Issue 10, October 2024

newsletter2024OCTUBRE

Our quarterly newsletter attempts to provide our latest news and also aims at becoming a forum for analysis of relevant topics on the field of NRF2 and provide comments to some of the most relevant articles published during the quarter. Previous newsletters can be accessed at:

https://benbedphar.org/our-first-newsletter/
https://benbedphar.org/issue-2-abril-2022/
https://benbedphar.org/issue-3-july-2022/
https://benbedphar.org/issue-4-october-2022/
https://benbedphar.org/issue-5-january-2023/
https://benbedphar.org/issue-6-april-2023/
https://benbedphar.org/issue-7-october-2023/
https://benbedphar.org/issue-8-january-2024/
https://benbedphar.org/issue-9-april-2024/

Antonio Cuadrado
Chair of COST Action 20121, BenBedPhar
Autonomous University of Madrid

It is well known that the nuclear factor erythroid 2–related factor 2 (NRF2) function can be activated by various synthetic and natural substances for example sulforaphane, resveratrol, quercetin, bardoxolone methyl, and even registered drugs metformin and statins. One of the most powerful NRF2 activators is dimethyl fumarate (DMF), originally isolated from Fumaria officinalis. DMF is approved for the treatment of psoriasis and sclerosis multiplex. In addition, several studies suggested that DMF (or other fumarates) also possesses the potential to be repurposed for the treatment of cardiovascular diseases, for example as a therapeutic agent in patients with atherosclerotic cardiovascular disease. However, less is known about the long-term effects of activation of NRF2 function by DMF in chronic social stress conditions, to which patients are, more or less, exposed during the diseased state and its treatment. Moreover, borderline elevated blood pressure (prehypertension) is widespread even in young adults and children, so the effect of elevated blood pressure should also be considered in cardiovascular studies investigating the role of NRF2 itself or in the conditions of activated NRF2 function by various activators.
In our recently published study (1), we investigated the effects of chronic social stress, DMF and their interaction in rats genetically predisposed to high blood pressure, so-called borderline hypertensive rats (BHR). Our study showed that chronic social stress produced by crowding significantly elevated blood pressure and plasma corticosterone while inhibiting body weight gain, as expected in this stress model. Furthermore, upregulation of Nfe2l2 gene expression was present in the hearts of crowding-exposed rats associated with elevated Sod1 and Hmox1 gene expressions and reduced lipid peroxidation. DMF alone did not exert a significant effect on blood pressure and the endothelium-dependent and independent relaxations in the femoral artery. However, DMF, similarly to stress, led to an increase in plasma corticosterone levels, Nfe2l2, Sod1, Hmox1 gene expressions in the heart, reduced oxidative damage to lipids and this was, in contrast to stress, associated with the increase in relative left heart ventricular mass, suggesting the development of left heart ventricular hypertrophy. When DMF was administered to stress-exposed BHR, it prevented the development of stress-induced hypertension and corticosterone elevations. This was accompanied by a reduction in noradrenaline-induced contractions in the femoral artery which we consider the primary mechanism by which DMF prevented stress-induced hypertension in the crowding stress model. On the other hand, a concomitant upregulation of inflammatory mediator genes (Tnf, Nos2) in the heart was found in rats treated with DMF in stress conditions. Collectively, the findings suggest that DMF can prevent chronic stress-induced hypertension by reducing vascular contractility without alterations in vasorelaxation. Moreover, DMF itself might produce reductive stress in the heart and induce inflammation when combined with stress. The study provides insights into the potential benefits and risks of using DMF in chronic social stress conditions in presence of prehypertension. It also suggests a need for careful consideration of long-term DMF treatment taking into account its impact on the heart.

Figure 1. Graphical abstract

Adapted from:
Kluknavsky M, Balis P, Liskova S, Micurova A, Skratek M, Manka J, Bernatova I. Dimethyl Fumarate Prevents the Development of Chronic Social Stress-Induced Hypertension in Borderline Hypertensive Rats. Antioxidants (Basel). 2024

Link to paper: https://doi.org/10.3390/antiox13080947

Iveta Bernatova
Principal investigator, Centre of Experimental Medicine, Sloval Academy of Sciences,
Bratislava, Slovakia
WG1 member, on behalf of authors

Artemisinin, a highly unusual endoperoxide-containing sesquiterpene lactone (Figure 1A) from the plant Artemisia annua, is the most effective antimalarial drug to date, which has saved millions of lives in South China, Southeast Asia, Africa, and South America. For its discovery, Tu Youyou shared the 2015 Nobel Prize in Physiology or Medicine. Like many natural products, artemisinin has been structurally ‘optimized’ during evolution to serve multiple biological functions, including boosting the endogenous defence mechanisms and facilitating the (often competitive) interactions with other organisms.

