be rational strategy for OA treatment. Bilirubin (BR) is a potent endogenous antioxidant that can scavenge various ROS and also exhibit anti‐inflammatory effects. However, whether BR could exert protection on chondrocytes for OA treatment has not yet been elucidated. Here, chondrocytes were exposed to hydrogen peroxide with or without BR treatment. The cell viability was assessed, and the intracellular ROS, inflammation cytokines were monitored to indicate the state of chondrocytes. In addition, BR was also tested on LPS‐treated Raw264.7 cells to test the anti‐inflammation property. An in vitro bimimic OA microenvironment was constructed by LPS‐treated Raw264.7 and chondrocytes, and BR also exert certain protection for chondrocytes by activating Nrf2/HO‐1 pathway and suppressing NF‐κB signalling. An ACLT‐induced OA model was constructed to test the in vivo therapeutic efficacy of BR. Compared to the clinical used HA, BR significantly reduced cartilage degeneration and delayed OA progression. Overall, our data shows that BR has a protective effect on chondrocytes and can delay OA progression caused by oxidative stress.
This article provides an overview of the background knowledge of ferroptosis in the nervous system, as well as the key role of nuclear factor E2-related factor 2 (Nrf2) in regulating ferroptosis. The article takes Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), and amyotrophic lateral sclerosis (ALS) as the starting point to explore the close association between Nrf2 and ferroptosis, which is of clear and significant importance for understanding the mechanism of neurodegenerative diseases (NDs) based on oxidative stress (OS). Accumulating evidence links ferroptosis to the pathogenesis of NDs. As the disease progresses, damage to the antioxidant system, excessive OS, and altered Nrf2 expression levels, especially the inhibition of ferroptosis by lipid peroxidation inhibitors and adaptive enhancement of Nrf2 signaling, demonstrate the potential clinical significance of Nrf2 in detecting and identifying ferroptosis, as well as targeted therapy for neuronal loss and mitochondrial dysfunction. These findings provide new insights and possibilities for the treatment and prevention of NDs.
Induction of phase 2 enzymes, which neutralize reactive electrophiles and act as indirect antioxidants, appears to be an effective means for achieving protection against a variety of carcinogens in animals and humans. Transcriptional control of the expression of these enzymes is mediated, at least in part, through the antioxidant response element (ARE) found in the regulatory regions of their genes. The transcription factor Nrf2, which binds to the ARE, appears to be essential for the induction of prototypical phase 2 enzymes such as glutathione S-transferases (GSTs) and NAD(P)H: quinone oxidoreductase (NQO1), Constitutive hepatic and gastric activities of CST and NQO1 were reduced by 50-80% in nrf2-deficient mice compared with wild-type mice. Moreover, the 2- to 5-fold induction of these enzymes in wild-type mice by the chemoprotective agent oltipraz, which is currently in clinical trials, was almost completely abrogated in the nrf2-deficient mice. In parallel with the enzymatic changes, nrf2-deficient mice had a significantly higher burden of gastric neoplasia after treatment with benzo[a]pyrene than did wild-type mice. Oltipraz significantly reduced multiplicity of gastric neoplasia in wild-type mice by 55%, but had no effect on tumor burden in nrf2-deficient mice. Thus, Nrf2 plays a central role in the regulation of constitutive and inducible expression of phase 2 enzymes in vivo and dramatically influences susceptibility to carcinogenesis. Moreover, the total loss of anticarcinogenic efficacy of oltipraz in the nrf2-disrupted mice highlights the prime importance of elevated phase 2 gene expression in chemoprotection by this and similar enzyme inducers.
Nrf1 is a member of the CNC-basic leucine zipper (CNC-bZIP) family of transcription factors. CNC bZIP factors, together with small Maf proteins, bind as heterodimers to the NF-E2/AP-1 element. Similarity between the NF-E2/AP-1 element and the antioxidant response element identified in a number of promoters of genes involved in detoxification and antioxidant response raises the possibility that Nrf1 plays a role in mediating the antioxidant response element response. In this study, we exploited the availability of cells from Nrf1 knockout mice to study the role of Nrf1 transcription factor in the regulation of antioxidant gene expression and in cellular antioxidant response. Fibroblast cells derived from Nrf1 null embryos showed lower levels of glutathione and enhanced sensitivity to the toxic effects of oxidant compounds. Our results indicate that Nrf1 plays a role in the regulation of genes involved in glutathione synthesis and suggest a basis for a correspondingly low GSH concentration and reduced stress response.
Nuclear factor erythroid derived 2-related factor-2 (Nrf2) is a master transcription regulator of antioxidant and cytoprotective proteins that mediate cellular defense against oxidative and inflammatory stresses. Disruption of cellular stress response by Nrf2 deficiency causes enhanced susceptibility to infection and related inflammatory diseases as a consequence of exacerbated immune-mediated hypersensitivity and autoimmunity. The cellular defense capacity potentiated by Nrf2 activation appears to balance the population of and of lymph node cells for proper innate immune responses. Nrf2 can negatively regulate the activation of pro-inflammatory signaling molecules such as p38 MAPK, NF-, and AP-1. Nrf2 subsequently functions to inhibit the production of pro-inflammatory mediators including cytokines, chemokines, cell adhesion molecules, matrix metalloproteinases, COX-2 and iNOS. Although not clearly elucidated, the antioxidative function of genes targeted by Nrf2 may cooperatively regulate the innate immune response and also repress the expression of pro-inflammatory mediators.
The molecular mechanisms by which a variety of naturally-occurring dietary compounds exert chemopreventive effects have been a subject of intense scientific investigations. Induction of phase II detoxification and anti-oxidant enzymes through activation of Nrf2/ARE-dependent gene is recognized as one of the major cellular defense mechanisms against oxidative or xenobiotic stresses and currently represents a critical chemopreventive mechanism of action. In the present review, the functional significance of Keap1/Nrf2 protein module in regulating ARE-dependent phase II detoxification and anti-oxidant gene expression is discussed. The biochemical mechanisms underlying the phosphorylation and expression of Keap1/Nrf2 proteins that are controlled by the intracellular signaling kinases and ubiquitin-mediated E3 ligase system as well as control of nucleocytoplasmic translocation of Nrf2 by its innate nuclear export signal (NES) are described.
Activated macrophages express high levels of Nrf2, a transcription factor that positively regulates the gene expression of
antioxidant and detoxication enzymes. In this study, we examined how Nrf2 contributes to the anti-inflammatory process. As
a model system of acute inflammation, we administered carrageenan to induce pleurisy and found that in Nrf2-deficient mice,
tissue invasion by neutrophils persisted during inflammation and the recruitment of macrophages was delayed. Using an antibody
against 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2), it was observed that macrophages from pleural lavage accumulate 15d-PGJ2. We show that in mouse peritoneal macrophages 15d-PGJ2 can activate Nrf2 by forming adducts with Keap1, resulting in an Nrf2-dependent induction of heme oxygenase 1 and peroxiredoxin
I (PrxI) gene expression. Administration of the cyclooxygenase 2 inhibitor NS-398 to mice with carrageenan-induced pleurisy
caused persistence of neutrophil recruitment and, in macrophages, attenuated the 15d-PGJ2 accumulation and PrxI expression. Administration of 15d-PGJ2 into the pleural space of NS-398-treated wild-type mice largely counteracted both the decrease in PrxI and persistence of
neutrophil recruitment. In contrast, these changes did not occur in the Nrf2-deficient mice. These results demonstrate that
Nrf2 regulates the inflammation process downstream of 15d-PGJ2 by orchestrating the recruitment of inflammatory cells and regulating the gene expression within those cells.
