Kelch-like ECH-associated Protein Research Articles

Evaluation of largemouth bass (Micropterus salmoide) fed selenium yeast diets: Growth, histopathology, antioxidant ability, and apoptosis

The objective of this study was to investigate the effects of selenium yeast on the growth, histomorphology, antioxidant capacity and apoptosis of largemouth bass (Micropterus salmoide). The five iso-nitrogen and iso-lipid feeds were supplemented with selenium yeast levels of 0, 0.5, 1.0, 1.5 and 2.0 mg/kg, respectively. The 525 largemouth bass (initial body weight: 11.48 ± 0.43 g) were equally divided into 15 buckets (three buckets per feed treatment) for a 67-day feeding experiment. The results showed that the addition of 1.5 and 2.0 mg/kg of selenium yeast significantly reduced weight gain rate, and specific growth rate (P < 0.05), disorganized the structure of liver cells and exacerbated the vacuolation of liver tissue in largemouth bass. The activities of glutathione peroxidase and superoxide dismutase in serum were significantly increased by 1.0 mg/kg selenium yeast (P < 0.05). After selenium yeast was supplemented, villus length and villus width were significantly increased (P < 0.05), NO contents and respiratory burst activities in hematocyte were not significantly changed (P > 0.05). With the addition of 1.5 mg/kg of selenium yeast, the relative expression of growth hormone 1, insulin-like growth factor-1, superoxide dismutase, catalase, Kelch-like ECH-associating protein 1, and nuclear factor erythroid 2 related factor 2 in hematocyte was significantly increased (P < 0.05). The significantly lowest percentage of apoptotic hematocyte was also seen in the 1.5 mg/kg selenium yeast addition group (P < 0.05). Overall, the supplementation of 1.5 mg/kg selenium yeast was not conducive to the growth and liver development of largemouth bass, but it could improve the antioxidant capacity of hematocyte and inhibit the apoptosis. It provided a theoretical basis for the enhancement of immune capacity during the farming of largemouth bass.

Dietary Histamine Impairs the Digestive Physiology Function and Muscle Quality of Hybrid Grouper (Epinephelus fuscoguttatus♀ × Epinephelus lanceolatus♂).

An 8-week feeding experiment was conducted to investigate the effect of dietary histamine on growth performance, digestive physiology function and muscle quality in a hybrid grouper (Epinephelus fuscoguttatus♀ × Epinephelus lanceolatus♂). Seven isoproteic (50%) and isolipidic (11%) diets were prepared with various histamine inclusion levels of 0, 30, 60, 120, 240, 480 and 960 mg/kg in diets (actual contents were 72.33, 99.56, 138.60, 225.35, 404.12, 662.12 and 1245.38 mg/kg), respectively. Each diet was randomly assigned to triplicates of 30 juveniles (average body weight 14.78 g) per tank in a flow-through mariculture system. The increase in the dietary histamine level up to 1245.38 mg/kg made no significant difference on the growth rate and feed utilization of the grouper. However, the increased histamine content linearly decreased the activities of digestive enzymes, while no differences were observed in groups with low levels of histamine (≤404.12 mg/kg). Similarly, high levels of histamine (≥404.12 mg/kg) significantly damaged the gastric and intestinal mucosa, disrupted the intestinal tight junction structure, and raised the serum diamine oxidase activity and endotoxin level. Meanwhile, high doses of histamine (≥662.12 mg/kg) significantly reduced the activities of antioxidant enzymes, upregulated the relative expression of Kelch-like ECH-associated protein 1, and hardened and yellowed the dorsal muscle of grouper. These results showed that dietary histamine was detrimental to the digestive physiology function and muscle quality of the grouper, although it did compromise its growth performance.

Dietary methionine restriction alleviates oxidative stress and inflammatory responses in lipopolysaccharide-challenged broilers at early age.

