Kelch-like ECH-associated Protein Research Articles

Oxidative Stress-regulating Enzymes and Endometrial Cancer Survival in Relation to Metformin Intake in Diabetic Patients.

Metformin inhibits tumorigenesis in endometrial carcinoma and interferes with the expression of oxidative stress-regulating proteins, such as nuclear factor erythroid 2-related factor 2 (Nrf2) and Kelch-like ECH-associated protein 1 (Keap1). Although manganese superoxide dismutase (MnSOD) is vital for withstanding mitochondrial oxidative stress, it has also been linked with chemoresistance and poorer outcomes in several cancer types. However, data on endometrial cancers are limited. This study aimed to highlight the relationship between mitochondrial redox regulation and endometrial cancer survival in relation to metformin consumption in women with type 2 diabetes mellitus (T2DM). Our retrospective hospital-based cohort study included 121 patients diagnosed with endometrial carcinoma and T2DM between 2007 and 2014. Fifty-eight patients were using metformin at the time of diagnosis. Nrf2 and Keap1 expression levels in the tumor samples were assessed immunohistochemically, and MnSOD levels were measured both immunohistochemically and from the serum samples. High MnSOD tissue expression was associated with better overall survival among metformin users in the univariate analysis (p=0.03). When adjusted for histology and stage, high serum MnSOD was associated with better overall survival (HR=0.22, 95%CI=0.07-0.71, p=0.01). No association was found between MnSOD, Nrf2, or Keap1 and overall survival among metformin non-users. Higher expression of MnSOD in patients with endometrial cancer and T2DM is associated with better overall survival if the patient is consuming metformin.

Amentoflavone mediated hepatoprotection to counteract paraquat instigated hepatotoxicity via modulating Nrf2/keap1 pathway: A biochemical, inflammatory, apoptotic and histopathological study

Paraquat (PQ) is a ubiquitous and water-soluble herbicide which has potential to cause systematic poisoning. PQ intoxication is known to be associated with various clinical complications including hepatotoxicity. Amentoflavone (AMF) is an active phenolic compound that exhibits a broad range of biological as well as pharmacological activities. This study was designed to determine the hepato-protective potential of AMF against PQ instigated hepatotoxicity in rats. Forty-eight rats were distributed into four groups such as control group, PQ-treated group (5 mg/kg), PQ (5 mg/kg) + AMF (40 mg/kg) exposed group and AMF (40 mg/kg) only supplemented group. It was revealed that PQ exposure reduced nuclear factor erythroid 2–related factor 2 (Nrf2) and antioxidative genes expression whereas increase the expression of Kelch-like ECH-associated protein 1(Keap1). Besides, PQ intoxication reduced the activities of superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GSR), glutathione peroxidase (GPx), Heme- oxygenase-1 (HO-1) & glutathione (GSH) content. Furthermore, the levels of reactive oxygen species (ROS) & malondialdehyde (MDA) were increased. In addition, PQ significantly increased the hepatic serum enzymes including alkaline phosphatase (ALP), aspartate transaminase (AST), & alanine transaminase (ALT) along with inflammatory biomarkers levels such as tumor necrosis- α (TNF- α), nuclear factor- κB (NF-κB), interleukin-6 (IL-6), interleukin 1beta (IL-1β), & cyclo‑oxygenase-2 (COX-2) activity. PQ intoxication increased the expressions of pro-apoptotic markers i.e., Bcl-2-associated X protein (Bax) & Cysteine-aspartic protease-3 (Caspase-3) while reducing the expression of anti-apoptotic protein B-cell lymphoma 2 (Bcl-2). Furthermore, PQ intoxication prompted various histopathological impairments. However, the co-administration of AMF significantly improved the abovementioned hepatic damages induced by PQ. The present study indicated that AMF may be an effective therapeutic candidate to mitigate PQ provoked hepatic impairments due to its anti-apoptotic, antioxidant & anti-inflammatory properties.

Attenuative effects of tamarixetin against polystyrene microplastics-induced hepatotoxicity in rats by regulation of Nrf-2/Keap-1 pathway.