Korean red ginseng (KRG) is used worldwide as a popular traditional herbal medicine. KRG has shown beneficial effects on cardiovascular diseases, such as atherosclerosis, diabetes, and hypertension. Up-regulation of a cytoprotective protein, heme oxygenase (HO)-1, is considered to augment the cellular defense against various agents that may induce cytotoxic injury. In the present study, we demonstrate that KRG water extract induces HO-1 expression in human umbilical vein endothelial cells (HUVECs) and possible involvement of the anti-oxidant transcription factor nuclear factor-eythroid 2-related factor 2 (Nrf2). KRG-induced HO-1 expression was examined by western blots, reverse transcriptase polymerase chain reaction and immunofluorescence staining. Specific silencing of Nrf2 genes with Nrf2-siRNA in HUVECs abolished HO-1 expression. In addition, the HO inhibitor zinc protoporphyrin blunted the preventive effect of KRG on H2O2-induced cell death, as demonstrated by terminal transferase dUTP nick end labeling assay. Taken together, these results suggest that KRG may exert a vasculoprotective effect through Nrf2- mediated HO-1 induction in human endothelial cell by inhibition of cell death.
The GABPB1 gene A/G polymorphism in Polish rowers
Nuclear respiratory factor 2 (NRF2), also referred to as the GA-binding protein (GABP) transcription factor, is a key transcriptional activator of many nuclear genes which encode a wide range of mitochondrial enzymes. The variants of the GABPB1 gene encoding the beta1 subunit of NRF2 protein have been associated with physical performance, particularly endurance. The aim of this study was to confirm the possible importance of the A/G polymorphism (rs7181866) in intron 3 of the GABPB1 gene in Polish rowers. The study was carried out on 55 Polish rowers and sedentary individuals, to evaluate the possible relationships between genotype and physical performance. DNA was extracted from buccal cells donated by the subjects. Genotyping was carried out by PCR-RFLP. The results revealed that the frequency of the GABPB1 A/G genotype (89.09% AA; 10.91% AG, 0% GG; vs. 97.69% AA; 2.31% AG; 0.00% GG) %; P = 0.012) and G allele (5.50% vs. 1.17%; P = 0.014) was significantly higher in the rowers compared to controls. The results suggest that the GABPB1 gene can be taken into consideration as a genetic marker in endurance athletes. However, these conclusions should be supported with more experimental studies on other GABPB1 polymorphisms and other genes in elite endurance athletes.
Induction of phase 2 enzymes, which neutralize reactive
electrophiles and act as indirect antioxidants, appears to be an
effective means for achieving protection against a variety of
carcinogens in animals and humans. Transcriptional control of the
expression of these enzymes is mediated, at least in part, through the
antioxidant response element (ARE) found in the regulatory regions of
their genes. The transcription factor Nrf2, which binds to the ARE,
appears to be essential for the induction of prototypical phase 2
enzymes such as glutathione S-transferases (GSTs) and
NAD(P)H:quinone oxidoreductase (NQO1). Constitutive hepatic and
gastric activities of GST and NQO1 were reduced by 50–80% in
nrf2-deficient mice compared with wild-type mice.
Moreover, the 2- to 5-fold induction of these enzymes in wild-type mice
by the chemoprotective agent oltipraz, which is currently in clinical
trials, was almost completely abrogated in the
nrf2-deficient mice. In parallel with the enzymatic
changes, nrf2-deficient mice had a significantly higher
burden of gastric neoplasia after treatment with
benzo[a]pyrene than did wild-type mice. Oltipraz
significantly reduced multiplicity of gastric neoplasia in wild-type
mice by 55%, but had no effect on tumor burden in
nrf2-deficient mice. Thus, Nrf2 plays a central role in
the regulation of constitutive and inducible expression of phase 2
enzymes in vivo and dramatically influences
susceptibility to carcinogenesis. Moreover, the total loss of
anticarcinogenic efficacy of oltipraz in the
nrf2-disrupted mice highlights the prime importance of
elevated phase 2 gene expression in chemoprotection by this and similar
enzyme inducers.
The mouse heme oxygenase-1 (HO-1) gene,ho-1, contains two inducible enhancers, E1 and E2. Of several cell lines tested, induction of an E1/luciferase fusion construct,
pE1-luc, by CdCl2 is most pronounced in MCF-7 cells. In these cells, E1, but not E2, is necessary and sufficient for ho-1 gene activation. Exposure of MCF-7 cells to 10 μm CdCl2 stimulates phosphorylation of ERK, JNK, and p38 mitogen-activated protein kinases, implicating one or more of these signaling
pathways in ho-1gene induction. SB203580, an inhibitor of p38, diminishes cadmium-stimulated pE1-luc expression and HO-1 mRNA levels by up
to 70–80%. PD098059, an ERK pathway inhibitor, does not affect HO-1 mRNA induction at the highest concentration (40 μm) tested. Similarly, co-expression of a dominant-negative mutant of p38α, but not of ERK1, ERK2, JNK1, or JNK2, reduces basal
and cadmium-induced pE1-luc activity. E1 contains binding sites for the activator protein-1 (Fos/Jun), Cap‘n’Collar/basic
leucine zipper (CNC-bZIP), and CCAAT/enhancer-binding protein (C/EBP) families of transcription factors. A dominant-negative
mutant of Nrf2 (a CNC-bZIP member), but not of c-Jun or C/EBPβ, inhibits pE1-luc activation by cadmium. Induction of the endogenous
ho-1gene is also inhibited by the Nrf2 mutant. Mutations of E1 that inhibit cadmium inducibility also suppress thetrans-activation and DNA binding activities of Nrf2, and SB203580, but not PD098059, attenuates Nrf2-mediatedtrans-activation of pE1-luc. Taken together, these results indicate that cadmium induces ho-1 gene expression via sequential activation of the p38 kinase pathway and Nrf2.
The NF-E2-binding sites or Mafrecognition elements (MARE) are essentialcis-acting elements in the regulatory regions of erythroid-specific genes recognized by the erythroid transcription factor NF-E2,
composed of p45 and MafK. Recently, two p45-related factors Nrf1 and Nrf2 were isolated, and they are now collectively grouped
as the Cap’n’ collar (CNC) family. CNC factors bind to MARE through heterodimer formation with small Maf proteins. We report
here the identification and characterization of a novel CNC factor, Nrf3, encoding a predicted 73-kDa protein with a basic
region-leucine zipper domain highly homologous to those of other CNC proteins. In vitro and in vivo analyses showed that Nrf3 can heterodimerize with MafK and that this complex binds to the MARE in the chicken β-globin enhancer
and can activate transcription. Nrf3 mRNA is highly expressed in human placenta and B cell and monocyte lineage. Chromosomal
localization of human Nrf3 is 7p14–15, which lies near the hoxA gene locus. As the genetic loci of p45, nrf1, andnrf2 have been mapped close to those ofhoxC, hoxB, and hoxD, respectively, the present study strongly argues for the idea that a single ancestral gene for the CNC family members may
have been localized near the ancestral Hox cluster and have diverged to give rise to four closely related CNC factors through
chromosome duplication.
The liver is a major organ responsible for most functions of cellular metabolism and a mediator between dietary and endogenous sources of energy for extrahepatic tissues. In this context, adenosine-monophosphate- (AMP-) activated protein kinase (AMPK) constitutes an intrahepatic energy sensor regulating physiological energy dynamics by limiting anabolism and stimulating catabolism, thus increasing ATP availability. This is achieved by mechanisms involving direct allosteric activation and reversible phosphorylation of AMPK, in response to signals such as energy status, serum insulin/glucagon ratio, nutritional stresses, pharmacological and natural compounds, and oxidative stress status. Reactive oxygen species (ROS) lead to cellular AMPK activation and downstream signaling under several experimental conditions. Thyroid hormone (L-3,3′,5-triiodothyronine, T
3
) administration, a condition that enhances liver ROS generation, triggers the redox upregulation of cytoprotective proteins affording preconditioning against ischemia-reperfusion (IR) liver injury. Data discussed in this work suggest that T
3
-induced liver activation of AMPK may be of importance in the promotion of metabolic processes favouring energy supply for the induction and operation of preconditioning mechanisms. These include antioxidant, antiapoptotic, and anti-inflammatory mechanisms, repair or resynthesis of altered biomolecules, induction of the homeostatic acute-phase response, and stimulation of liver cell proliferation, which are required to cope with the damaging processes set in by IR.
