The role of nuclear factor erythroid 2-related factor 2 (NRF2) in arsenic toxicity.

Arsenic, a naturally occurring toxic element, manifests in various chemical forms and is widespread in the environment. Exposure to arsenic is a well-established risk factor for an elevated incidence of various cancers and chronic diseases. The crux of arsenic-mediated toxicity lies in its ability to induce oxidative stress, characterized by an unsettling imbalance between oxidants and antioxidants, accompanied by the rampant generation of reactive oxygen species and free radicals. In response to this oxidative turmoil, cells deploy their defense mechanisms, prominently featuring the redox-sensitive transcription factor known as nuclear factor erythroid 2-related factor 2 (NRF2). NRF2 stands as a primary guardian against the oxidative harm wrought by arsenic. When oxidative stress activates NRF2, it orchestrates a symphony of downstream antioxidant genes, leading to the activation of pivotal antioxidant enzymes like glutathione-S-transferase, heme oxygenase-1, and NAD(P)H: quinone oxidoreductase 1. This comprehensive review embarks on the intricate and diverse ways by which various arsenicals influence the NRF2 antioxidant pathway and its downstream targets, shedding light on their roles in defending against arsenic exposure toxic effects. It offers valuable insights into targeting NRF2 as a strategy for safeguarding against or treating the harmful and carcinogenic consequences of arsenic exposure.

Epigallocatechin-3-Gallate Inhibits Oxidative Stress Through the Keap1/Nrf2 Signaling Pathway to Improve Alzheimer Disease.

Alzheimer disease (AD) is a common neurodegenerative disease with an intricate pathophysiological mechanism. Oxidative stress has been shown in several investigations as a significant factor in AD progression. For instance, studies have confirmed that oxidative stress inhibition may considerably improve AD symptoms, with potent antioxidants being touted as a possible interventional strategy in the search for AD treatment. Epigallocatechin-3-gallate (EGCG) acts as a natural catechin that has antioxidant effect. It activates the kelch-like epichlorohydrin-associated proteins (Keap1)/nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway to inhibit oxidative stress. The Keap1/Nrf2 signal pathway is not only an upstream signaling target for a variety of antioxidant enzymes, but also minimizes high levels of reactive oxygen species. This report analyzes the antioxidant effect of EGCG in AD, elaborates its specific mechanism of action, and provides a theoretical basis for its clinical application in AD.

Vitamin D receptor alleviates lipid peroxidation in diabetic nephropathy by regulating ACLY/Nrf2/Keap1 pathway.

The membrane lipid damage caused by reactive oxygen species(ROS) and various peroxides, namely lipid peroxidation, plays an important role in the progression of diabetic nephropathy (DN).We previously reported that vitamin D receptor(VDR) plays an active role in DN mice by modulating autophagy disorders. However, it is unclear whether the ATP-citrate lyase (ACLY)/NF-E2-related factor-2 (Nrf2)/Kelch-like ECH-associated protein 1 (Keap1) pathway is associated with the reduction of lipid peroxidation by VDR in the DN model. We found that in the DN mouse model, VDR knockout significantly aggravated mitochondrial morphological damage caused by DN, increased the expression of ACLY, promoted the accumulation of ROS, lipid peroxidation products Malondialdehyde(MDA) and 4-hydroxy-2-nonenal (4-HNE),consumed the Nrf2/Keap1 system, thus increasing lipid peroxidation. However, the overexpression of VDR and intervention with the VDR agonist paricalcitol (Pari) can reduce the above damage. On the other hand, cellular experiments have shown that Pari can significantly reduce the elevated expression of ACLY and ROS induced by advanced glycation end products (AGE). However, ACLY overexpression partially eliminated the positive effects of the VDR agonist. Next, we verified the transcriptional regulation of ACLY by VDR through chromatin immunoprecipitation (ChIP)-qPCR and dual luciferase experiments. Moreover, in AGE models, knockdown of ACLY decreased lipid peroxidation and ROS production, while intervention with Nrf2 inhibitor ML385 partially weakened the protective effect of ACLY downregulation. In summary, VDR negatively regulates the expression of ACLY through transcription, thereby affecting the state of Nrf2/Keap1 system and regulating lipid peroxidation, thereby inhibiting kidney injury induced by DN.