More than 10 years ago, extracts from Saudi Arabian Artemisia plants were found to induce the NRF2-transcriptional target NAD(P)H:quinone oxidoreductase 1 (NQO1), and it was further shown that artemisinin, but not the closely structurally related derivative deoxyartemisinin (Figure 1B), which lacks the endoperoxide moiety, is an NQO1 inducer (1). Since then, several reports have demonstrated that artemisinin and its derivatives affect multiple signalling pathways in mammalian cells, including KEAP1/NRF2 (2). In a recent study, Deng et al. (3) have shown that artemisinin induces the nuclear translocation of NRF2 and the increased transcription of SLC7A11 and GPX4 in the immortalized mouse hippocampal cell line HT-22. Moreover, pretreatment with artemisinin protects against erastin-induced cell death, with potency comparable to the ferroptosis inhibitor ferrostatin-1, and the protective effect of artemisinin is lost upon knockdown of NRF2. Molecular docking and surface plasmon resonance analysis suggest that artemisinin binds to the Kelch domain of KEAP1. Artemisinin also inhibited ferroptosis in primary hippocampal neurons, and in vivo, in Alzheimer’s disease models. This study highlights the role of ferroptosis in the pathology of Alzheimer’s disease and the protective effects of NRF2 activation.

References:

  1. Shahat AA, Alsaid MS, Alyahya MA, Higgins M, Dinkova-Kostova AT. NAD(P)H: quinone oxidoreductase 1 inducer activity of some Saudi Arabian medicinal plants. Planta Med. 2013 Apr;79(6):459-64. 
  2. Ho WE, Peh HY, Chan TK, Wong WS. Artemisinins: pharmacological actions beyond anti-malarial. Pharmacol Ther. 2014 Apr;142(1):126-39.
  3. Deng PX, Silva M, Yang N, Wang Q, Meng X, Ye KQ, Gao HC, Zheng WH. Artemisinin inhibits neuronal ferroptosis in Alzheimer’s disease models by targeting KEAP1. Acta Pharmacol Sin. 2024 Sep 9. doi: 10.1038/s41401-024-01378-6.

Ana I Rojo
Autonomous University of Madrid, Spain
Albena T Dinkova-Kostova
University of Dundee, United Kingdom
WG2 Leaders, on behalf of authors

The cytoprotective transcription factor NRF2 regulates the expression of several hundred genes in mammalian cells and is a promising therapeutic target in a number of diseases associated with oxidative stress and inflammation. Hence, an ability to monitor basal and inducible NRF2 signalling is vital for mechanistic understanding in translational studies. Due to some caveats related to the direct measurement of NRF2 levels, the modulation of NRF2 activity is typically determined by measuring changes in the expression of one or more of its target genes and/or the associated protein products. However, there is a lack of consensus regarding the most relevant set of these genes/proteins that best represents NRF2 activity across cell types and species. We present the findings of a comprehensive literature search that according to stringent criteria identifies GCLC, GCLM, HMOX1, NQO1, SRXN1 and TXNRD1 as a robust panel of markers that are directly regulated by NRF2 in multiple cell and tissue types. We assess the relevance of these markers in clinically accessible biofluids and highlight future challenges in the development and use of NRF2 biomarkers in humans.

Figure 1. Workflow showing the literature-based strategy of defining biomarkers of NRF2. 

Adapted from:
Morgenstern C, Lastres-Becker I, Demirdöğen BC, Costa VM, Daiber A, Foresti R, Motterlini R, Kalyoncu S, Arioz BI, Genc S, Jakubowska M, Trougakos IP, Piechota-Polanczyk A, Mickael M, Santos M, Kensler TW, Cuadrado A, Copple IM. Biomarkers of NRF2 signalling: Current status and future challenges. Redox Biol. 2024.