Polystyrene microplastics (PS-MPs) are environmental contaminants due to their potential to induce damages in multiple organs specifically liver. Tamarixetin (TMT) is a naturally occurring flavonoid present in Tamarix ramosissima plant that exhibits multiple pharmacological properties. Therefore, the present research was designed to evaluate the palliative role of TMT against PS-MPs instigated liver dysfunction in rats. The exposure to PS-MPs reduced the expressions of nuclear factor erythroid 2-related factor 2and antioxidant genes, while increasing the expression of Kelch-like ECH-associated protein 1. PS-MPs exposed rats exhibited considerably (p < .05) higher alkaline phosphatase (ALP), aspartate aminotransferase (AST) as well as alanine aminotransferase (ALT) contents. Additionally, PS-MPs treatment resulted in a notable decrease in anti-oxidants activity, that is, glutathione S-transferase (GST), superoxide dismutase (SOD), heme oxygenase-1 (HO-1), glutathione reductase (GSR), glutathione peroxidase (GPx), catalase (CAT) and glutathione (GSH) content, whereas upregulating reactive oxygen species (ROS) and malondialdehyde (MDA) contents. Moreover, PS-MPs intoxication noticeably increased (p < .05) the inflammatory indices (interleukin-1ß [IL-1ß],nuclear factor kappa B [NF-κB], interleukin-6 [IL-6], tumor necrosis factor-α [TNF-α] levels, and cyclooxygenase-2 [COX-2] activity). Besides, Caspase-3 and Bax expressions were upregulated and Bcl-2 expression was decreased after PS-MPs exposure. Additionally, the histomorphological examination revealed notable hepatic damage in PS-MPs treated group. However, TMT treatment substantially (p < .05) recovered all the PS-MPs-induced damages and histopathological changes. Taken together, it can be deduced that TMT might be used as a pharmacological agent to ameliorate hepatic damage.

Dissecting molecular mechanisms underlying ferroptosis in human umbilical cord mesenchymal stem cells: Role of cystathionine γ-lyase/hydrogen sulfide pathway.

Ferroptosis can induce low retention and engraftment after mesenchymal stem cell (MSC) delivery, which is considered a major challenge to the effectiveness of MSC-based pulmonary arterial hypertension (PAH) therapy. Interestingly, the cystathionine γ-lyase (CSE)/hydrogen sulfide (H2S) pathway may contribute to mediating ferroptosis. However, the influence of the CSE/H2S pathway on ferroptosis in human umbilical cord MSCs (HUCMSCs) remains unclear. To clarify whether the effect of HUCMSCs on vascular remodelling in PAH mice is affected by CSE/H2S pathway-mediated ferroptosis, and to investigate the functions of the CSE/H2S pathway in ferroptosis in HUCMSCs and the underlying mechanisms. Erastin and ferrostatin-1 (Fer-1) were used to induce and inhibit ferroptosis, respectively. HUCMSCs were transfected with a vector to overexpress or inhibit expression of CSE. A PAH mouse model was established using 4-wk-old male BALB/c nude mice under hypoxic conditions, and pulmonary pressure and vascular remodelling were measured. The survival of HUCMSCs after delivery was observed by in vivo bioluminescence imaging. Cell viability, iron accumulation, reactive oxygen species production, cystine uptake, and lipid peroxidation in HUCMSCs were tested. Ferroptosis-related proteins and S-sulfhydrated Kelch-like ECH-associating protein 1 (Keap1) were detected by western blot analysis. In vivo, CSE overexpression improved cell survival after erastin-treated HUCMSC delivery in mice with hypoxia-induced PAH. In vitro, CSE overexpression improved H2S production and ferroptosis-related indexes, such as cell viability, iron level, reactive oxygen species production, cystine uptake, lipid peroxidation, mitochondrial membrane density, and ferroptosis-related protein expression, in erastin-treated HUCMSCs. In contrast, in vivo, CSE inhibition decreased cell survival after Fer-1-treated HUCMSC delivery and aggravated vascular remodelling in PAH mice. In vitro, CSE inhibition decreased H2S levels and restored ferroptosis in Fer-1-treated HUCMSCs. Interestingly, upregulation of the CSE/H2S pathway induced Keap1 S-sulfhydration, which contributed to the inhibition of ferroptosis. Regulation of the CSE/H2S pathway in HUCMSCs contributes to the inhibition of ferroptosis and improves the suppressive effect on vascular remodelling in mice with hypoxia-induced PAH. Moreover, the protective effect of the CSE/H2S pathway against ferroptosis in HUCMSCs is mediated via S-sulfhydrated Keap1/nuclear factor erythroid 2-related factor 2 signalling. The present study may provide a novel therapeutic avenue for improving the protective capacity of transplanted MSCs in PAH.