The Kelch-like ECH-associated protein 1 (Keap1)-NF-E2-related factor 2 (Nrf2) system is essential for cytoprotection against oxidative and electrophilic insults. Under unstressed conditions, Keap1 serves as an adaptor for ubiquitin E3 ligase and promotes proteasomal degradation of Nrf2, but Nrf2 is stabilized when Keap1 is inactivated under oxidative/electrophilic stress conditions. Autophagy-deficient mice show aberrant accumulation of p62, a multifunctional scaffold protein, and develop severe liver damage. The p62 accumulation disrupts the Keap1-Nrf2 association and provokes Nrf2 stabilization and accumulation. However, individual contributions of p62 and Nrf2 to the autophagy-deficiency-driven liver pathogenesis have not been clarified. To examine whether Nrf2 caused the liver injury independent of p62, we crossed liver-specific Atg7::Keap1-Alb double-mutant mice into p62- and Nrf2-null backgrounds. Although Atg7::Keap1-Alb::p62(-/-) triple-mutant mice displayed defective autophagy accompanied by the robust accumulation of Nrf2 and severe liver injury, Atg7::Keap1-Alb::Nrf2(-/-) triple-mutant mice did not show any signs of such hepatocellular damage. Importantly, in this study we noticed that Keap1 accumulated in the Atg7- or p62-deficient mouse livers and the Keap1 level did not change by a proteasome inhibitor, indicating that the Keap1 protein is constitutively degraded through the autophagy pathway. This finding is in clear contrast to the Nrf2 degradation through the proteasome pathway. We also found that treatment of cells with tert-butylhydroquinone accelerated the Keap1 degradation. These results thus indicate that Nrf2 accumulation is the dominant cause to provoke the liver damage in the autophagy-deficient mice. The autophagy pathway maintains the integrity of the Keap1-Nrf2 system for the normal liver function by governing the Keap1 turnover.
Background Nrf2 belongs to the Cap-N-Collar (CNC) transcription factor family and is essential for the antioxidant responsive element (ARE)-mediated expression of a group of detoxifying and antioxidant genes. The forced expression of Nrf2 in mammalian cells activates the expression of target genes through the ARE, with Nrf2 showing the highest transactivation activity among the CNC family of transcription factors. To elucidate the molecular mechanisms generating this potent transactivation activity, we examined the functions of the domains within Nrf2.
Result We found that Nrf2 contains two transcription activation domains, Neh4 and Neh5, which act synergistically to attain maximum a activation of reporter gene expression. Neh4 and Neh5 both individually and cooperatively bind to CBP (CREB (cAMP Responsive Element Binding protein) Binding Protein). In fact, the specific inhibitor of CBP, adenovirus E1A protein, significantly reduced Nrf2 activity. Importantly, the CBP-binding activity of Nrf2 deletion mutants positively correlated with their transactivation activity. Neh5 contains a motif which is commonly conserved among the CNC factors, whereas Neh4 contains the novel CBP-interacting motif recently identified in p53 and E2F.
Conclusions Our results indicate that Nrf2 exploits the cooperative binding of two independent transactivation domains to CBP in the acquisition of a potent transactivation activity.
Recent studies have revealed two new functions of prothymosin α (ProTα), a well-known protein and a subject of intense research.
In addition to acting as an immunomodulator and stimulating cell proliferation, ProTα is involved in protecting the cell from
apoptosis and regulating the expression of oxidative stress defense genes. The review considers the methods and approaches
used to demonstrate the two new functions of ProTα.
A model for the redox regulation of the functional state of the cell has been constructed on the basis of representation of
electron transfer processes by equivalent electric circuits. The mechanism of action of redox-active molecules on biosystems
has been discussed in terms of circuit theory. A method for determining the parameters of cellular redox sensors has been
proposed. It has been established that the concentration and redox potential of compounds entering the cell are the main regulatory
parameters of redox signals for the cell. It has been experimentally shown that the calcium response to hydrogen peroxide
in rat C6 glioma cells and human FL amnion cells depends on the redox-buffer capacity of cells.
Keywordsredox state–redox regulation–oxidative stress–effective redox potential–redox-buffer capacity–reactive oxygen species–2′,7′-dichlorodihydrofluorescein
Oxidative stress is integral to the development of endothelial dysfunction and cardiovascular disease. As NRF2 is a key transcription factor in antioxidant defense, we aimed to determine whether polymorphisms within the promoter region of the gene encoding NRF2 (NFE2L2) would significantly modify vasodilator responses in humans.
Associations between the -653A/G (rs35652124), -651G/A (rs6706649), and -617C/A (rs6721961) polymorphisms within the NFE2L2 promoter and vascular function were evaluated in healthy African-American (n=64) and white (n=184) individuals. Forearm blood flow (FBF) was measured by strain-gauge venous occlusion plethysmography at baseline and in response to incremental doses of bradykinin or sodium nitroprusside. Forearm vascular resistance (FVR) was calculated as the mean arterial pressure/FBF.
In African Americans, -653G variant allele carriers had significantly lower FBF and higher FVR under basal conditions as well as in response to bradykinin or sodium nitroprusside compared with wild-type individuals (P<0.05 for each comparison). In whites, although no significant associations were observed with the -653A/G genotype, -617A variant allele carriers had significantly higher FVR at baseline and in response to bradykinin or sodium nitroprusside compared with wild-type individuals (P<0.05 for each comparison). The -651G/A polymorphism was not associated with vasodilator responses in either racial group.
Polymorphisms within the NFE2L2 promoter were associated with impaired forearm vasodilator responses in an endothelial-independent manner, suggesting an important role of NRF2 in the regulation of vascular function in humans.
L-3,3',5-triiodothyronine (T(3)) administration upregulates nuclear factor-E2-related factor 2 (Nrf2) in rat liver, which is redox-sensitive transcription factor mediating cytoprotection. In this work, we studied the role of Kupffer cell respiratory burst activity, a process related to reactive oxygen species generation and liver homeostasis, in Nrf2 activation using the macrophage inactivator gadolinium chloride (GdCl(3); 10 mg/kg i.v. 72 h before T(3) [0.1 mg/kg i.p.]) or NADPH oxidase inhibitor apocynin (1.5 mmol/L added to the drinking water for 7 days before T(3)), and determinations were performed 2 h after T(3). T(3) increased nuclear/cytosolic Nrf2 content ratio and levels of heme oxygenase 1 (HO-1), catalytic subunit of glutamate cysteine ligase, and thioredoxin (Western blot) over control values, proteins whose gene transcription is induced by Nrf2. These changes were suppressed by GdCl(3) treatment prior to T(3), an agent-eliciting Kupffer-cell depletion, inhibition of colloidal carbon phagocytosis, and the associated respiratory burst activity, with enhancement in nuclear inhibitor of Nrf2 kelch-like ECH-associated protein 1 (Keap1)/Nrf2 content ratios suggesting Nrf2 degradation. Under these conditions, T(3)-induced tumor necrosis factor-α (TNF-α) response was eliminated by previous GdCl(3) administration. Similar to GdCl(3), apocynin given before T(3) significantly reduced liver Nrf2 activation and HO-1 expression, a NADPH oxidase inhibitor eliciting abolishment of colloidal carbon-induced respiratory burst activity without altering carbon phagocytosis. It is concluded that Kupffer cell functioning is essential for upregulation of liver Nrf2-signaling pathway by T(3). This contention is supported by suppression of the respiratory burst activity of Kupffer cells and the associated reactive oxygen species production by GdCl(3) or apocynin given prior to T(3), thus hindering Nrf2 activation.