Investigation of Nrf2-Keap-1 pathway, Sestrin 2 and oxidative stress markers in serum of patients with placenta Accreata spectrum.

Placenta accreta spectrum (PAS) is an important disease group with risks such as maternal bleeding, hysterectomy, and death, which expresses the pathological adhesion of the placenta to the uterine myometrium, including placenta accreta, increta, and percreta, with an increased incidence with an increase in cesarean section rates. In this study, we aimed to investigate the Nuclear factor erythroid 2-related factor 2 (Nrf2)-Kelch-like ECH-related protein 1 (Keap1) pathway in these patients. Serum Sestrin 2, Nrf2, Keap1, glycogen synthase kinase 3ß (GSK-3ß), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) activities and malondialdehyde-modified low-density lipoprotein (MDA-LDL) levels were performed by the Enzyme-Linked Immunosorbent Assay (ELISA) method. In the findings obtained, Nrf2, Keap1, GSK-3ß, MDA-LDL levels, SOD and GSH-Px activities were statistically significantly different in the patient group compared to the control group. While MDA-LDL values were found to be high in the patient group, Nrf2, Keap1, GSK-3ß levels, SOD and GSH-Px activities were significantly lower, except for Sestrin 2 values. In addition, when grouped according to the degree of invasion, Nrf2 levels were found to be lower and Keap1 levels higher. As a result, it was determined that the Nrf2-Keap1 pathway was disrupted in PAS patients, and the oxidant/antioxidant balance was impaired in the oxidant direction. The results show that Nrf2 and Keap1 parameters can be useful in determining the degree of placental invasion.

New insights into evodiamine attenuates IPEC-J2 cells pyroptosis induced by T-2 toxin - Activating Keap1-Nrf2/NF-κB signaling pathway through binding with Keap1.

T-2 toxin (T-2) is a highly toxic mycotoxin with a molecular weight of 466.52 g/mol. Evodiamine (EV), an alkaloid component of Evodia, has anti-inflammation and antioxidant properties. As a receptor of oxidative stress, Keap1 with a molecular weight of 70 kDa, is a molecular switch that controls the Nrf2 signaling pathway. In this paper, the effect of EV on Keap1-Nrf2/NF-κB pathway was investigated. Based on our research outcomes, it was observed that T-2 exposure substantially increased IPEC-J2 cells intracellular ROS levels and MDA accumulation, decreased SOD and CAT activities, disrupted intestinal tight junction (ZO-1, occludin, and claudin-1), and up-regulated pyroptosis-related protein (ASC, NLRP3, caspase-1, GSDMD, IL-1ß, and IL-18). Additionally, EV could bind well with Keap1, the separating it from Nrf2, promoting Nrf2 into the nucleus, enhanced antioxidant enzyme activities, reduced the production of ROS, down-regulated NF-κB expression, alleviated T-2-induced pyroptosis, and restored tight junction protein expression. However, after treatment with the Nrf2 inhibitor ML385, ML385 reversed the protective effect of EV on IPEC-J2 cells. Collectively, EV can activate the Keap1-Nrf2/NF-κB signaling pathway via binding to Keap1, exert anti-inflammatory and antioxidant effects, inhibit the pyroptosis of IPEC-J2 cells triggered by T-2, and retore intestinal barrier function.

Nrf2 pathways in neuroprotection: Alleviating mitochondrial dysfunction and cognitive impairment in aging.