Link to paper: https://doi.org/10.1016/j.redox.2024.103134

Christina Morgenstern
Karl-Franzens Universität, Austria
WG3 Leader​, on behalf of authors

Liver steatosis is a growing global health issue. Sex differences may play a role in the etiopathogenesis and severity of metabolic dysfunction-associated steatotic liver disease (MASLD). Sex differences have been observed to influence the KEAP1/NRF2 axis in the early phases of MASLD in a rat model orally supplemented with high-fat diet and liquid fructose.
The study showed that females are more prone to liver steatosis, which is attributed to impaired autophagic flux and reduced nuclear translocation of NRF2. Females displayed increased cytosolic levels of NRF2 but impaired nuclear translocation, limiting their ability to manage oxidative damage compared to males. By contrast, male rats exhibited greater resilience, showing better abilities to cope with oxidative stress through more effective autophagy and endoplasmic reticulum stress responses.
Understanding the biological differences in disease progression between sexes can pave the way for targeted therapeutic strategies, especially for women, who may be more vulnerable to advanced liver damage. These findings suggest that future treatments for MASLD may need to consider sex-specific therapies, particularly focusing on enhancing the NRF2 pathway in females to improve their response to oxidative and proteotoxic stress.

Figure 1. Graphical abstract.

Adapted from:
Di Veroli B, Bentanachs R, Roglans N, Alegret M, Giona L, Profumo E, Berry A, Saso L, Laguna JC, Buttari B. Sex Differences Affect the NRF2 Signaling Pathway in the Early Phase of Liver Steatosis: A High-Fat-Diet-Fed Rat Model Supplemented with Liquid Fructose. Cells. 2024

Link to paper: https://doi.org/10.3390/cells13151247

Brigitta Buttari
Istituto Superiore di Sanità, IT
WG5 Leader, on behalf of authors

Nrf2, a crucial protein involved in defense mechanisms, particularly oxidative stress, plays a significant role in neurological diseases (NDs) by reducing oxidative stress and inflammation. NDs, including Alzheimer’s, Parkinson’s, Huntington’s, amyotrophic lateral sclerosis, stroke, epilepsy, schizophrenia, depression, and autism, exhibit ferroptosis, iron-dependent regulated cell death resulting from lipid and iron-dependent reactive oxygen species (ROS) accumulation. Nrf2 has been shown to play a critical role in regulating ferroptosis in NDs. Age-related decline in Nrf2 expression and its target genes (HO-1, Nqo-1, and Trx) coincides with increased iron-mediated cell death, leading to ND onset. The modulation of iron-dependent cell death and ferroptosis by Nrf2 through various cellular and molecular mechanisms offers a potential therapeutic pathway for understanding the pathological processes underlying these NDs. This review emphasizes the mechanistic role of Nrf2 and ferroptosis in multiple NDs, providing valuable insights for future research and therapeutic approaches.

Access to the original article: https://pubmed.ncbi.nlm.nih.gov/39350404/

Oxidative stress is a consequence of the disruption of the balance between the generation of reactive nitrogen and oxygen species and the biological system’s ability to neutralize those reactive products. Oxidative stress is involved in the generation of many disorders, including epilepsy, which is a prevalent chronic neurological disease that affects the lives of millions of people around the world. Epilepsy is characterized by unforeseeable and repeated seizures that can be very disturbing. Studies have reported that oxidative stress occurs before and after seizures. A transcription factor named Nuclear factor erythroid-derived 2-related factor 2 (Nrf2) controls genes related to the induction of oxidative stress and defends cells against oxidative stress. The Nrf2 protein has seven different domains, ranging from Neh1 to Neh7. Each domain is responsible for a distinctive function of this protein. Keap1 binds to Nrf2, but during oxidative stress, Nrf2 detaches from the Keap1 protein, moves to the nucleus, and binds to DNA. The result of this translocation and binding is the initiation of transcription of detoxifying genes to control the harmful effects of oxidative stress. There is some evidence of oxidative stress involvement in epilepsy. In this review, we have listed potential Nrf2-related therapeutic targets for treating and controlling epilepsy, such as Berberis alkaloids, pentoxifylline, lovastatin, progesterone, and chrysin nanoparticles. These activators were tested in animals (in vivo) and cells (in vitro), and most of these experiments showed promising results in different epilepsy models. Finally, the results have suggested that the activation of Nrf2 can be an option for controlling epilepsy.