The role of cellular redox potential in the regulation of protein activity is becoming increasingly appreciated and characterized. In this paper we put forward a new hypothesis relating to redox regulation of cellular physiology. We have exemplified our hypothesis using apoptosis since its redox phenomenology is well established, but believe that it is equally applicable to several other pathways. Our hypothesis is that since multiple proteins in the apoptosis pathway are thought to be regulated via oxidation/reduction reactions and since cellular redox potentials have been shown to become progressively more oxidative during apoptosis, that the proteins could be arranged in an electrochemical series where the protein's standard potential correlates with its position in the pathway. Since the most stable oxidation state of the protein is determined by its standard potential and the redox potential of its environment (in a way predictable by the Nernst equation), a quantitative model of the redox regulation of the pathway could be developed. We have outlined our hypothesis, illustrating it using a pathway map which assembles a selection of the literature on apoptosis into a readable graphical format. We have also outlined experimental approaches suitable for testing our hypothesis.
Abstract Nuclear-factor-E2-related transcription factor 2 (Nrf2) regulates a large panel of Phase II genes and plays an important role in cell survival. Nrf2 activation has been shown as preventing cigarette smoke-induced alveolar enlargement in mice. Therefore, activation of the Nrf2 protein by small-molecule activators represents an attractive therapeutic strategy that is used for chronic obstructive pulmonary disease. In this article, we describe a cell-based luciferase enzyme fragment complementation assay that identifies Nrf2 activators. This assay is based on the interaction of Nrf2 with its nuclear partner MafK or runt-related transcription factor 2 (RunX2) and is dependent on the reconstitution of a "split" luciferase. Firefly luciferase is split into two fragments, which are genetically fused to Nrf2 and MafK or RunX2, respectively. BacMam technology was used to deliver the fusion constructs into cells for expression of the tagged proteins. When the BacMam-transduced cells were treated with Nrf2 activators, the Nrf2 protein was stabilized and translocated into the nucleus where it interacted with MafK or RunX2. The interaction of Nrf2 and MafK or RunX2 brought together the two luciferase fragments that form an active luciferase. The assay was developed in a 384-well format and was optimized by titrating the BacMam concentration, transduction time, cell density, and fetal bovine serum concentration. It was further validated with known Nrf2 activators. Our data show that this assay is robust, sensitive, and amenable to high throughput screening of a large compound collection for the identification of novel Nrf2 activators.
NF-E2 binding sites, located in distant regulatory sequences, may be important for high level α- and β-globin gene expression. Surprisingly, targeted disruption of each subunit of NF-E2 has either little or no effect on erythroid maturation in mice. For p18 NF-E2, this lack of effect is due, at least in part, to the presence of redundant proteins. For p45 NF-E2, one possibility is that NF-E2–related factors, Nrf-1 or Nrf-2, activate globin gene expression in the absence of NF-E2. To test this hypothesis for Nrf-2, we disrupted the Nrf-2 gene by homologous recombination. Nrf-2–deficient mice had no detectable hematopoietic defect. In addition, no evidence was found for reciprocal upregulation of NF-E2 or Nrf-2 protein in fetal liver cells deficient for either factor. Fetal liver cells deficient for both NF-E2 and Nrf-2 expressed normal levels of α- and β-globin. Mature mice with combined deficiency of NF-E2 and Nrf-2 did not exhibit a defect in erythroid maturation beyond that seen with loss of NF-E2 alone. Thus, the presence of a mild erythroid defect in NF-E2–deficient mice is not the result of compensation by Nrf-2.
γ-Glutamylcysteine synthetase (GCS; also referred to as glutamate-cysteine ligase, GLCL) catalyzes the rate-limiting reaction in glutathione (GSH) biosynthesis. The GCS holoenzyme is composed of a catalytic and regulatory subunit, each encoded by a unique gene. In addition to some conditions which specifically upregulate the catalytic subunit gene, expression of both genes is increased in response to many Phase II enzyme inducers including oxidants, heavy metals, phenolic antioxidants and GSH-conjugating agents. Electrophile Response Elements (EpREs), located in 5′-flanking sequences of both the GCSh and GCSl subunit genes, are hypothesized to at least partially mediate gene induction following xenobiotic exposure. Recent experiments indicate that the bZip transcription factor Nrf2 participates in EpRE-mediated GCS subunit gene activation in combination with other bZip proteins. An AP-1-like binding sequence and an NF-κB site have also been implicated in regulation of the catalytic subunit gene following exposure to certain pro-oxidants. Potential signaling mechanisms mediating GCS gene induction by the diverse families of Phase II enzyme inducers include thiol modification of critical regulatory sensor protein(s) and the generation of the reactive oxygen species. This review summarizes recent progress in defining the molecular mechanisms operative in transcriptional control of the genes encoding the two GCS subunits, identifying areas of agreement and controversy. The mechanisms involved in GCS regulation might also be relevant to the transcriptional control of other components of the antioxidant defense battery.
Nrf2-deficient female mice develop lupus-like autoimmune nephritis.BackgroundNF-E2–related factor 2 (Nrf2) is a basic leucine zipper transcriptional activator essential for the coordinate transcriptional induction of antioxidant enzymes and phase II drug metabolizing enzymes through the antioxidant response element/electrophile response element. The Nrf2-deficient mice were found to develop normally under standard laboratory conditions. However, upon closer examination, we found that aged female Nrf2-deficient mice displayed a shortened lifespan and developed severe glomerulonephritis. The present study investigated the glomerulonephritis findings in Nrf2-deficient mice.Methods
To evaluate glomerular lesions of Nrf2-deficient mice, histological and functional analyses were performed. The amounts of serum immunoglobulins, anti-double-stranded (ds) DNA antibody, and lipid peroxidation using thiobarbituric acid reactive substances (TBARS) also were measured.ResultsNrf2-deficient female mice over 60 weeks of age developed severe nephritis characterized by cellular proliferation, lobular formation, crescent formation, and subepithelial electron-dense deposits. In immunofluorescent assays, Nrf2-deficient female mice showed mesangial deposits and massive granular deposits of IgG, IgM, and C3 along the capillary walls. Higher serum levels of IgG, anti-dsDNA antibody, lower creatinine clearance, and slight splenomegaly also were found in Nrf2-deficient female mice. A higher concentration of TBARS also was found in Nrf2-deficient female mice.Conclusions
These data indicate that the aged Nrf2-deficient female mice develop lupus-like autoimmune nephritis and suggest that nrf2 is one of the genes determining susceptibility to autoimmune disease. Analysis of nephritis in the Nrf2-deficient female mouse may clarify the mechanisms leading to the development of lupus disease.
The antioxidant responsive element (ARE) mediates transcriptional regulation of phase II detoxification en- zymes and antioxidant proteins such as NAD(P)H:qui- none oxidoreductase (NQO1), glutathione S-trans- ferases, and glutamate-cysteine ligase. In this study, we demonstrate that NF-E2-related factor-2 (Nrf2) plays a major role in transcriptional activation of ARE-driven genes and identify Nrf2-dependent genes by oligonu- cleotide microarray analysis using primary cortical as- trocytes from Nrf2/ and Nrf2/ mice. Nrf2/ astro- cytes had decreased basal NQO1 activity and no induction by tert-butylhydroquinone compared with Nrf2/ astrocytes. Similarly, both basal and induced levels of human NQO1-ARE-luciferase expression in Nrf2/ astrocytes were significantly lower than in Nrf2/ astrocytes. Furthermore, human NQO1-ARE-lu- ciferase expression in Nrf2/ astrocytes was restored by overexpression of Nrf2, whereas ARE activation in Nrf2/ astrocytes was completely blocked by dominant- negative Nrf2. In addition, we observed that Nrf2-de- pendent genes protected primary astrocytes from H2O2- or platelet-activating factor-induced apoptosis. In sup- port of these observations, we identified Nrf2-depend- ent genes encoding detoxification enzymes, glutathi- one-related proteins, antioxidant proteins, NADPH- producing enzymes, and anti-inflammatory genes using oligonucleotide microarrays. Proteins within these functional categories are vital to the maintenance and responsiveness of a cell defense system, suggesting that an orchestrated change in gene expression via Nrf2 and the ARE gives a synergistic protective effect against oxidative stress.
Transcription factor AP-1 is a dimer formed by DNA-binding proteins of the Jun, Fos, and ATF families (activating transcription
factor). AP-1 mediates the cell response to growth factors, cytokines, neurotransmitters, and other intercellular signaling
molecules. Regulation of AP-1 activity involves G-proteins, adapter proteins, MAP kinases, and other components of cell regulatory
systems. Genes responsive to AP-1 play an important role in regulating cell proliferation, morphogenesis, apoptosis, and differentiation.