Mitochondrial dysfunction and cognitive impairment are widespread phenomena among the elderly, being crucial factors that contribute to neurodegenerative diseases. Nuclear factor erythroid 2-related factor 2 (Nrf2) is an important regulator of cellular defense systems, including that against oxidative stress. As such, increased Nrf2 activity may serve as a strategy to avert mitochondrial dysfunction and cognitive decline. Scientific data on Nrf2-mediated neuroprotection was collected from PubMed, Google Scholar, and Science Direct, specifically addressing mitochondrial dysfunction and cognitive impairment in older people. Search terms included "Nrf2", "mitochondrial dysfunction," "cognitive impairment," and "neuroprotection." Studies focusing on in vitro and in vivo models and clinical investigations were included to review Nrf2's therapeutic potential comprehensively. The relative studies have demonstrated that increased Nrf2 activity could improve mitochondrial performance, decrease oxidative pressure, and mitigate cognitive impairment. To a large extent, this is achieved through the modulation of critical cellular signalling pathways such as the Keap1/Nrf2 pathway, mitochondrial biogenesis, and neuroinflammatory responses. The present review summarizes the recent progress in comprehending the molecular mechanisms regarding the neuroprotective benefits mediated by Nrf2 through its substantial role against mitochondrial dysfunction and cognitive impairment. This review also emphasizes Nrf2-target pathways and their contribution to cognitive function improvement and rescue from mitochondria-related abnormalities as treatment strategies for neurodegenerative diseases that often affect elderly individuals.

Inhibition of ubiquitin-specific protease 7 ameliorates ferroptosis-mediated myocardial infarction by contrasting oxidative stress: An in vitro and in vivo analysis.

BACKGROUND: Our prior research determined that USP7 exacerbates myocardial injury. Additionally, existing studies indicate a strong connection between USP7 and ferroptosis. However, the influence of USP7 on ferroptosis-mediated myocardial infarction (MI) remains unclear. Given these findings, we are particularly interested in USP7's regulatory role in ferroptosis-mediated MI and its underlying mechanisms. METHODS: In this study, we established MI models and lentivirus-transfected groups to inhibit USP7 expression both in vivo and in vitro. Cardiac function was detected with Echocardiography. TTC and HE staining were employed to assess myocardial alterations. The expression of ferroptosis markers (PTGS2, ACSL4, GPX4) were analyzed by RT-qPCR and Western blotting. Flow cytometry and ELISA were used for measuring Fe2+, lipid ROS, GSH, and GSSG levels. TEM and Prussian blue staining were used to observe mitochondrial alterations and iron deposition. RT-qPCR, Western blotting, and immunofluorescence were conducted to analyze Keap1, Nrf2, and nuclear Nrf2 expression in vitro and in vivo. RESULTS: In the MI model group, USP7 expression significantly increased, worsening ferroptosis-mediated MI. Conversely, in the USP7-inhibited group, activation of the Keap1-Nrf2 signaling pathway improved ferroptosis-mediated MI outcomes. In vitro, the MI model exhibited a marked decline in cardiomyocyte viability and notable mitochondrial damage. However, these issues improved in the USP7-inhibited groups. In vivo, USP7 intensified MI and iron deposition within the MI model group, with decreased values of LVEF, LVFS, SV, LVAWd, and LVPWs, all of which showed improvement in the USP7-inhibited group, except for LVPWd and LVPWs, which showed no significant variation. Importantly, both the in vitro and in vivo experiments revealed analogous results: a reduction in Keap1 expression and an increase in both Nrf2 and nuclear Nrf2 post USP7 inhibition. Additionally, GPX4 expression decreased while PTGS2 and ACSL4 expressions increased. Notably, concentrations of Fe2+, lipid ROS, GSH, and GSSG significantly decreased. CONCLUSION: In vitro and in vivo studies have found that inhibition of USP7 attenuates iron deposition and suppresses oxidative stress, resulting in amelioration of ferroptosis-induced MI.

miR-141-3p promotes paclitaxel resistance by attenuating ferroptosis via the Keap1-Nrf2 signaling pathway in breast cancer.