Age-induced aortic modifications are accompanied by alterations in the antioxidant defense system in female rats

IntroductionAging leads to significant structural and functional changes in blood vessels, which disrupt their normal function and impact cardiovascular health. Current research is actively exploring the NRF2 antioxidative pathway, recognizing its role in protecting cells by preserving their antioxidant defenses against damage. However, there has been limited exploration into the role of the NRF2 pathway in vascular aging. The primary objective of this study was to determine whether age-related changes in the aorta are associated with variations in the baseline levels of antioxidant enzymes, with a particular emphasis on how the NRF2 pathway operates in the aortic wall.MethodsA group of healthy aging female SD rats was compared with their younger counterparts. Various assessments were conducted, including measuring blood pressure, analyzing serum lipid profiles, examining aortic tissue, and assessing the expression of antioxidant enzymes.ResultsThe results revealed significant differences in both blood pressure and serum lipid levels between the aged and younger rats. The examination of the aorta in older rats showed structural alterations, increased apoptosis, and the accumulation of fatty deposits. In the older rats, levels of SOD-1 (superoxide dismutase) and GSS (glutathione synthetase) were lower, whereas NRF2, KEAP-1 (Kelch-like ECH-associated protein 1), and HO-1 (Heme oxygenase 1) were higher.DiscussionThis study advances our understanding of how aging affects the antioxidant system in blood vessels, particularly in relation to the regulation of the NRF2/HO-1 pathway in the aorta. These findings suggest that targeting the NRF2/HO-1 pathway could present anovel therapeutic approach for addressing age-related vascular issues.

Sevoflurane alleviates oxygen-glucose deprivation/reoxygenation-induced damage in HT22 cells by activating the Keap1/Nrf2/ARE pathway to inhibit oxidative stress

Objective: This study aimed to investigate the impact of sevoflurane on oxygen-glucose deprivation/reoxygenation-induced damage in HT22 cells and its associated mechanisms. Methods: HT22 cells were treated with sevoflurane, and an oxygen-glucose deprivation/reoxygenation injury model was established. The HT22 cells were randomly divided into the control group, oxygen-glucose deprivation/reoxygenation group, sevoflurane low-dose group, sevoflurane medium-dose group, and sevoflurane high-dose group. The proliferation of HT22 cells was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The apoptosis rate and mitochondrial membrane potential of HT22 cells were determined by flow cytometry. Protein expression levels of B-cell lymphoma-2-associated X protein (Bax), B-cell lymphoma-2 (Bcl-2), nuclear factor erythroid 2-related factor 2 (Nrf2), Kelch-like ECH-associated protein 1 (Keap1), and heme oxygenase-1 (HO-1) in HT22 cells were examined using Western blot. Reactive oxygen species (ROS) levels were measured with 2',7'-dichlorofluorescin diacetate (DCFH-DA). Malondialdehyde (MDA), glutathione peroxidase (GSH-Px) levels, and superoxide dismutase (SOD) enzyme activity in HT22 cells were determined using assay kits. Results: Compared to controls, OGD/R group had reduced cell viability, mitochondrial potential, Bcl-2, nuclear Nrf2, HO-1, GSH-Px levels, and SOD enzyme activity (p < 0.05), with increased apoptosis, Bax, cytoplasmic Nrf2, ROS, and MDA levels. Sevoflurane groups showed opposite trends (p < 0.05). Conclusion: Sevoflurane can mitigate oxygen-glucose deprivation/reoxygenation-induced damage in HT22 cells, and its mechanism may be related to the activation of the Keap1/Nrf2/ARE pathway to inhibit oxidative stress.

Sedanolide Activates KEAP1-NRF2 Pathway and Ameliorates Hydrogen Peroxide-Induced Apoptotic Cell Death.

Sedanolide is a bioactive compound with anti-inflammatory and antitumor activities. Although it has been recently suggested that sedanolide activates the nuclear factor E2-related factor 2 (NRF2) pathway, there is little research on its effects on cellular resistance to oxidative stress. The objective of the present study was to investigate the function of sedanolide in suppressing hydrogen peroxide (H2O2)-induced oxidative damage and the underlying molecular mechanisms in human hepatoblastoma cell line HepG2 cells. We found that sedanolide activated the antioxidant response element (ARE)-dependent transcription mediated by the nuclear translocation of NRF2. Pathway enrichment analysis of RNA sequencing data revealed that sedanolide upregulated the transcription of antioxidant enzymes involved in the NRF2 pathway and glutathione metabolism. Then, we further investigated whether sedanolide exerts cytoprotective effects against H2O2-induced cell death. We showed that sedanolide significantly attenuated cytosolic and mitochondrial reactive oxygen species (ROS) generation induced by exposure to H2O2. Furthermore, we demonstrated that pretreatment with sedanolide conferred a significant cytoprotective effect against H2O2-induced cell death probably due to preventing the decrease in the mitochondrial membrane potential and the increase in caspase-3/7 activity. Our study demonstrated that sedanolide enhanced cellular resistance to oxidative damage via the activation of the Kelch-like ECH-associated protein 1 (KEAP1)-NRF2 pathway.