Access to the original article: https://pubmed.ncbi.nlm.nih.gov/39350402/

Glucocorticoids (GCs) are widely prescribed for various medical conditions, but prolonged use can result in osteonecrosis of the femoral head (ONFH), a serious condition characterized by bone tissue death due to reduced blood flow. Alpha-2-macroglobulin (A2M) is known to regulate oxidative stress and has been implicated in numerous biological processes. However, its role in GCs-induced ONFH has not been fully elucidated. This study investigates the involvement of A2M in ONFH by examining its activation of the Keap1/Nrf2 signaling pathway. Transcriptomic and proteomic analyses of patient samples with GCs-induced ONFH revealed a significant downregulation of A2M. A rat model of GCs-induced ONFH was then used to overexpress A2M, with subsequent evaluation through histopathological staining. Single-cell RNA sequencing and proteomic analysis indicated that A2M overexpression promotes the proliferation of anti-inflammatory macrophage clusters. Both in vivo and in vitro experiments demonstrated that A2M overexpression significantly alleviated ONFH symptoms by modulating oxidative stress and apoptosis via the Keap1/Nrf2 pathway. These findings underscore the critical role of A2M in mitigating GCs-induced ONFH, providing new therapeutic strategies and targets for future research.

Access to the original article: https://pubmed.ncbi.nlm.nih.gov/39343183/

Cardiac ischaemia/reperfusion (I/R) impairs mitochondrial function, resulting in excessive oxidative stress and cardiomyocyte ferroptosis and death. Nuclear factor E2-related factor 2 (Nrf2) is a key regulator of redox homeostasis and has cardioprotective effects against various stresses. Here, we tested whether CBR-470-1, a noncovalent Nrf2 activator, can protect against cardiomyocyte death caused by I/R stress. Compared with vehicle treatment, the administration of CBR-470-1 (2 mg/kg) to mice significantly increased Nrf2 protein levels and ameliorated the infarct size, the I/R-induced decrease in cardiac contractile performance, and the I/R-induced increases in cell apoptosis, ROS levels, and inflammation. Consistently, the beneficial effects of CBR-470-1 on cardiomyocytes were verified in a hypoxia/reoxygenation (H/R) model in vitro, but this cardioprotection was dramatically attenuated by the GPX4 inhibitor RSL3. Mechanistically, CBR-470-1 upregulated Nrf2 expression, which increased the expression levels of antioxidant enzymes (NQO1, SOD1, Prdx1, and Gclc) and antiferroptotic proteins (SLC7A11 and GPX4) and downregulated the protein expression of p53 and Nlrp3, leading to the inhibition of ROS production and inflammation and subsequent cardiomyocyte death (apoptosis, ferroptosis and pyroptosis). In summary, CBR-470-1 prevented I/R-mediated cardiac injury possibly through inhibiting cardiomyocyte apoptosis, ferroptosis and pyroptosis via Nrf2-mediated inhibition of p53 and Nlrp3 and activation of the SLC7A11/GPX4 pathway. Our data also highlight that CBR-470-1 may serve as a valuable agent for treating ischaemic heart disease.
Access to the original article: https://pubmed.ncbi.nlm.nih.gov/39343043/

In the KEAP1-NRF2 stress response system, KEAP1 acts as a sensor for oxidative and electrophilic stresses through formation of S-S bond and C-S bond, respectively. Of the many questions left related to the sensor activity, following three appear important; whether these KEAP1 sensor systems are operating in vivo, whether oxidative and electrophilic stresses are sensed by the similar or distinct systems, and how KEAP1 equips highly sensitive mechanisms detecting oxidative and electrophilic stresses in vivo. To address these questions, we conducted a series of analyses utilizing KEAP1-cysteine substitution mutant mice, conditional selenocysteine-tRNA (Trsp) knockout mice, and human cohort whole genome sequence (WGS) data. Firstly, the Trsp-knockout provokes severe deficiency of selenoproteins and compensatory activation of NRF2. However, mice lacking homozygously a pair of critical oxidative stress sensor cysteine residues of KEAP1 fail to activate NRF2 in the Trsp-knockout livers. Secondly, this study provides evidence for the differential utilization of KEAP1 sensors for oxidative and electrophilic stresses in vivo. Thirdly, theoretical calculations show that the KEAP1 dimer equips quite sensitive sensor machinery in which modification of a single molecule of KEAP1 within the dimer is sufficient to affect the activity. WGS examinations of rare variants identified seven non-synonymous variants in the oxidative stress sensors in human KEAP1, while no variant was found in electrophilic sensor cysteine residues, supporting the fail-safe nature of the KEAP1 oxidative stress sensor activity. These results provide valuable information for our understanding how mammals respond to oxidative and electrophilic stresses efficiently.