Purpose: Breast cancer poses a huge threat to the lives and health of women worldwide. However, drug resistance makes the treatment of breast cancer challenging. This study aims to investigate the effect of miR-141-3p on paclitaxel resistance and its underlying mechanisms in breast cancer. Methods: Using bioinformatics analysis and qRT-PCR to explore the potential molecule miR-141-3p. Specific binding of miR-141-3p to Keap1 was determined by using a dual luciferase reporter assay. qRT-PCR and Western blot were utilized to observe the expression of miR-141-3p, Keap1, Nrf2, SLC7A11 and GPX4. GSH/GSSG content, MDA content and JC-1 assays were used to observe the ferroptosis levels of breast cancer cells. CCK-8 assay was used to observe the cell viability of breast cancer cells. Tumor subcutaneous transplantation experiment was used to understand the effect of miR-141-3p on paclitaxel resistance in breast cancer in vivo. Results: In the present study, miR-141-3p was found to be highly expressed and associated with poor prognosis in breast cancer. miR-141-3p inhibited Keap1 expression, promoted Nrf2 expression, and facilitated paclitaxel resistance in breast cancer cells. Inhibition of miR-141-3p promoted Keap1 expression, inhibited Nrf2 and its downstream SLC7A11-GSH-GPX4 signaling pathway, as well as promoted ferroptosis in cancer cells, and inhibited paclitaxel and RSL3 resistance. ML385 blocks the effect of miR-141-3p on paclitaxel resistance and ferroptosis resistance in breast cancer cells. In vivo, miR-141-3p mimics promoted paclitaxel resistance, whereas miR-141-3p inhibitors inhibited paclitaxel resistance in breast cancer cells. Conclusion: This work revealed that modulation of the Keap1-Nrf2 signaling pathway by miR-141-3p promoted paclitaxel resistance via regulating ferroptosis in breast cancer cells.

Allosteric regulation of Keap1 by 8ß-hydroxy-α-cyclocostunolide for the treatment of acute lung injury.

Image 1.

The role of Keap1-Nrf2 signaling pathway in the treatment of respiratory diseases and the research progress on targeted drugs.

Lungs are exposed to external oxidants from the environment as in harmful particles and smog, causing oxidative stress in the lungs and consequently respiratory ailment. The NF-E2-related factor 2 (Nrf2) is the one with transcriptional regulatory function, while its related protein Kelch-like ECH-associated protein 1 (Keap1) inhibits Nrf2 activity. Together, they form the Keap1-Nrf2 pathway, which regulates the body's defense against oxidative stress. This pathway has been shown to maintain cellular homeostasis during oxidative stressing, inflammation, oncogenesis, and apoptosis by coordinating the expression of cytoprotective genes and making it a potential therapeutic target for respiratory diseases. This paper summarizes this point in detail in Chapter 2. In addition, this article summarizes the current drug development and clinical research progress related to the Keap1-Nrf2 signaling pathway, with a focus on the potential of Nrf2 agonists in treating respiratory diseases. Overall, the article reviews the regulatory mechanisms of the Keap1-Nrf2 signaling pathway in respiratory diseases and the progress of targeted drug research, aiming to provide new insights for treatment.

Nuclear factor erythroid 2-related factor 2 (Nrf2) signaling in heavy metals-induced oxidative stress.

Organisms encounter reactive oxidants through intrinsic metabolism and environmental exposure to toxicants. Reactive oxygen and nitrogen species (ROS, RNS) are generally considered detrimental because they induce oxidative stress. In order to combat oxidative stress, a potential modulator of cellular defense nuclear factor erythroid 2-related factor 2 (Nrf2) and its endogenous inhibitor Kelch-like ECH-associated protein 1 (Keap1) operate as a common, genetically preserved intrinsic defense system. There has been a significant increase in the amount of harmful metalloids and metals that individuals are exposed to through their food, water, and air, primarily due to human activities. Many studies have looked at the connection between the emergence of different ailments in humans and ecological exposure to metalloids, i.e., arsenic (As) and metals viz., chromium (Cr), mercury (Hg), cadmium (Cd), cobalt (Co), and lead (Pb). It is known that they can produce ROS in several organs by both direct and indirect means. Studies suggest that Nrf2 signaling is a crucial mechanism in maintaining antioxidant balance and can have two roles, depending on the particular biological setting. From one perspective, Nrf2 is an essential defense mechanism against metal-induced toxicity. Still, it may also operate as a catalyst for metal-induced carcinogenesis in situations involving protracted exposure and persistent activation. Therefore, this review aims to provide an overview of the antioxidant defense mechanism of Nrf2-Keap1 signaling and the interrelation between Nrf2 signaling and the toxic elements.

Lutein protects senescent ciliary muscle against oxidative stress through the Keap1/Nrf2/ARE pathway.