α-Lipoic acid alleviates dextran sulfate sodium salt-induced ulcerative colitis via modulating the Keap1-Nrf2 signaling pathway and inhibiting ferroptosis.

Inflammatory bowel disease (IBD) is a chronically relapsing inflammatory disease with severe diarrhea, fatigue and weight loss. α-Lipoic acid (LA), a well-known antioxidant, is able to scavenge reactive oxygen species (ROS) and maintain a healthy cellular redox state. However, the role of LA in protecting IBD is still unclear. Hence the aim of this research was to investigate the protective effect of LA on dextran sulfate sodium salt-induced ulcerative colitis (UC) and its underlying mechanism. Here, our findings showed that LA significantly alleviated UC symptoms and the overproduction of pro-inflammatory cytokines in UC mice. In addition, LA treatment inhibited intestinal cell apoptosis by regulating the expression levels of p53/caspase-3 pathway-related protein in UC mice. Meanwhile, the inhibitory effects of LA on colonic oxidative stress and ferroptosis were revealed. Our study further demonstrated that LA treatment could regulate the Kelch-like ECH-associating protein 1 (Keap1)-nuclear factor E2-related factor 2 (Nrf2) signaling pathway. Interestingly, we confirmed that LA inhibited ferroptosis by attenuating endoplasmic reticulum stress and suppressing apoptosis in erastin-induced ferroptosis model in vitro. Taken together, this study's findings suggest that LA could be considered as a therapeutic agent protecting against IBD. © 2023 Society of Chemical Industry.

The role of MOTS-c-mediated antioxidant defense in aerobic exercise alleviating diabetic myocardial injury.

Myocardial remodeling and dysfunction are commonly observed in type 2 diabetes mellitus (T2DM). Aerobic exercise can partly alleviate diabetes-induced myocardial dysfunction through its antioxidant actions. MOTS-c is a potential exercise mimic. This study aimed to investigate the effects of MOTS-c on improving diabetic heart function and its mechanism and to identify whether MOTS-c improved antioxidant defenses due to aerobic exercise. Herein, we established a rat model of T2DM induced by high-fat diet combined with a low-dose streptozotocin injection. Interventions were performed using intraperitoneal injections of MOTS-c (i.p. 0.5mg/kg/day, 7days/week) or aerobic exercise training (treadmill, 20 m/min, 60 min/day, 5days/week) for 8 weeks. Myocardial ultrastructure was assessed using transmission electron microscopy (TEM), myocardial lipid peroxidation levels (MDA), superoxide dismutase (SOD), glutathione (GSH), and catalase (CAT) levels were assessed using colorimetric methods, and molecular analyses including MOTS-c, Kelch-like ECH-associated protein 1 (Keap1), Nuclear factor E2-related factor 2 (Nrf2), adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK)and phospho-AMPK (p-AMPK) were examined using Western blot. The results showed that MOTS-c, with or without exercise, reduced myocardial ultrastructural damage and improved glucolipid metabolism and cardiac function in T2DM. Furthermore, MOTS-c increased antioxidant markers such as SOD, CAT, and the protein expression of myocardial MOTS-c, Keap1, Nrf2, and p-AMPK. MOTS-c with exercise treatment reduced myocardial MDA and increased p-AMPK significantly comparing to only exercise or MOTS-c alone. Our findings suggest that MOTS-c may be a helpful supplement for overcoming exercise insufficiency and improving myocardial structure and function in diabetes.

Bardoxolone methyl inhibits the infection of rabies virus via Nrf2 pathway activation in vitro