Access to the original article: https://pubmed.ncbi.nlm.nih.gov/39307045/

Friedreich ataxia (FA) is a rare neurodegenerative disease caused by decreased levels of the mitochondrial protein frataxin. Frataxin has been related in iron homeostasis, energy metabolism, and oxidative stress. Ferroptosis has recently been shown to be involved in FA cellular degeneration; however, its role in dorsal root ganglion (DRG) sensory neurons, the cells that are affected the most and the earliest, is mostly unknown. In this study, we used primary cultures of frataxin-deficient DRG neurons as well as DRG from the FXNI151F mouse model to study ferroptosis and its regulatory pathways. A lack of frataxin induced upregulation of transferrin receptor 1 and decreased ferritin and mitochondrial iron accumulation, a source of oxidative stress. However, there was impaired activation of NRF2, a key transcription factor involved in the antioxidant response pathway. Decreased total and nuclear NRF2 explains the downregulation of both SLC7A11 (a member of the system Xc, which transports cystine required for glutathione synthesis) and glutathione peroxidase 4, responsible for increased lipid peroxidation, the main markers of ferroptosis. Such dysregulation could be due to the increase in KEAP1 and the activation of GSK3β, which promote cytosolic localization and degradation of NRF2. Moreover, there was a deficiency in the LKB1/AMPK pathway, which would also impair NRF2 activity. AMPK acts as a positive regulator of NRF2 and it is activated by the upstream kinase LKB1. The levels of LKB1 were reduced when frataxin decreased, in agreement with reduced pAMPK (Thr172), the active form of AMPK. SIRT1, a known activator of LKB1, was also reduced when frataxin decreased. MT-6378, an AMPK activator, restored NRF2 levels, increased GPX4 levels and reduced lipid peroxidation. In conclusion, this study demonstrated that frataxin deficiency in DRG neurons disrupts iron homeostasis and the intricate regulation of molecular pathways affecting NRF2 activation and the cellular response to oxidative stress, leading to ferroptosis.

Access to the original article: https://pubmed.ncbi.nlm.nih.gov/39243573/

Arsenic is a toxic environmental pollutant heavy metal, and one of its critical target tissues in the body is the liver. Carvacrol is a natural phytocompound that stands out with its antioxidant, anti-inflammatory, and antiapoptotic properties. The current study aims to investigate the protective feature of carvacrol against sodium arsenite-induced liver toxicity. Thirty-five Sprague-Dawley male rats were divided into five groups: Control, Sodium arsenite (SA), CRV, SA + CRV25, and SA + CRV50. Sodium arsenite was administered via oral gavage at a dose of 10 mg/kg for 14 days, and 30 min later, CRV 25 or 50 mg/kg was administered via oral gavage. Oxidative stress, inflammation, apoptosis, autophagy damage pathways parameters, and liver tissue integrity were analyzed using biochemical, molecular, western blot, histological, and immunohistological methods. Carvacrol decreased sodium arsenite-induced oxidative stress by suppressing malondialdehyde levels and increasing superoxide dismutase, catalase, glutathione peroxidase activities, and glutathione levels. Carvacrol reduced inflammation damage by reducing sodium arsenite-induced increased levels of NF-κB and the cytokines (TNF-α, IL-1β, IL-6, RAGE, and NLRP3) it stimulates. Carvacrol also reduced sodium arsenite-induced autophagic (Beclin-1, LC3A, and LC3B) and apoptotic (P53, Apaf-1, Casp-3, Casp-6, Casp-9, and Bax) parameters. Carvacrol preserved sodium arsenite-induced impaired liver tissue structure. Carvacrol alleviated toxic damage by reducing sodium arsenite-induced increases in oxidative stress, inflammation, apoptosis, and autophagic damage parameters in rat liver tissues. Carvacrol was also beneficial in preserving liver tissue integrity.