BACKGROUND: Aging-induced decline in ciliary muscle function is an important factor in visual accommodative deficits in elderly adults. With this study, we provide an innovative investigation of the interaction between ciliary muscle aging and oxidative stress. METHODS: Tricolor guinea pigs were used for the experiments in vivo and primary guinea pig ciliary smooth muscle cells were used for the experiments in vitro. RESULTS: We enriched for genes associated with muscle-aging-lutein relationship using bioinformatics, including Nuclear factor-erythroid 2-related factor-2 (Nrf2), Glutathione Peroxidase (GPx) gene family, Superoxide Dismutase (SOD) gene family, NAD(P)H: Quinone Oxidoreductase 1 (NQO1) and Heme Oxygenase-1 (HO-1). After gavage to aged guinea pigs, lutein reduced Reactive Oxygen Species (ROS) and P21 levels in senescent ciliary muscle; lutein decreased refractive error and restored accommodation of the eye. In addition, lutein increased GPx, SOD, and Catalase (CAT) levels in serum; lutein increased GPx and CAT levels in ciliary bodies. Lutein regulated the expression of proteins such as Nrf2, Kelch-like ECH-associated protein 1 (Keap1), and downstream proteins in senescent ciliary bodies. Similarly, guinea pig ciliary muscle cell senescence was associated with oxidative stress. In vitro, 100 µM lutein reversed the damage caused by 800 µM H2O2; it reduced Senescence-Associated ß-galactosidase (SA-ß-Gal) and ROS activites, cell apoptosis and cell migration. Also, lutein increased the expression of smooth muscle contractile proteins. Lutein also increased the expression of Nrf2, GPx2, NQO1 and HO-1, decreased the expression of Keap1. A reduction in Nrf2 activity led to a reduction in the ability of lutein to activate antioxidant enzymes in the cells, thus reducing its inhibitory effect on cell senescence. CONCLUSION: lutein improved resistance to oxidative stress in senescent ciliary muscle in vivo and in vitro by regulating the Keap1/Nrf2/Antioxidant Response Element pathway. We have innovatively demonstrated the molecular pharmacological mechanism by which lutein reverse age-related ciliary muscle systolic and diastolic deficits.

Circ_001653 alleviates sepsis associated-acute kidney injury by recruiting BUD13 to regulate KEAP1/NRF2/HO-1 signaling pathway.

BACKGROUND: The kidney is exceptionally vulnerable during sepsis, often resulting in sepsis-associated acute kidney injury (SA-AKI), a condition that not only escalates morbidity but also significantly raises sepsis-related mortality rates. Circular RNA circ_001653 has been previously reported to be upregulated in the serum of SA-AKI patients, while the role and underlying mechanism of circ_001653 in SA-AKI remains unknown. In this study, we aimed to explore the functional role and the molecular mechanism of circ_001653 in the pathogenesis of SA-AKI. METHODS: LPS-stimulated HK-2 cells and ligation and perforation of cecum (CLP)-induced rats were used as in vitro and in vivo models of SA-AKI. The target gene expression levels were measured using qRT-PCR and western blot. Renal function (BUN, sCr, uNGAL, and uKIM-1), and renal pathological changes were detected in septic mice. TUNEL and EdU assays were conducted to measure apoptosis and proliferation rates in vitro. DCFH-DA staining was used to detect ROS levels in vitro and in vivo. Oxidative stress markers (SOD, GSH-Px, MDA, and SOD), and inflammation markers (IL-1ß, IL-6, and TNF-α) were determined using commercial kits both in vitro and in vivo. Additionally, gain-and-loss-of-function assays and mechanistic experiments were conducted to explore the regulatory role of circ_001653 in SA-AKI pathogenesis. RESULTS: Data showed that circ_001653 expression was high in LPS-stimulated HK-2 cells and CLP-induced rat renal tissue and was mainly localized in the cytoplasm. Notably, circ_001653 silencing alleviated SA-AKI by reducing apoptosis and alleviating oxidative stress and inflammation in HK-2 cells and renal tissue of rats. Mechanistically, it was found that circ_001653 alleviated SA-AKI by recruiting BUD13 to activate the KEAP1/Nrf2/HO-1 signaling pathway. CONCLUSIONS: To summarize, our study is the first to reveal elevated expression of circ_001653 in sepsis-associated AKI, and its downregulation effectively attenuates AKI by reducing apoptosis, inflammation, and oxidative stress. Mechanistically, circ_001653 exerts its effects by recruiting BUD13 to activate the KEAP1/Nrf2/HO-1 signaling pathway. These findings suggest circ_001653 as a potential therapeutic target for the drug development of sepsis-associated AKI.