BackgroundRabies is a widespread, fatal, infectious disease. Several antivirals against rabies virus (RABV) infection have been reported, but no approved, RABV-specific antiviral drugs that inhibit RABV infection in the clinic after symptom onset are available. Therefore, more effective drugs to reduce rabies fatalities are urgently needed. Bardoxolone methyl (CDDO-Me), an FDA-approved compound that has long been known as an antioxidant inflammatory modulator and one of the most potent nuclear factor erythroid-derived 2-like 2 (Nrf2) activators, protects myelin, axons, and CNS neurons by Nrf2 activation. Therefore, we investigated the potency of its anti-RABV activity in vitro.MethodsThe mouse neuroblastoma cell line Neuro2a (N2a) and three RABV strains of different virulence were used; the cytotoxicity and anti-RABV activity of CDDO-Me in N2a cells were evaluated by CCK-8 assay and direct fluorescent antibody (DFA) assay. Pathway activation in N2a cells infected with the RABV strains SC16, CVS-11 or CTN upon CDDO-Me treatment was evaluated by western blotting (WB) and DFA assay.ResultsCDDO-Me significantly inhibited infection of the three RABV strains of differing virulence (SC16, CVS-11 and CTN) in N2a cells. We also examined whether CDDO-Me activates the Nrf2-associated pathway upon infection with RABV strains of differing virulence. Nrf2, phosphorylated sequestosome (SQSTM1), SQSTM1, hemoglobin oxygenase (HO-1) and NAD(P)H dehydrogenase quinone 1 (NQO1) expression in N2a cells increased to varying degrees with CDDO-Me treatment, accompanied by Kelch-like ECH-associated protein 1 (Keap1) dissociation, upon infection with SC16, CVS-11 or CTN. The activation of SQSTM1 phosphorylation was significantly associated with the degradation of Keap-1 in CDDO-Me-treated N2a cells upon RABV infection. Furthermore, N2a cells pretreated with the Nrf2-specific inhibitor ATRA showed a significant decrease in HO-1 and NQO1 expression and a decrease in the anti-RABV efficacy of CDDO-Me. These inhibitory effects were observed upon infection with three RABV strains of differing virulence.ConclusionCDDO-Me inhibited RABV infection via Nrf2 activation, promoting a cytoprotective defense response in N2a cells. Our study provides a therapeutic strategy for RABV inhibition and neuroprotection during viral infection.

Taurine alleviates high-fat-high-glucose-induced pancreatic islet β-cell oxidative stress and apoptosis in rat

The effect of taurine (TAU) as a specific regulatory mediator on pancreatic function in obese rats induced by a high-fat-high-glucose (HFHG) diet was investigated. We fed male Sprague-Dawley rats under different conditions, namely the control, HFHG, TAU, and HFHG + TAU treatment groups for 4 months. Compared with the HFHG group, TAU supplementation significantly reduced malondialdehyde levels and increased superoxide dismutase, total antioxidant capacity, and glutathione levels in the rat pancreas. In addition, TAU significantly decreased the level of reactive oxygen species, and markedly increased the activity of heme oxygenase 1 (HO-1), Kelch-like ECH-associated protein 1 (KEAP-1), and nuclear factor erythrocyte-2-related factor 2 (Nrf2) in the rat pancreas. Notably, HFHG diet could induce pancreatic injury in the rats through the Nrf2/HO-1 signaling pathway and activate the mitochondrial channel-mediated apoptotic signaling pathway. The addition of TAU significantly improved the pancreatic tissue injury induced by the HFHG diet in the rats and reduced the protein expression of Caspase-3, Cleaved-caspase-3, Caspase-9 and Bcl-2 associated protein X (BAX), and increased the protein expression of B-cell lymphoma-2 (Bcl-2). In conclusion, this experiment confirmed that TAU could alleviate the oxidative stress and apoptosis induced by the HFHG diet in rat pancreatic β-cells.

Demethylcalabaxanthone from Garcinia mangostana Exerts Antioxidant Effects through the Activation of the Nrf2 Pathway as Assessed via Molecular Docking and Biological Evaluation.

Nuclear factor erythroid 2-related factor 2 (Nrf2) pathway activation promotes the expression of antioxidant enzymes in response to rising oxidative stress, resulting in reactive oxygen species (ROS) detoxification and playing a central role in the maintenance of intracellular redox homeostasis and regulation of inflammation. Moreover, the biological effects of Nrf2 pathway activation contribute to reducing apoptosis and enhancing cell survival. The activity of Nrf2 is negatively regulated by Kelch-like ECH-associated protein 1 (Keap1). Prompted by the recent results reporting the impact of xanthone metabolites on oxidative stress, cancer, and inflammation, the antioxidant properties of xanthones isolated from Garcinia mangostana (γ-mangostin, α-mangostin, 8-deoxygartanin, demethylcalabaxanthone, garcinone D) were assessed. In particular, the capability of these natural products to disrupt the interaction between Kelch-like ECH-associated protein 1 (Keap1) and nuclear factor erythroid 2-related factor 2 (Nrf2), triggering the activation of the Nrf2-mediated pathway, was evaluated using molecular docking experiments and in vitro tests. The modulation of some key Nrf2-related mediators like glutathione (GSH) and lactate dehydrogenase (LDH) to highlight a possible direct antioxidant effect was investigated. Among the tested compounds, demethylcalabaxanthone showed an indirect antioxidant effect, as corroborated by a Western blot assay, displaying a significant increase in the translocated protein upon its administration.