Access to the original article: https://pubmed.ncbi.nlm.nih.gov/39318027/

p-Coumaric acid is a significant phenolic compound known for its potent antioxidant activity. Thus, this study investigated the effects of p-coumaric acid on the behavioral and neurochemical changes induced in Drosophila melanogaster by exposure to rotenone in a Parkinson disease (PD)-like model. The flies were divided into four groups and maintained for seven days on different diets: a standard diet (control), a diet containing rotenone (500 μM), a control diet to which p-coumaric acid was added on the fourth day (0.3 μM), and a diet initially containing rotenone (500 μM) with p-coumaric acid added on the fourth day (0.3 μM). Exposure to p-coumaric acid ameliorated locomotor impairment and reduced mortality induced by rotenone. Moreover, p-coumaric acid normalized oxidative stress markers (ROS, TBARS, SOD, CAT, GST, and NPSH), mitigated oxidative damage, and reflected in the recovery of dopamine levels, AChE activity, and cellular viability post-rotenone exposure. Additionally, p-coumaric acid restored the immunoreactivity of Parkin and Nrf2. The results affirm that p-coumaric acid effectively mitigates PD-like model-induced damage, underscoring its antioxidant potency and potential neuroprotective effect.

Access to the original article: https://pubmed.ncbi.nlm.nih.gov/39276910/

Recently, non-small cell lung cancer (NSCLC) has been the prime concern of cancer clinicians due to its high mortality rate worldwide. Cisplatin, a platinum derivative, has been used as a therapeutic option for treating metastatic NSCLC for several years. However, acquired, or intrinsic drug resistance to Cisplatin is the major obstacle to the successful treatment outcome of patients. Dysregulation of Nrf2 (nuclear factor erythroid 2-related factor 2) and EGFR (epidermal growth factor receptor) signaling have been associated with cellular proliferation, cancer initiation, progression and confer drug resistance to several therapeutic agents including Cisplatin in various cancers. To dissect the molecular mechanism of EGFR activation in resistant cells, we developed Cisplatin-resistant (CisR) human NSCLC cell lines (A549 and NCIH460) with increasing doses of Cisplatin treatment over a 3-month period. CisR cells demonstrated increased proliferative capacity, clonogenic survivability and drug efflux activity compared to the untreated parental (PT) cells. These resistant cells also showed higher levels of Nrf2 and EGFR expression. Here, we found that Nrf2 upregulates both basal and inducible expression of EGFR in these CisR cells at the transcriptional level. Moreover, genetic inhibition of Nrf2 with siRNA in CisR cells showed increased sensitivity towards the EGFR tyrosine kinase inhibitor (TKIs), AG1478. Our study, therefore suggests the use of Nrf2 inhibitors in combinatorial therapy with EGFR TKIs for the treatment of resistant NSCLC.

Access to the original article: https://pubmed.ncbi.nlm.nih.gov/39179137/

Neurodegenerative diseases constitute a global health issue and a major economic burden. They significantly impair both cognitive and motor functions, and their prevalence is expected to rise due to ageing societies and continuous population growth. Conventional therapies provide symptomatic relief, nevertheless, disease-modifying treatments that reduce or halt neuron death and malfunction are still largely unavailable. Amongst the common hallmarks of neurodegenerative diseases are protein aggregation, oxidative stress, neuroinflammation and mitochondrial dysfunction. Transcription factor nuclear factor-erythroid 2-related factor 2 (NRF2) constitutes a central regulator of cellular defense mechanisms, including the regulation of antioxidant, anti-inflammatory and mitochondrial pathways, making it a highly attractive therapeutic target for disease modification in neurodegenerative disorders. Here, we describe the role of NRF2 in the common hallmarks of neurodegeneration, review the current pharmacological interventions and their challenges in activating the NRF2 pathway, and present alternative therapeutic approaches for disease modification.

Access to the original article: https://pubmed.ncbi.nlm.nih.gov/39119604/

Joana Miranda
Faculty of Pharmacy, University of Lisbon
Portugal


Summary