[Suppression effect of secoisolariciresinol diglucoside against trans fatty acids-induced oxidative damage and inflammatory in brain of offspring mice].

OBJECTIVE: To probe into the protective effect of different dose of secoisolariciresinol diglucoside(SDG) on brain of offspring of mice anainst oxidative damage and inflammatory reaction induced by maternal exposure to trans fatty acids(TFA) during gestation, and observe the the changes of regulating Nrf2/Keap1 pathway in the course. METHODS: 30 healthy female mice(C57BL/6) were divided into 5 groups randomly, they are respectively control group, TFA-exposed group, and three SDG-intervention groups(low-(TFA+LSDG), medium-(TFA+MSDG) and high-(TFA+HSDG)). The pregnancy mice of control group and TFA group were treated with distilled water and 60 mg/kg·d TFA by gavage, in the same time, the mice of three SDG-intervention groups were treated with 60 mg/kg·d TFA by gavage and fed with feed included SDG(10, 20 and 30 mg/kg). The treatment to pregnancy mice continued to birth of offspring. After 21 days of lactation, the offspring were killed under anesthesia and the experiment was ended. The coefficient of brain was calculated. The levels of superoxide dismutase(SOD), glutathione peroxidase(GSH-Px), malondialdehyde(MDA), tumor necrosis factor-α(TNF-α), interferon-γ(IFN-γ) and amyloid-ß(Aß)of brain were detected. RT-PCR and Western Blot was used to detected gene expression and protein levels of nuclear factor erythroid-2 related factor 2(Nrf2), kelch-like ECH-associated protein 1(Keap1), quinone oxidoreductase 1(NQO1) and hemeoxygenase-l(HO-1). RESULTS: Compared with control group, the brain coefficient and Aß1-40 of offspring of TFA-group had no significant changes(P>0.05), the activity of SOD and GSH-Px reduced, the content of MDA, IFN-γ, TNF-α and Aß1-42 increased, the level of mRNA and protein expression of Nrf2, NQO1 and HO-1 decreased and the level of mRNA and protein expression of Keap1 increase because of the exposion to TFA during gestation and all the differences were statistically significant(P<0.05). Compared with TFA-group, the brain coefficient, Aß1-40 and the level of NQO1 mRNA of offspring of three SDG-intervention groups had no significant changes(P>0.05), the activity of SOD(the middle and high dose SDG intervention groups) and GSH-Px(three SDG-intervention groups) increased, the content of MDA(the middle and high dose SDG intervention groups), IFN-γ(the middle and high dose SDG intervention groups), TNF-α(three SDG-intervention groups) and Aß1-42(the middle and high dose SDG intervention groups) decreased, the mRNA expression of Nrf2 and HO-1(the middle and high dose SDG intervention groups) was up-regulated, the mRNA expression of Keap1(the middle and high dose SDG intervention group) decreased, proteic expression of Nrf2, NQO1 and HO-1 of three SDG-intervention groups increase and the level of protein of Keap1 decreased because of the intervention of SDG during gestation(P<0.05). CONCLUSION: These result suggest that maternal TFA exposure during gestation can result in oxidative stress and inflammation to brain of offspring in a way. SDG can protect brain of mice of offspring from TFA-induced oxidative injury by up-regulating the expression of mRNA and protein of Nrf2, down-regulating the expression of Keap1, accelerating expression of protein of NQO1 and HO-1 which are antioxidant protein lying downstream of pathway of Nrf2/Keap1.

KEAP1-NRF2 pathway as a novel therapeutic target for EGFR-mutant non-small cell lung cancer.