Transcription factor NF-E2-related factor 2 (NRF2) plays a crucial role in the cellular defense against oxidative/electrophilic stress by up-regulating multiple antioxidant genes. Numerous studies with genetically modified animals have demonstrated that Nrf2 is a sensitivity determining factor upon the exposure to environmental chemicals including carcinogens. Moreover, recent studies have demonstrated that polymorphisms in the human NRF2 promoter is associated with higher risks for developing acute lung injury, gastric mucosal inflammatory, and nephritis. Therefore, the identification of reliable and effective human target genes of NRF2 may allow the monitoring of NRF2 activity and to predict individual sensitivity to environmental stress-induced damage. For this purpose, we investigated genes that are tightly controlled by NRF2 to establish markers for NRF2 activity in human cells. Firstly, in the normal human renal epithelial HK-2 cells, the measurement of the expression of 30 previously reported NRF2 target genes in response to NRF2 inducers (sulforaphane, tert-butylhydroquinone, cinnamic aldehyde, and hydrogen peroxide) showed that the aldo-keto reductase (AKR) 1C1 is highly inducible by all treatments. Accordantly, the basal and inducible expression of AKRs was significantly attenuated in NRF2-silenced HK-2 cells. Whereas, cells with stable KEAP1 knockdown, which causes a modest NRF2 activation, demonstrated substantially increased levels of AKR1A1, 1B1, 1B10, 1C1, 1C2, and 1C3. Secondly, the linkage between NRF2 and the AKRs was confirmed in human monocytic leukemia cell line U937, which can be a model of peripherally available blood cells. The treatment of U937 cells with NRF2 inducers including sulforaphane effectively elevated the expression of AKR1B1, 1B10, 1C1, 1C2, and 1C3. Whereas, the levels of both the basal and sulforaphane-inducible expression of AKR1C1 were significantly reduced in NRF2-silenced stable U937 cells compared to the control cells. Similarly, the inducible expression of AKR1C1 was observed in another human monocytic leukemia cell line THP-1 as well as in human primary blood CD14(+) monocytes. In conclusion, together with the high inducibility and NRF2 dependency shown in renal epithelial cells as well as in peripherally available blood cells, current findings suggest that AKRs can be utilized as a marker of NRF2 activity in human cells.
Lead is linked to many reproductive problems. This study was to explore the chronic effects of low lead level on expressions of Nrf2 and Mrp1 in rats' testes. Maternal SD rats were administered lead acetate from 10 days before gestation to weaning at three doses respectively after randomization. From each group, 15 male offsprings were then chosen and administrated lead acetate from weaning to six months old at the doses of 0, 0.3 and 0.9g/L respectively. The dose administrations were through drinking water freely. The methods of RT-PCR, Western blotting and immunohistochemistry were used for Mrp1 and Nrf2 of the testes. Compared with control group, significant increases were observed in the expressions of Mrp1 and Nrf2 in two lead groups (P<0.05); nucleus translocation of Nrf2 was observed; both GST and GSH was decreased with increasing the lead dose. In conclusion, Mrp1 might play important roles in lead detoxification by Nrf2.
Cysteine residues on proteins play key roles in catalysis and regulation. These functional cysteines serve as active sites for nucleophilic and redox catalysis, sites of allosteric regulation and metal-binding ligands on proteins from diverse classes including proteases, kinases, metabolic enzymes and transcription factors. In this article, we focus on a few select examples that serve to highlight the multiple functions performed by cysteines, with an emphasis on cysteine-mediated protein activities implicated in cancer. The enhanced reactivity of functional cysteines renders them susceptible to modification by electrophilic species. Towards this end, we discuss recent advancements and future prospects for utilizing cysteine-reactive small molecules as drugs and imaging agents for the treatment and diagnosis of cancer.
Chemoprevention refers to the use of agents to inhibit, reverse or retard tumorigenesis. Numerous phytochemicals derived from edible plants have been reported to interfere with a specific stage of the carcinogenic process. Many mechanisms have been shown to account for the anticarcinogenic actions of dietary constituents, but attention has recently been focused on intracellular-signalling cascades as common molecular targets for various chemopreventive phytochemicals.
Activation of protein kinase C (PKC) is a critical intracellular signaling triggered by ischemic preconditioning (IPC), but the precise mechanisms underlying the actions of PKC in IPC-mediated cardioprotection remain unclear. Here, we investigated the role of PKC activation on the antioxidant activity by IPC in rabbit hearts. Isolated rabbit hearts were subjected to 60 min of global ischemia by cold cardioplegic arrest (4 °C) and 60 min of reperfusion (37 °C). IPC was induced by three cycles of 2-min ischemia following 3 min of reperfusion (37 °C) before cardioplegic arrest. IPC resulted in a better recovery of mechanical function, increased tissue reduced glutathione-to-oxidized glutathione ratio (GSH/GSSG), superoxide dismutase and catalase content, and decreased tissue malondialdehyde (MDA) content compared to control hearts subjected to 60 min of cardioplegic ischemia and 60 min of reperfusion. IPC also significantly induced activation of nuclear factor erythroid 2-related factor 2 (Nrf2) and the inductions of antioxidant genes heme oxygenase-1 (HO-1) and manganese superoxide dismutase (MnSOD). Injection of phorbol 12-myristate 13 acetate, an activator of PKC, before cardioplegic ischemia induced translocation of PKC-δ and -ε isoforms to membrane fraction, nuclear accumulation of Nrf2, and conferred cardioprotection similar to IPC. Polymyxin B, an inhibitor of PKC, blocked the membrane translocation of PKC-δ and -ε during IPC, inhibited Nrf2 nuclear accumulation, and significantly diminished the IPC-induced cardioprotection when administrated before IPC. These results indicate that the activation of PKC induces the translocation of Nrf2 and the enhancement of endogenous antioxidant defenses in the IPC hearts and suggest that PKC may target Nrf2 to confer cardioprotection.
The role of oxidative stress in ischaemic heart disease has been thoroughly investigated in humans. Increased levels of ROS (reactive oxygen species) and RNS (reactive nitrogen species) have been demonstrated during ischaemia and post-ischaemic reperfusion in humans. Depending on their concentrations, these reactive species can act either as benevolent molecules that promote cell survival (at low-to-moderate concentrations) or can induce irreversible cellular damage and death (at high concentrations). Although high ROS levels can induce NF-κB (nuclear factor κB) activation, inflammation, apoptosis or necrosis, low-to-moderate levels can enhance the antioxidant response, via Nrf2 (nuclear factor-erythroid 2-related factor 2) activation. However, a clear definition of these concentration thresholds remains to be established. Although a number of experimental studies have demonstrated that oxidative stress plays a major role in heart ischaemia/reperfusion pathophysiology, controlled clinical trials have failed to prove the efficacy of antioxidants in acute or long-term treatments of ischaemic heart disease. Oral doses of vitamin C are not sufficient to promote ROS scavenging and only down-regulate their production via NADPH oxidase, a biological effect shared by vitamin E to abrogate oxidative stress. However, infusion of vitamin C at doses high enough to achieve plasma levels of 10 mmol/l should prevent superoxideproduction and the pathophysiological cascade of deleterious heart effects. In turn, n-3 PUFA (polyunsaturated fatty acid) exposure leads to enhanced activity of antioxidant enzymes. In the present review, we present evidence to support themolecular basis for a novel pharmacological strategy using these antioxidant vitamins plus n-3 PUFAs for cardioprotection in clinical settings, such as post-operative atrial fibrillation, percutaneous coronary intervention following acutemyocardial infarction and other events that are associated with ischaemia/reperfusion.