Screening approaches for the identification of Nrf2-Keap1 protein-protein interaction inhibitors targeting hot spot residues

Protein-protein interactions (PPIs) play a crucial role in most biological processes and are important targets in the development of therapeutic agents. However, small molecule drug discovery that targets PPIs remains very challenging. Targeting hot spot residues is considered the best option for inhibiting such interactions, but there are few examples of how knowledge of hot spots can be used in high throughput screening to find hit compounds. A substrate adaptor protein for a ubiquitin ligase complex, Kelch-like ECH-associated protein 1 (Keap1), negatively modulates the expression of genes involved in cellular protection against oxidative stress. Here, we focused on three arginine hot spot residues in the Keap1 substrate binding pocket (Arg380, Arg415, and Arg483), and screened the carboxylic acid library owned by Japan Tobacco Inc. for compounds that interact with the arginine residues in differential scanning fluorescence assays. Furthermore, we identified several small molecule compounds that specifically bind to the Keap1 Kelch domain hot spots by comparing binding to alanine mutant proteins (R380A, R415A, and R483A) with binding to the wild-type protein using surface plasmon resonance (SPR) screening. These compounds inhibited the protein-protein interaction between the Keap1 Kelch domain and the nuclear factor erythroid 2-related factor 2 (Nrf2) peptide, and the ubiquitination of Nrf2 catalyzed by the Cul3/RINGBox 1 E3 ligase. In addition, the binding mode of one compound (Compound 4) was determined by X-ray crystallography after validation of binding by isothermal titration calorimetry, native mass spectrometry, and nuclear magnetic resonance. Compound 4 had favorable thermodynamic properties, and noncovalently bound to Keap1 with a stoichiometry of 1:1. Our results suggest that Compound 4 could potentially be developed into effective therapeutic or preventive agents for a variety of diseases and conditions such as oxidative stress response, inflammation, and carcinogenesis. We believe that the use of a set of complementary biophysical techniques including the SPR assay with single alanine mutant of hot spots provides opportunities to identify hit compounds for developing inhibitors of PPIs.

Mild Oxidative Stress Induced by Sodium Arsenite Reduces Lipocalin-2 Expression Levels in Cortical Glial Cells.

Astrocytes and microglia, the most abundant glial cells in the central nervous system, are involved in maintaining homeostasis in the brain microenvironment and in the progression of various neurological disorders. Lipocalin-2 (LCN2) is a small secretory protein that can be transcriptionally upregulated via nuclear factor kappa B (NF-κB) signaling. It is synthesized and secreted by glial cells, resulting in either the restoration of damaged neural tissues or the induction of neuronal apoptosis in a context-dependent manner. It has recently been reported that when glial cells are under lipopolysaccharide-induced inflammatory stress, either reduced production or accelerated degradation of LCN2 can alleviate neurotoxicity. However, the regulatory mechanisms of LCN2 in glial cells are not yet fully understood. In this study, we used primary astroglial-enriched cells which produce LCN2 and found that the production of LCN2 could be reduced by sodium arsenite treatment. Surprisingly, the reduced LCN2 production was not due to the suppression of NF-κB signaling. Mild oxidative stress induced by sodium arsenite treatment activated antioxidant responses and downregulated Lcn2 expression without reducing the viability of astroglial-enriched cells. Intriguingly, reduced LCN2 production could not be achieved by simple activation of the nuclear factor erythroid-2-related factor 2 (Nrf2)-Kelch-like ECH-associated protein 1 (Keap1) pathway in astroglial-enriched cells. Thus, it appears that mild oxidative stress, occurring in an Nrf2-independent manner, is required for the downregulation of Lcn2 expression. Taken together, our findings provide new insights into the regulatory mechanisms of LCN2 and suggest that mild oxidative stress may alter LCN2 homeostasis, even under neuroinflammatory conditions.

CXX-3, a Norditerpene Lactones From Podocarpus nakaii, Inhibits Cancer Cell Viability by Suppressing the NRF2/KEAP1 Antioxidant Pathway

Objective: The role of Nuclear factor E2-related factor 2 (NRF2) in cells is a double-edged sword, as it can maintain the stability of the inner environment for normal cells by resisting oxidation, but can also confer drug adaptability to cancer cells. Recent reports suggest that inhibiting NRF2 can weaken the drug resistance of cells, making it a potential target for cancer therapy. Our research on CXX-3, a norditerpenoid isolated from Podocarpus nakaii, demonstrated its ability to combat cancer cells. Methods: We observe for the first time that an active ingredient from P. nakaii induces cancer cell death accompanied by the accumulation of reactive oxygen species (ROS) and arrests the cell cycle in the S phase. The ROS inhibitor NAC(N-acetyl-l-cysteine) can partially rescue cell death. Additionally, we identified the NRF2/KEAP1 (Kelch-like ECH-associated protein 1, KEAP1) complex as the critical site conquered by CXX-3. Dysfunction of this complex ruined the antioxidation ability of glioma and colon carcinoma. Interestingly, CXX-3 seems to disable the KEAP1/NRF2 complex through a separate mechanism. Results: In conclusion, CXX-3 can disrupt the antioxidation ability of cancer cells, leading to the accumulation of ROS, thereby arresting the cell cycle, and inducing cell death. Conclusion: As such, CXX-3 holds promise as a prospective compound for cancer therapy.