BACKGROUND: Kelch-like ECH-associated protein 1 (KEAP1)-nuclear factor erythroid-2-related factor 2 (NRF2) pathway is a major regulator protecting cells from oxidative and metabolic stress. Studies have revealed that this pathway is involved in mediating resistance to cytotoxic chemotherapy and immunotherapy, however, its implications in oncogene-addicted tumors are largely unknown. This study aimed to elucidate whether this pathway could be a potential therapeutic target for epidermal growth factor receptor (EGFR)-mutant non-small cell lung cancer. METHODS: We measured the baseline expression of NRF2 using EGFR-mutant parental cells and acquired gefitinib resistant cells. We investigated whether NRF2 inhibition affected cell death in vitro and tumor growth in vivo using a xenograft mouse model, and compared the transcriptional changes before and after NRF2 inhibition. RESULTS: Baseline NRF2 expression was enhanced in PC9 and PC9 with gefitinib resistance (PC9/GR) cells than in other cell lines, with a more prominent expression in PC9/GR. The NRF2 inhibitor induced NRF2 downregulation and cell death in a dose-dependent manner. Co-treatment with an NRF2 inhibitor enhanced osimertinib-induced cell death in vitro, and potentiated tumor growth inhibition in a PC9/GR xenograft model. Finally, RNA sequencing revealed that NRF2 inhibition resulted in the altered expression of multiple genes involved in various signaling pathways. CONCLUSION: We identified that NRF2 inhibition enhanced cell death and inhibited tumor growth in TKI-resistant lung cancer with EGFR-mutation. Thus, NRF2 modulation may be a novel therapeutic strategy to overcome the resistance to EGFR-tyrosine kinase inhibitors.

Natural Linoleic Acid from Marine Fungus Eutypella sp. F0219 Blocks KEAP1/NRF2 Interaction and Ameliorates MASLD by Targeting FABP4.

Ectopic lipid accumulation induced lipotoxicity plays a crucial role in exacerbating the development of metabolic dysfunction-associated steatotic liver disease (MASLD), which affects over 30% of the worldwide population and 85% of the obese population. The growing demand for effective therapeutic agents highlights the need for high-efficacy lipotoxicity ameliorators and relevant therapeutic targets in the fight against MASLD. This study aimed to discover natural anti-lipotoxic and anti-MASLD candidates and elucidate the underlying mechanism and therapeutic targets. Utilizing palmitic acid (PA)-induced HepG-2 and primary mouse hepatocyte models, we identified linoleic acid (HN-002), a ligand of fatty acid binding protein 4 (FABP4), from the marine fungus Eutypella sp. F0219. HN-002 dose-dependently prevented lipid overload-induced hepatocyte damage and lipid accumulation, inhibited fatty acid esterification, and ameliorated oxidative stress. These beneficial effects were associated with improvements in mitochondrial adaptive oxidation. HN-002 treatment enhanced lipid transport into mitochondria and oxidation, inhibited mitochondrial depolarization, and reduced mitochondrial ROS (mtROS) level in PA-treated hepatocytes. Mechanistically, HN-002 treatment disrupted the interaction between KEAP1 and NRF2, leading to NRF2 deubiquitylation and nuclear translocation, which activated beneficial metabolic regulation. In vivo, HN-002 treatment (20 mg/kg/per 2 days, i. p.) for 25 days effectively reversed hepatic steatosis and liver injury in the fast/refeeding plus high-fat/high-cholesterol diet induced MASLD mice. These therapeutic effects were associated with enhanced mitochondrial adaptive oxidation and activation of NRF2 signaling in the liver. These data suggest that HN-002 would be an interesting candidate for MASLD by improving mitochondrial oxidation via the FABP4/KEAP1/NRF2 axis. The discovery offers new insights into developing novel anti- MASLD agents derived from marine sources.

Artemisinin inhibits neuronal ferroptosis in Alzheimer's disease models by targeting KEAP1.

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 Aß1-42-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.

Quercetin inhibited LPS-induced cytokine storm by interacting with the AKT1-FoxO1 and Keap1-Nrf2 signaling pathway in macrophages.