In this study, we investigated the effect of dietary methionine restriction (MR) on the antioxidant function and inflammatory responses in lipopolysaccharide (LPS)-challenged broilers reared at high stocking density. A total of 504 one-day-old male Arbor Acre broiler chickens were randomly divided into four treatments: 1) CON group, broilers fed a basal diet; 2) LPS group, LPS-challenged broilers fed a basal diet; 3) MR1 group, LPS-challenged broilers fed a methionine-restricted diet (0.3% methionine); and 4) MR2 group, LPS-challenged broilers fed a methionine-restricted diet (0.4% methionine). LPS-challenged broilers were intraperitoneally injected with 1mg/kg body weight (BW) of LPS at 17, 19, and 21days of age, whereas the CON group was injected with sterile saline. The results showed that: LPS significantly increased the liver histopathological score (p < 0.05); LPS significantly decreased the serum total antioxidant capacity (T-AOC), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px) activity at 3h after injection (p < 0.05); the LPS group had a higher content of Interleukin (IL)-1β, IL-6, and tumor necrosis factor-α (TNF)-α, but a lower content of IL-10 than the CON group in serum (p < 0.05). Compared with the LPS group, the MR1 diet increased catalase (CAT), SOD, and T-AOC, and the MR2 diet increased SOD and T-AOC at 3h after injection in serum (p < 0.05). Only MR2 group displayed a significantly decreased liver histopathological score (p < 0.05) at 3h, while MR1 and MR2 groups did so at 8h. Both MR diets significantly decreased serum LPS, CORT, IL-1β, IL-6, and TNF-α contents, but increased IL-10 content (p < 0.05). Moreover, the MR1 group displayed significantly increased expression of nuclear factor erythroid 2-related factor 2 (Nrf2), CAT, and GSH-Px at 3 h; the MR2 group had a higher expression of Kelch-like ECH-associated protein 1 (Keap1), SOD, and GSH-Px at 8h (p < 0.05). In summary, MR can improve antioxidant capacity, immunological stress, and liver health in LPS-challenged broilers. The MR1 and MR2 groups experienced similar effects on relieving stress; however, MR1 alleviated oxidative stress more rapidly. It is suggested that precise regulation of methionine levels in poultry with stress may improve the immunity of broilers, reduce feed production costs, and increase production efficiency in the poultry industry.

Carnosol inhibits KGN cells oxidative stress and apoptosis and attenuates polycystic ovary syndrome phenotypes in mice through Keap1-mediated Nrf2/HO-1 activation.

Excessive oxidative stress and apoptosis of ovarian granulosa cells lead to abnormal follicular development and ovulation disorders in polycystic ovary syndrome (PCOS). Carnosol is a plant-derived polyphenol that has been proven to exhibit several cell protective effects. In this study, we established hyperandrogenic PCOS models both in vitro and in vivo. In the humanovariangranulosacellline,KGN cells, decreased viability and mitochondrial membrane potential, and upregulated reactive oxygen species (ROS) level and apoptosis induced by DHT were partly reversed by carnosol. Western blotting results showed that carnosol treatment inhibited the DHT-activated mitochondrial apoptotic pathway by activating nuclear factor-erythroid 2-related factor (Nrf2)/heme oxygenase 1 (HO-1). Knockdown of Nrf2 by transfecting with siRNA or inhibiting HO-1 by zinc protoporphyrin (ZnPP) blocked the protective effects of carnosol. Computational modeling and pull-down assay results confirmed the direct binding of carnosol to kelch-like ECH-associated protein 1 (Keap1). In vivo results showed that the intraperitoneal administration of carnosol (50 and 100 mg/kg) improved estrous cycle disorders, polycystic ovary, and decreased elevated androgen in the PCOS mice. In summary, Carnosol has an effective role in inhibiting oxidative stress and apoptosis in DHT-treated KGN cells and protecting against mouse PCOS phenotypes through the Keap1-mediated activation of Nrf2/HO-1 signaling.

A Multifunctional (-)-Meptazinol-Serotonin Hybrid Ameliorates Oxidative Stress-Associated Apoptotic Neuronal Death and Memory Deficits via Activating the Nrf2/Antioxidant Enzyme Pathway.

The pathogenesis of Alzheimer's disease (AD) involves multiple pathophysiological processes. Oxidative stress is a major cause of AD-associated neuronal injury. The current research was designed to examine whether a novel (-)-meptazinol-serotonin hybrid (Mep-S) with potent antioxidant activity and additional inhibitory properties for acetylcholinesterase (AChE) activity could attenuate oxidative neuronal damage and cognitive deficits. In human SH-SY5Y cells, Mep-S suppressed H2O2-induced apoptosis by restoring mitochondrial membrane potential and inhibiting caspase-3 activation. Meanwhile, it attenuated oxidative stress elicited by H2O2 through lessening generation of reactive oxygen species as well as enhancing production of glutathione (GSH) and activity of superoxide dismutase (SOD). Mechanistically, Mep-S promoted nuclear translocation of a transcription factor nuclear factor E2-related factor-2 (Nrf2) in H2O2-challenged cells. This effect was accompanied by reduction in Kelch-like ECH-associated protein-1 (Keap1) levels as well as augmentation of Akt phosphorylation and expression of heme oxygenase-1 (HO-1) and NAD(P)H quinine oxidoreductase-1 (NQO-1). Molecular docking analysis revealed that Mep-S may disrupt the protein-protein interactions between Keap1 and Nrf2. In an in vivo mouse model, Mep-S attenuated scopolamine-caused cognitive deficits with inhibition of apoptotic neuronal death and brain AChE activity. Furthermore, the scopolamine-induced impairment of total antioxidant capacity and reduction in SOD1, SOD2, and γ-glutamate-cysteine ligase expression in the brain were counteracted by Mep-S, accompanied by decreased Keap1 levels, increased Akt catalytic subunit and Nrf2 phosphorylation, and decreased Nrf2, HO-1, and NQO-1 expression. Collectively, our results suggest that Mep-S ameliorates apoptotic neuronal death and memory dysfunction associated with oxidative stress by regulating the Nrf2/antioxidant enzyme pathway through inactivating Keap1 and phosphorylating Nrf2 via Akt activation. Therefore, Mep-S may be a potential lead for multitarget neuroprotective agents to treat AD-like symptoms.