Prenatal arsenic exposure accelerates atherosclerosis in ApoE(-/-) mice by unknown mechanism. Arsenic is an hepatotoxicant, and liver disease increases atherosclerosis risk. Prenatal arsenic exposure may predispose to liver disease by priming for susceptibility to other environmental insults. Earlier microarray analyses showed prenatal arsenic exposure increased Hsc70 (HspA8) and Hsp70 (HspA1a) mRNAs in livers of 10 week old mice. We determined effects of prenatal arsenic exposure on hepatic Hsp70 and Hsc70 expression by western blot and on DNA methylation by methyl acceptance assay during prenatal and postnatal development. Pregnant ApoE(-/-) mice were given drinking water containing 85 mg/L NaAsO(2) (49 ppm arsenic) from gestation day (GD) 8 - 18. Hsp70 and Hsc70 expression and DNA methylation were determined in GD18 fetuses, and 3, 10 and 24 week old mice. Hsc70 expression was unchanged at all ages. Hsp70 induction was observed at 3 and 10 weeks, but was unchanged in GD18 and 24 week livers. Global DNA methylation increased with age; arsenic had no effects. Bisulfite sequencing of DNA from livers of 10 week old mice showed Hsp70 promoter region methylation was unchanged, but methylation was increased within the transcribed region. Hsf1 and Nrf2 nuclear translocation were investigated as potential mechanisms of Hsp70 induction, and found unaltered. Putative binding sites were identified in HSP70 for in utero arsenic exposure suppressed microRNAs suggesting a possible mechanism. Thus, prenatal arsenic exposure causes delayed temporal hepatic Hsp70 induction, suggesting a transient state of stress in livers which can predispose the mice to developing liver disease.
Enhanced level of nuclear erythroid-related factor-2 (Nrf2) has been associated with cancer chemo/radioresistance. Therefore, the role of Nrf2 in radiosensitization of malignant cells induced by a combination of 2-deoxy-D-Glucose (2-DG) and 6-aminonicotinamide (6-AN) was investigated. Two established human malignant cells lines namely KB (head and neck squamous carcinoma) and BMG-1 (cerebral glioma) were used. Following treatment with a combination of 2-DG (5 mM) and 6-AN (5 μM), irradiated (2Gy) KB and BMG-1 cells were assessed for protein level of Nrf2, Keap1 and γ-glutamylcysteine synthetase (γ-GCS) by western blotting and mRNA expression of γ-GCS, glutathione reductase (GR) and glutathione peroxidase (GPx1) by RT-PCR at 24 hours post treatment. A significant decrease in the level of Nrf2 with a concomitant increase in Keap1 was observed in both the irradiated malignant cells at 24 hours following treatment with combination (2-DG + 6-AN). Down regulation of γ-GCS, GR and GPx1 at 24 hours following treatment with combination (2-DG + 6-AN) resulted in abrogation of glutathione (GSH)-mediated defense in both the irradiated malignant cells. Eventual accumulation of ROS led to radiosensitization of both the malignant cells. These results indicate that deregulated Nrf2-Keap1 signalling leads to the radiosensitization of malignant cells due to abrogated glutathione defense. Metabolic modification-mediated down regulation of Nfr2 and its downstream signalling may have a potential of improving tumour radiotherapy.
Oxidative stress can result in insulin resistance, a primary cause of type-2 diabetes. Methylglyoxal (MG), a highly reactive dicarbonyl metabolite generated during glucose metabolism, has also been confirmed to cause pancreatic injury and induce inflammation, thereby resulting in insulin resistance. Recently, resveratrol has been reported to exert antioxidant properties, protecting cells from the generation of reactive oxygen species (ROS). The aim of this study was to evaluate resveratrol activation of nuclear factor erythroid 2-related factor 2 (Nrf2) to attenuate MG-induced insulin resistance in Hep G2 cells. Therefore, the molecular signaling events affecting resveratrol-mediated heme oxygenase-1 (HO-1) and glyoxalase expression levels were further investigated in this study. Our findings indicated that resveratrol activated the extracellular signal-regulated kinase (ERK) pathway but not the p38 or c-Jun N-terminal kinase (JNK) pathways, subsequently leading to Nrf2 nuclear translocation and elevation of HO-1 and glyoxalase expression levels. Moreover, resveratrol significantly elevated glucose uptake and protected against MG-induced insulin resistance in Hep G2 cells. In contrast, depletion of Nrf2 by small interfering RNA (si-RNA) resulted in the abrogation of HO-1 and glyoxalase expression in the MG-treated resveratrol group in Hep G2 cells. Administration of an appropriate chemopreventive agent, such as resveratrol, may be an alternative strategy for protecting against MG-induced diabetes.
Nuclear erythroid 2-related factor 2 (Nrf2) is an oxidative stress-mediated transcription factor with a variety of downstream targets aimed at cytoprotection. Nrf2 has recently been implicated as a new therapeutic target for the treatment of liver disease. Here, we focus on the most common liver diseases-nonalcoholic fatty liver disease/steatohepatitis, alcoholic liver disease, and drug-induced liver injury-and highlight areas in the development of these conditions where activation of Nrf2 may alleviate disease progression.
Background and purpose:
Cannabis extracts and several cannabinoids have been shown to exert broad anti-inflammatory activities in experimental models of inflammatory CNS degenerative diseases. Clinical use of many cannabinoids is limited by their psychotropic effects. However, phytocannabinoids like cannabidiol (CBD), devoid of psychoactive activity, are, potentially, safe and effective alternatives for alleviating neuroinflammation and neurodegeneration.
Experimental approach:
We used experimental autoimmune encephalomyelitis (EAE) induced by myelin oligodendrocyte glycoprotein (MOG) in C57BL/6 mice, as a model of multiple sclerosis. Using immunocytochemistry and cell proliferation assays we evaluated the effects of CBD on microglial activation in MOG-immunized animals and on MOG-specific T-cell proliferation.
Key results:
Treatment with CBD during disease onset ameliorated the severity of the clinical signs of EAE. This effect of CBD was accompanied by diminished axonal damage and inflammation as well as microglial activation and T-cell recruitment in the spinal cord of MOG-injected mice. Moreover, CBD inhibited MOG-induced T-cell proliferation in vitro at both low and high concentrations of the myelin antigen. This effect was not mediated via the known cannabinoid CB(1) and CB(2) receptors.
Conclusions and implications:
CBD, a non-psychoactive cannabinoid, ameliorates clinical signs of EAE in mice, immunized against MOG. Suppression of microglial activity and T-cell proliferation by CBD appeared to contribute to these beneficial effects.
Triptolide is a major active ingredient of the Chinese herb Tripterygium wilfordii Hook f. (TWHF) and has been shown to possess multiple biological activities, such as anti-inflammatory, anti-fertility, anti-neoplastic and immunosuppressive activities. However, severe adverse effects, especially nephrotoxicity, limit its clinical use. Oxidative stress has been reported to be involved in triptolide-induced renal injury, but the existence of other mechanisms remains unclear. This study aimed to investigate whether NF-E2-related factor 2 (Nrf2), which is an antioxidant nuclear transcription factor, plays a protective role in defense against triptolide-induced toxicity in a normal rat kidney cell line (NRK-52E). Triptolide induced oxidative stress in NRK-52E cells by induction of reactive oxygen species (ROS) and depletion of glutathione (GSH), which resulted in a rapid increase in Nrf2 nuclear accumulation, as well as an induction of antioxidant response element (ARE)-driven genes. In addition, overexpression of Nrf2 protected against triptolide-induced cell death, whereas knockdown of Nrf2 by its specific small interfering RNA resulted in increased cytotoxicity. We also found that Nrf2 knockdown enhanced both the production of ROS and the depletion of GSH. Taken together, these results indicate that activation of Nrf2 plays a protective role against triptolide-induced cytotoxicity in NRK-52E cells through the counteraction of oxidative stress.
Aims:
Nuclear factor-erythroid-related factor 2 (Nrf2) is a critical transcriptional factor that is used in regulating cellular defense against oxidative stress. This study is aimed at investigating new interacting protein partners of Nrf2 using One-strep tag pull-down coupled with LTQ Orbitrap LC/MS/MS, and at examining the impact on Nr2 signaling by the newly identified IQ motif containing GTPase activating protein 1 (IQGAP1).