Cytokine storm (CS) emerges as an exacerbated inflammatory response triggered by various factors such as pathogens and excessive immunotherapy, posing a significant threat to life if left unchecked. Quercetin, a monomer found in traditional Chinese medicine, exhibits notable anti-inflammatory and antiviral properties. This study endeavors to explore whether quercetin intervention could mitigate CS through a combination of network pharmacology analysis and experimental validation. First, common target genes and potential mechanisms affected by quercetin and CS were identified through network pharmacology, and molecular docking experiments confirmed quercetin and core targets. Subsequently, in vitro experiments of Raw264.7 cells stimulated by lipopolysaccharide (LPS) showed that quercetin could effectively inhibit the overexpression of pro-inflammatory mediators and regulate the AKT1-FoxO1 signaling pathway. At the same time, quercetin can reduce ROS through the Keap1-Nrf2 signaling pathway. In addition, in vivo studies of C57BL/6 mice injected with LPS further confirmed quercetin's inhibitory effect on CS. In conclusion, this investigation elucidated novel target genes and signaling pathways implicated in the therapeutic effects of quercetin on CS. Moreover, it provided compelling evidence supporting the efficacy of quercetin in reversing LPS-induced CS, primarily through the regulation of the AKT1-FoxO1 and Keap1-Nrf2 signaling pathways.

Activation of the Nrf2/Keap1 signaling pathway mediates the neuroprotective effect of Perillyl alcohol against cerebral hypoxic-ischemic damage in neonatal rats.

Neonatal hypoxic-ischemic encephalopathy (HIE) is a severe disease with a poor prognosis, whose clinical treatment is still limited to therapeutic hypothermia with limited efficacy. Perillyl alcohol (POH), a natural monoterpene found in various plant essential oils, has shown neuroprotective properties, though its effects on HIE are not well understood. This study investigates the neuroprotective effects of POH on HIE both in vitro and in vivo. We established an in vitro model using glucose deprivation and hypoxia/reperfusion (OGD/R) in PC12 cells, alongside an in vivo model via the modified Rice-Vannucci method. Results indicated that POH acted as an indirect antioxidant, reducing inducible nitric oxide synthase and malondialdehyde production, maintaining content of antioxidant molecules and enzymes in OGD/R-induced PC12 cells. In vivo, POH remarkably lessened infarct volume, reduced cerebral edema, accelerated tissue regeneration, and blocked reactive astrogliosis after hypoxic-ischemic brain injury. POH exerted antiapoptotic activities through both the intrinsic and extrinsic apoptotic pathways. Mechanistically, POH activated Nrf2 and inactivated its negative regulator Keap1. The use of ML385, a Nrf2 inhibitor, reversed these effects. Overall, POH mitigates neuronal damage in HIE by combating oxidative stress, reducing inflammation, and inhibiting apoptosis via the Nrf2/Keap1 pathway, suggesting its potential for HIE treatment.

Serine ubiquitination of SQSTM1 regulates NFE2L2-dependent redox homeostasis.

The KEAP1-NFE2L2 axis is essential for the cellular response against metabolic and oxidative stress. KEAP1 is an adaptor protein of CUL3 (cullin 3) ubiquitin ligase that controls the cellular levels of NFE2L2, a critical transcription factor of several cytoprotective genes. Oxidative stress, defective autophagy and pathogenic infections activate NFE2L2 signaling through phosphorylation of the autophagy receptor protein SQSTM1, which competes with NFE2L2 for binding to KEAP1. Here we show that phosphoribosyl-linked serine ubiquitination of SQSTM1 catalyzed by SidE effectors of Legionella pneumophila controls NFE2L2 signaling and cell metabolism upon Legionella infection. Serine ubiquitination of SQSTM1 sterically blocks its binding to KEAP1, resulting in NFE2L2 ubiquitination and degradation. This reduces NFE2L2-dependent antioxidant synthesis in the early phase of infection. Levels of serine ubiquitinated SQSTM1 diminish in the later stage of infection allowing the expression of NFE2L2-target genes; causing a differential regulation of the host metabolome and proteome in a NFE2L2-dependent manner.