Combinatory analysis of immune cell subsets and tumor-specific genetic variants predict clinical response to PD-1 blockade in patients with non-small cell lung cancer.

Immunotherapy by blocking programmed death protein-1 (PD-1) or programmed death protein-ligand1 (PD-L1) with antibodies (PD-1 blockade) has revolutionized treatment options for patients with non-small cell lung cancer (NSCLC). However, the benefit of immunotherapy is limited to a subset of patients. This study aimed to investigate the value of combining immune and genetic variables analyzed within 3-4 weeks after the start of PD-1 blockade therapy to predict long-term clinical response. Blood collected from patients with NSCLC were analyzed for changes in the frequency and concentration of immune cells using a clinical flow cytometry assay. Next-generation sequencing (NGS) was performed on DNA extracted from archival tumor biopsies of the same patients. Patients were categorized as clinical responders or non-responders based on the 9 months' assessment after the start of therapy. We report a significant increase in the post-treatment frequency of activated effector memory CD4+ and CD8+ T-cells compared with pre-treatment levels in the blood. Baseline frequencies of B cells but not NK cells, T cells, or regulatory T cells were associated with the clinical response to PD-1 blockade. NGS of tumor tissues identified pathogenic or likely pathogenic mutations in tumor protein P53, Kirsten rat sarcoma virus, Kelch-like ECH-associated protein 1, neurogenic locus notch homolog protein 1, and serine/threonine kinase 11, primarily in the responder group. Finally, multivariate analysis of combined immune and genetic factors but neither alone, could discriminate between responders and non-responders. Combined analyses of select immune cell subsets and genetic mutations could predict early clinical responses to immunotherapy in patients with NSCLC and after validation, can guide clinical precision medicine efforts.

Modulating SQSTM1/p62-dependent selective autophagy of neurons by activating Nrf2 with multifunctional nanoparticles to eliminate α-synuclein aggregates and boost therapy of Parkinson’s disease

The misfolding and aggregation of α-synuclein is one of the key pathogeneses in Parkinson's disease (PD). Development of innovative and effective approaches to eliminate α-synuclein aggregates is crucial for PD treatment. Herein, we report that quercetin-modified ultrasmall Cu2-xSe antioxidative nanoparticles (abbreviated as CSPQ) can promote neurons to efficiently scavenge α-synuclein aggregates and boost PD therapy by activating their nuclear factor-erythroid 2-related factor 2 (Nrf2) to enhance the SQSTM1/p62-dependent selective autophagy. We reveal that CSPQ nanoparticles can promote the degradation of Kelch-like ECH-associated protein 1 (Keap1) and thus activate Nrf2/p62 pathway of neurons by stimulating their nuclear translocation of transcription factor EB (TFEB). Moreover, these nanoparticles can successfully reduce α-synuclein associated pathology to alleviate neuronal damage in vivo and improve motor disorder and exploration ability of α-synuclein preformed fibrils (PFFs)-induced PD mice. Our work demonstrates the importance and novelty of modulating the selective autophagy of neurons by nanoparticles in clearance of α-synuclein aggregates for PD therapy, and highlights the great potential of CSPQ nanoparticles in PD therapy.

Racial differences in transcriptomics and reactive oxygen species burden in myometrium and leiomyoma.