Results:
Using the One-strep tag pull-down and LTQ Orbitrap LC/MS/MS, we identified IQGAP1 as a new Nrf2 interacting partner. Direct interactions between IQGAP1 and Nrf2 proteins were verified using in vitro glutathione S-transferase (GST) pull-down, transcription/translation assays, and in vivo utilizing Nrf2 overexpressing cells. Coexpression of Dsredmono-IQGAP1 and eGFP-Nrf2 increased the stability of eGFP-Nrf2 and enhanced the expression of Nrf2-target gene heme oxygenase-1 (HO-1). To confirm the functional role of IQGAP1 on Nrf2, knock-downed IQGAP1 using siIQGAP1 attenuated the expression of endogenous Nrf2, HO-1 proteins, and Nrf2-target genes GSTpi, GCLC, and
Nad(p)h:
quinone oxidoreductase 1 (NQO-1). Furthermore, the stability of Nrf2 was dramatically decreased in IQGAP1-deficient mouse embryonic fibroblast (MEF) cells. Since IQGAP1 signaling could be mediated by calcium, treating the cells with calcium showed the translocation of IQGAP1/Nrf2 complex into the nucleus, suggesting that IQGAP1 may play a critical role in Nrf2 stability. Interestingly, consistent with calcium signaling for IQGAP1, treating the cells with calcium functionally enhanced Nrf2-mediated antioxidant responsive element-transcription activity and enhanced the expression of the endogenous Nrf2-target gene HO-1.
Innovation:
In the aggregate, our current study identifies and functionally characterizes a new Nrf2 partner protein IQGAP1, which may contribute to Nrf2's regulation of antioxidant enzymes such as HO-1.
Conclusion:
IQGAP1 may play a critical role in the stability and transactivation of Nrf2.
Oxidative stress, implicated in the etiology of cancer, results from an imbalance in the production of Reactive Oxygen Species (ROS) and cell's own antioxidant defenses. As a oxidative stress sensor, Keap1 functions as both an adaptor for Cul3⋅Rbx1 E3 ligase complex mediated degradation of the transcription factor Nrf2, and a master regulator of cytoprotective gene expression. Although Nrf2 is a well known substrate for Keap1, the DGR domain of Keap1 has been reported also to bind other proteins directly or indirectly. IKKβ as positive regulator of NF-κB is also destabilized by Keap1, which resulted in inhibiting NF-κB-derived tumor promotion. In addition, anti-apoptotic Bcl-2/Bcl-xL protein was identified as another substrate for the Keap1-Cul3-E3 ligase complex. Keap1 led to the repression and destabilization of Bcl-2, decreased Bcl-2:Bax heterodimers and facilitated cancer cells apoptosis. Given that Keap1 might function as a tumor suppressor protein to mitigate tumor progression, the different kinds of Keap1 somatic mutations were detected in numerous cancer cells. Therefore, it is important to understand the Keap1-involved signaling cascades. This review primarily focuses on the prevention of tumorigenesis role of Keap1 through negative regulation of three substrates Nrf2, IKKβ and Bcl-2/Bcl-xL, with emphasis on the recent findings indicating the cancer guarder function of Keap1.
The Keap1-Nrf2 system plays a critical role in cellular defense against electrophiles and reactive oxygen species. Keap1 possesses a number of cysteine residues, some of which are highly reactive and serves as sensors for these insults. Indeed, point mutation of Cys151 abrogates the response to certain electrophiles. However, this mutation does not affect the other set of electrophiles, suggesting that multiple sensor systems reside within the cysteine residues of Keap1. The precise contribution of each reactive cysteine to the sensor function of Keap1 remains to be clarified. To elucidate the contribution of Cys151 in vivo, in this study we adopted transgenic complementation rescue assays. Embryonic fibroblasts and primary peritoneal macrophages were prepared from mice expressing the Keap1-C151S mutant. These cells were challenged with various Nrf2 inducers. We found that some of the inducers triggered only marginal responses in Keap1-C151S-expressing cells, while others evoked responses in a comparable magnitude to those observed in the wild-type cells. We found that tert-butyl hydroquinone, diethylmaleate, sulforaphane, and dimethylfumarate were Cys151 preferable, whereas 15-deoxy-Δ(12,14)-prostaglandin J(2) (15d-PG-J(2)), 2-cyano-3,12 dioxooleana-1,9 diene-28-imidazolide (CDDO-Im), ebselen, nitro-oleic acid, and cadmium chloride were Cys151 independent. Experiments with embryonic fibroblasts and primary macrophages yielded consistent results. Experiments testing protective effects against the cytotoxicity of 1-chloro-2,4-dinitrobenzene of sulforaphane and 15d-PG-J(2) in Keap1-C151S-expressing macrophages revealed that the former inducer was effective, while the latter was not. These results thus indicate that there exists distinct utilization of Keap1 cysteine residues by different chemicals that trigger the response of the Keap1-Nrf2 system, and further substantiate the notion that there are multiple sensing mechanisms within Keap1 cysteine residues.
Paraquat (1,1'-dimethyl-4,4'-bipyridinium chloride; PQ) is widely and commonly used as a herbicides in the world. PQ has been reported to be a major hazard because it causes lung injury. However, the molecular mechanisms underlying PQ-induced lung toxicity still need to be elucidated. Here, we found that PQ significantly decreases cell viability, increases sub-G1 hypodiploids DNA contents and caspase 3/7 activity in lung alveolar epithelial cell-derived L2 cells, which also caused mitochondrial dysfunction, and decreased the mRNA expression of Bcl-2 and increased that of Bax, Bak, and p53. Moreover, the protein expressions of Bax and Bak were increased in PQ-treated cells. In addition, when PQ was exposed to L2 cells, the expressions of ER stress-related signaling genes (including Grp78, CHOP, and caspase-12 mRNA) and proteins (including phospho-eIF-2α, CHOP, Grp78, calpain I and -II, and caspase-12) were significantly increased. PQ also decreased the protein expressions of pro-caspase-9/7/3. Next, we investigated the role of Nrf-2 in PQ-induced alveolar epithelial cell toxicity. In L2 cells, PQ induced Nrf-2 translocation from the cytosol to the nucleus. Cells transfected with Nrf-2 siRNA significantly reversed the PQ-induced toxicity, including depolarization of MMP, increased the Bax, Bak, p53 mRNAs expression, decreased the Bcl-2 mRNA expression, increased the caspase 3/7 activity, Grp78, CHOP, and caspase-12 mRNAs and protein expression, and decreased that of pro-caspase-3. Taken together, these results suggest that Nrf-2-regulated mitochondria and ER stress-related pathways are involved in the PQ-induced alveolar epithelial cell injury.
Haeme oxygenase-1 (HO-1) is often viewed as a cytoprotective gene. Toxic heavy metals induce HO-1, but it is unclear whether particular metal micronutrients also induce HO-1. Hence, the ability of exogenously-added copper, iron and zinc to influence HO-1 expression in HCT-116 cells was evaluated. Under the chosen experimental conditions, only zinc noticeably increased the expression of HO-1 mRNA and protein. Concurrently, zinc decreased non-protein thiol levels to a certain extent, but zinc did not increase the production of reactive oxygen species (ROS). Moreover, ascorbate and Trolox did not inhibit zinc-induced HO-1 upregulation. In contrast, deferoxamine blunted the induction of HO-1 mRNA, protein, and enzymatic activity caused by zinc. Additionally, N-acetylcysteine and Tiron inhibited zinc-induced HO-1 upregulation and also nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2). Collectively, these findings suggest that zinc at above normal levels upregulates HO-1 expression in HCT-116 cells in a ROS-independent manner.