Antioxidant System and Endoplasmic Reticulum Stress in Cataracts.

The primary underlying contributor for cataract, a leading cause of vision impairment and blindness worldwide, is oxidative stress. Oxidative stress triggers protein damage, cell apoptosis, and subsequent cataract formation. The nuclear factor-erythroid 2-related factor 2 (Nrf2) serves as a principal redox transcriptional factor in the lens, offering a line of defense against oxidative stress. In response to oxidative challenges, Nrf2 dissociates from its inhibitor, Kelch-like ECH-associated protein 1 (Keap1), moves to the nucleus, and binds to the antioxidant response element (ARE) to activate the Nrf2-dependent antioxidant system. In parallel, oxidative stress also induces endoplasmic reticulum stress (ERS). Reactive oxygen species (ROS), generated during oxidative stress, can directly damage proteins, causing them to misfold. Initially, the unfolded protein response (UPR) activates to mitigate excessive misfolded proteins. Yet, under persistent or severe stress, the failure to rectify protein misfolding leads to an accumulation of these aberrant proteins, pushing the UPR towards an apoptotic pathway, further contributing to cataractogenesis. Importantly, there is a dynamic interaction between the Nrf2 antioxidant system and the ERS/UPR mechanism in the lens. This interplay, where ERS/UPR can modulate Nrf2 expression and vice versa, holds potential therapeutic implications for cataract prevention and treatment. This review explores the intricate crosstalk between these systems, aiming to illuminate strategies for future advancements in cataract prevention and intervention. The Nrf2-dependent antioxidant system communicates and cross-talks with the ERS/UPR pathway. Both mechanisms are proposed to play pivotal roles in the onset of cataract formation.

Exogenous H2S initiating Nrf2/GPx4/GSH pathway through promoting Syvn1-Keap1 interaction in diabetic hearts

Excessive ROS accumulation contributes to cardiac injury in type 2 diabetes mellitus. Hydrogen sulfide (H2S) is a vital endogenous gasotransmitter to alleviate cardiac damage in diabetic cardiomyopathy (DCM). However, the underlying mechanisms remain unclear. In this study, we investigated the effects of NaHS administration in db/db mice via intraperitoneal injection for 20 weeks and the treatment of high glucose (HG), palmitate (PA) and NaHS in HL-1 cardiomyocytes for 48 h, respectively. H2S levels were decreased in hearts of db/db mice and HL-1 cardiomyocytes exposed to HG and PA, which were restored by NaHS. Exogenous H2S activated the nuclear factor erythroid 2-related factor 2 (Nrf2)/glutathione peroxidase 4 (GPx4)/glutathione (GSH) pathway, suppressed ferroptosis and mitigated mitochondrial apoptosis in db/db mice. However, these effects were abrogated after Nrf2 knockdown. NaHS treatment elevated the ubiquitination level of Kelch-like ECH-associated protein (Keap1) by preserving its E3 ligase synoviolin (Syvn1), resulting in Nrf2 nuclear translocation. H2S facilitated the sulfhydration of Syvn1-cys115 site, a post-translational modification. Transfecting Syvn1 C115A in cardiomyocytes exposed to HG and PA partially attenuated the effects of NaHS on Nrf2 and cell death. Our findings suggest that exogenous H2S regulates Nrf2/GPx4/GSH pathway by promoting the Syvn1-Keap1 interaction to reduce ferroptosis and mitochondrial apoptosis in DCM.

KEAP1 polymorphisms and neurodevelopmental outcomes in children with exposure to prenatal MeHg from the Seychelles Child Development Study Nutrition Cohort 2