Kelch-like ECH-associated protein 1 (Keap1)-nuclear factor erythroid 2-related factor 2 (Nrf2)-antioxidant response elements (ARE) pathway represents one of the most important cellular defense mechanisms against oxidative stress and xenobiotic damage. Activation of Nrf2 signaling induces the transcriptional regulation of ARE-dependent expression of various detoxifying and antioxidant defense enzymes and proteins. Keap1-Nrf2-ARE signaling has become an attractive target for the prevention and treatment of oxidative stress-related diseases and conditions including cancer, neurodegenerative, cardiovascular, metabolic, and inflammatory diseases. Over the last few decades, numerous Nrf2 inducers have been developed and some of them are currently undergoing clinical trials. Recently, overactivation of Nrf2 has been implicated in cancer progression as well as in drug resistance to cancer chemotherapy. Thus, Nrf2 inhibitors could potentially be used to improve the effectiveness of cancer therapy. Herein, we review the signaling mechanism of Keap1-Nrf2-ARE pathway, its disease relevance, and currently known classes of small molecule modulators. We also discuss several aspects of Keap1-Nrf2 interaction, Nrf2-based peptide inhibitor design, and the screening assays currently used for the discovery of direct inhibitors of Keap1-Nrf2 interaction.
Silver nanoparticles (nAg) are known to evoke reactive oxygen species (ROS) generation and consequent cell damage. The transcription factor NF-E2-related factor 2 (NRF2) controls both the basal and inducible expression of multiple antioxidant genes. This study was aimed to investigate the role of NRF2 in nAg-induced renal epithelial cell damage. nAg treatment intensified DNA damage and G2/M cell cycle arrest by nAg in NRF2 knockdown HK-2 (NRF2i) compared with the control cells. As a signaling mechanism associated with nAg-mediated growth arrest, the levels of phospho-CDC25C and phospho-CDC2 were significantly increased in NRF2i. Target gene analysis revealed that nAg-mediated increase in γ-glutamate cysteine ligase expression is NRF2-dependent: nAg-treated NRF2i showed a reduction in glutathione (GSH) content and elevation in ROS level in comparison with the control cells. Additionally, pretreatment of N-acetylcystein in nAg-treated NRF2i alleviated ROS-mediated DNA damage and G2/M cell cycle arrest, while GSH depletion exacerbated DNA damage and cell cycle arrest in the control cells. Taken together, these results suggest that NRF2-mediated GSH increase plays a protective role in nAg-induced DNA damage and subsequent G2/M cell cycle arrest in human renal epithelial cells.
Oxidative stress has been hypothesized to contribute to the development of age-related macular degeneration (AMD), the most common cause of blindness in the United States. At present, there is no treatment for early disease. Reactive oxygen species (ROS) play a physiological role in the retinal pigment epithelium (RPE), a key cell type in this disease, but with excessive ROS, oxidative damage or excessive innate immune system activation can result. The RPE has developed a robust antioxidant system driven by the transcription factor Nrf2. Impaired Nrf2 signaling can lead to oxidative damage or activate the innate immune response, both of which can lead to RPE apoptosis, a defining change in AMD. Several mouse models simulating environmental stressors or targeting specific antioxidant enzymes such as superoxide dismutase or Nrf2, have simulated some of the features of AMD. While ROS are short-lived, oxidatively damaged molecules termed oxidation specific epitopes (OSEs), can be long-lived and a source of chronic stress that activates the innate immune system through pattern recognition receptors (PRRs). The macula accumulates a number of OSEs including carboxyethylpyrrole, malondialdehyde, 4-hydroxynonenal, and advanced glycation endproducts, as well as their respective neutralizing PRRs. Excessive accumulation of OSEs results in pathologic immune activation. For example, mice immunized with the carboxyethylpyrrole develop cardinal features of AMD. Regulating ROS in the RPE by modulating antioxidant systems or neutralizing OSEs through an appropriate innate immune response are potential modalities to treat or prevent early AMD.
S-nitrosothiol formation and protein S-nitrosation is an important nitric oxide (NO)-dependent signaling paradigm that is relevant to almost all aspects of cell biology, from proliferation, to homeostasis, to programmed cell death. However, the mechanisms by which S-nitrosothiols are formed are still largely unknown, and there are gaps of understanding between the known chemical biology of S-nitrosothiols and their reported functions. RECENT ADVANCES: This review attempts to describe the biological chemistry of S-nitrosation and to point out where the challenges lie in matching the known chemical biology of these compounds with their reported functions. The review will detail new discoveries concerning the mechanisms of the formation of S-nitrosothiols in biological systems.
Although S-nitrosothiols may be formed with some degree of specificity on particular protein thiols, through un-catalyzed chemistry, and mechanisms for their degradation and redistribution are present, these processes are not sufficient to explain the vast array of specific and targeted responses of NO that have been attributed to S-nitrosation. Elements of catalysis have been discovered in the formation, distribution, and metabolism of S-nitrosothiols, but it is less clear whether these represent a specific network for targeted NO-dependent signaling.
Much recent work has uncovered new targets for S-nitrosation through either targeted or proteome-wide approaches There is a need to understand which of these modifications represent concerted and targeted signaling processes and which is an inevitable consequence of living with NO. There is still much to be learned about how NO transduces signals in cells and the role played by protein S-nitrosation.
Diesel exhaust (DE) has been recognized as a noxious mutagen and/or carcinogen, because its components can form DNA adducts. Mechanisms governing the susceptibility to DE and the efficiency of such DNA adduct formation require clarification. The transcription factor Nrf2 is essential for inducible and/or constitutive expression of a group of detoxification and antioxidant enzymes, and we hypothesized that the nrf2 gene knockout mouse might serve as an excellent model system for analyzing DE toxicity. To address this hypothesis, lungs from nrf2(−/−) and nrf2(+/−) mice were examined for the production of xenobiotic–DNA adducts after exposure to DE (3 mg/m3 suspended particulate matter) for 4 weeks. Whereas the relative adduct levels (RAL) were significantly increased in the lungs of both nrf2(+/−) and nrf2(−/−) mice upon exposure to DE, the increase of RAL in the lungs from nrf2(−/−) mice exposed to DE were approximately 2.3-fold higher than that of nrf2(+/−) mice exposed to DE. In contrast, cytochrome P4501A1 mRNA levels in the nrf2(−/−) mouse lungs were similar to those in the nrf2(+/−) mouse lungs even after exposure to DE, suggesting that suppressed activity of phase II drug-metabolizing enzymes is important in giving rise to the increased level of DNA adducts in the Nrf2-null mutant mouse subjected to DE. Importantly, severe hyperplasia and accumulation of the oxidative DNA adduct 8-hydroxydeoxyguanosine were observed in the bronchial epidermis of nrf2(−/−) mice following DE exposure. These results demonstrate the increased susceptibility of the nrf2 germ line mutant mouse to DE exposure and indicate the nrf2 gene knockout mouse may represent a valuable model for the assessment of respiratory DE toxicity.
The induction of phase II detoxifying enzymes is an important defense mechanism against intake of xenobiotics. While this group of enzymes is believed to be under the transcriptional control of antioxidant response elements (AREs), this contention is experimentally unconfirmed. Since the ARE resembles the binding sequence of erythroid transcription factor NF-E2, we investigated the possibility that the phase II enzyme genes might be regulated by transcription factors that also bind to the NF-E2 sequence. The expression profiles of a number of transcription factors suggest that an Nrf2/small Maf heterodimer is the most likely candidate to fulfill this rolein vivo.To directly test these questions, we disrupted the murinenrf2 genein vivo.While the expression of phase II enzymes (e.g., glutathione S-transferase and NAD(P)H: quinone oxidoreductase) was markedly induced by a phenolic antioxidantin vivoin both wild type and heterozygous mutant mice, the induction was largely eliminated in the liver and intestine of homozygousnrf2-mutant mice. Nrf2 was found to bind to the ARE with high affinity only as a heterodimer with a small Maf protein, suggesting that Nrf2/small Maf activates gene expression directly through the ARE. These results demonstrate that Nrf2 is essential for the transcriptional induction of phase II enzymes and the presence of a coordinate transcriptional regulatory mechanism for phase II enzyme genes. Thenrf2-deficient mice may prove to be a very useful model for thein vivoanalysis of chemical carcinogenesis and resistance to anti-cancer drugs.