BackgroundHumans differ in the metabolism of the neurotoxicant methyl mercury (MeHg). This variation may be partially due to variation in genes encoding the transcription factor Nuclear factor E2-related factor 2 (NRF2) and its negative regulator Kelch-like ECH-Associated Protein 1 (KEAP1), which regulate glutathione and related transporter and antioxidant proteins that play a role in the metabolism and neurotoxicity of MeHg. AimTo elucidate a potential risk from genetic variation in NFE2L2 (encoding NRF2) and KEAP1 toward prenatal mercury exposure and child neurodevelopmental outcomes at 20 months and 7 years of age in a population with variable prenatal exposure to MeHg from maternal fish consumption. Material and MethodsNutrition Cohort 2 is a mother–child cohort in the Republic of Seychelles. Children were genotyped for NFE2L2 (rs2364723, rs13001694) and KEAP1 (rs8113472, rs9676881) polymorphisms (N = 1285 after removing siblings). Total mercury (Hg) was measured in cord blood as a biomarker for prenatal MeHg exposure. Child neurodevelopmental outcomes included the Bayley Scales of Infant Development II administered at 20 months of age, and outcomes across multiple neurodevelopmental domains from 14 tests administered in children and 3 instruments completed by parents when children were 7 years of age. ResultsThe mean cord blood MeHg concentration was 34 (95% CI 11, 75) µg/L. None of the four polymorphisms had a significant association (p < 0.05) with either cord MeHg or neurodevelopmental test results at 20 months. There were no significant associations between either NFE2L2 polymorphism and any developmental test scores. At 7 years, children carrying KEAP1 rs8113472 CA showed significantly worse performance on psychomotor function than children with the CC variant (finger tapping, dominant hand: β − 1.19, SE 0.34; finger tapping, non-dominant hand: β − 0.92, SE 0.31) and worse social communication (SCQ Total: β 0.65, SE 0.27). Children carrying rs8113472 AA, versus children with CC, showed significantly better performance on social communication (SRS Total: β − 8.88, SE 3.60). Children carrying KEAP1 rs9676881 AG, versus children with GG, showed significantly worse performance on psychomotor function (trailmaking A time: β 8.66, SE 3.37) and cognition (KBIT Matrices: β − 0.96, SE 0.36). ConclusionNo associations between NFE2L2 and KEAP1 polymorphisms and MeHg concentration were identified. However, at 7 years, KEAP1 polymorphisms were associated with differences in neurodevelopmental outcomes in children from a population with high fish intake.

Nrf2-mediated protective effect of alpha-lipoic acid on synaptic oxidative damage and inhibition of PKC/ERK/CREB pathway in bisphenol A-exposed HT-22 cells

The harmful effects of bisphenol A (BPA) on learning and memory may involve hippocampal oxidative damage; however, the underlying mechanism remains unclear. Antioxidants that antagonize BPA-induced neuronal oxidative damage lack research. This study aimed to develop an in vitro model using the HT-22 mouse hippocampal neuronal cell line to investigate the neurotoxic mechanism of BPA and the protective effect of alpha-lipoic acid (ALA) on nuclear factor erythroid 2-related factor 2 (Nrf2) inhibition. The results showed that ALA reduced BPA-induced reactive oxygen species and neuronal nitric oxide synthase (nNOS) levels; however, inhibiting Nrf2 weakened the protective effects of ALA. BPA reduced mitochondrial complex I/III activity and ATP levels, but ALA ameliorated this damage. ALA improved the BPA-induced downregulation of the kelch-like ECH-associated protein 1 (keap1)/Nrf2 system, synaptic-related proteins, and the protein kinase C (PKC)/extracellular signal-regulated kinase (ERK)/cAMP response element binding protein (CREB) pathway; however, the protective effects of ALA were weakened when Nrf2 was inhibited. Our results suggest that BPA causes oxidative damage to HT-22 cells by damaging mitochondrial function, nNOS, and the keap1/Nrf2 system, thereby impairing synaptic-related proteins and the PKC/ERK/CREB pathway. ALA counters BPA-induced damage via Nrf2, which may be a significant target for the protective action of ALA.

Identification of antioxidant peptides targeting Keap1−Nrf2−ARE pathway from in vitro digestion of pork sausage with partial substitution of NaCl by KCl

The Kelch-like ECH-associated protein 1 (Keap1)–nuclear factor erythroid 2–related factor 2 (Nrf2)–antioxidant response elements (ARE) pathway is one of the most important cellular defense mechanisms against oxidative stress. This study focuses on finding antioxidant peptides from in vitro digestion products of pork sausage with partial substitution of NaCl by KCl by virtual screening. Six antioxidant peptides, LIVGFPAYGH, DWWGSTVR, WNSLLIR, IVGFPAYGH, FDNLWDQGL, and LRSPSWDPF, could activate the Keap1–Nrf2–ARE pathway and protect cells from oxidative stress. DWWGSTVR exhibits the most robust activity among them. Further studies indicated that DWWGSTVR could increase the expression of many antioxidant enzymes by enabling the transfer of Nrf2 from the cytoplasm to the nucleus. In summary, these six peptides are proven to be Nrf2 activators and could be used as functional foods to prevent and treat various oxidative stress-induced diseases.