Are there differences in Mediator Complex Subunit 12 mutations (MED12) mutation, transcriptomics, and protein expression in uterine myometrium and leiomyomas of Black and White women? RNA sequencing, tissue microarray, and immunohistochemistry data revealed that Black and White women have significant differences in their myometrium and leiomyoma profiles. Black women develop uterine leiomyoma earlier than White women, and are more likely to be anemic, have multiple tumors, undergo hysterectomy at an earlier age, have a higher uterine weight, and report very severe pelvic pain. Uterine tissues were collected from premenopausal women undergoing hysterectomy or myomectomy at Northwestern University Prentice Women's Hospital (Chicago, IL) from 2010 to 2021. Tissues were collected from a total of 309 women, including from 136 Black women, 135 White women, and 38 women from other racial groups. A total of 529 uterine leiomyomas (290 from Black women, 184 from White women, and 55 from women of other racial groups) were subjected to molecular analysis. Leiomyoma and matched myometrium from a total of 118 cases including 60 Black women and 58 White women, were used for tissue microarrays, along with 34 samples of myometrium without leiomyoma from White women. Tissues from the above patient cohorts were analyzed by tissue microarray, immunohistochemistry, RNA sequencing, and mutation analysis. The results indicated that leiomyoma from Black women have a higher rate of MED12 mutations (79.0%) than those from White women (68.5%) (*P ≤ 0.05). RNA-sequencing analysis in myometrium revealed differentially expressed genes (270 upregulated, 374 downregulated) dependent on race, wherein reactive oxygen species, hypoxia, and oxidative phosphorylation pathways were positively correlated with samples derived from Black patients. The levels of proteins associated with oxidative DNA damage and repair, 8-hydroxyguanosine (8-OHdG), 8-oxoguanine glycosylase (OGG1), heme oxygenase-1 (HO-1), and kelch-like ECH-associated protein 1 (KEAP1), were higher in leiomyoma and matched myometrium, particularly those from Black patients, compared to the control myometrium (with leiomyoma) (***P ≤ 0.001). The datasets are available in the NCBI (The BioProject number: PRJNA859428). Myometrium without leiomyoma derived from White patients was used as a control in the tissue microarray analysis, as myometrium without leiomyoma from Black patients was not accessible in large numbers. The RNA sequencing was performed on myometrium tissue with leiomyoma present from 10 White and 10 Black women. However, one sample from a Black woman yielded low-quality RNA-sequencing data and was excluded from further analysis. Women with symptomatic leiomyomas have a considerable loss in their quality of life. This study provides information on underlying genetic and molecular defects that may be necessary for future therapeutics targeted at leiomyomas. This work was supported by grants from NCI (R01CA254367) and NICHD (P01HD057877). The authors declare no conflict of interest.

Effects of Tributyrin Supplementation on Growth Performance, Intestinal Digestive Enzyme Activity, Antioxidant Capacity, and Inflammation-Related Gene Expression of Large Yellow Croaker (Larimichthys crocea) Fed with a High Level of Clostridium autoethanogenum Protein.

An 8-week growth experiment was conducted to investigate effects of tributyrin (TB) supplementation on growth performance, intestinal digestive enzyme activity, antioxidant capacity, and inflammation-related gene expression of juvenile large yellow croaker (Larimichthys crocea) (initial weight of 12.90 ± 0.02 g) fed diets with high level of Clostridium autoethanogenum protein (CAP). In the negative control diet, 40% fish meal was used as the major source of protein (named as FM), while 45% fish meal protein of FM was substituted with CAP (named as FC) to form a positive control diet. Based on the FC diet, grade levels of 0.05%, 0.1%, 0.2%, 0.4%, and 0.8% tributyrin were added to formulate other five experimental diets. Results showed that fish fed diets with high levels of CAP significantly decreased the weight gain rate (WGR) and specific growth rate (SGR) compared with fish fed the FM diet (P < 0.05). WGR and SGR were significantly higher than in fish fed diets with 0.05% and 0.1% tributyrin that fed the FC diet (P < 0.05). Supplementation of 0.1% tributyrin significantly elevated fish intestinal lipase and protease activities compared to FM and FC diets (P < 0.05). Meanwhile, compared to fish fed the FC diet, fish fed diets with 0.05% and 0.1% tributyrin showed remarkably higher intestinal total antioxidant capacity (T-AOC). Malondialdehyde (MDA) content in the intestine of fish fed diets with 0.05%-0.4% tributyrin was remarkably lower than those in the fish fed the FC diet (P < 0.05). The mRNA expressions of tumor necrosis factor α (tnfα), interleukin-1β (il-1β), interleukin-6 (il-6), and interferon γ (ifnγ) were significantly downregulated in fish fed diets with 0.05%-0.2% tributyrin, and the mRNA expression of il-10 was significantly upregulated in fish fed the 0.2% tributyrin diet (P < 0.05). In regard to antioxidant genes, as the supplementation of tributyrin increased from 0.05% to 0.8%, the mRNA expression of nuclear factor erythroid 2-related factor 2 (nrf2) demonstrated a trend of first rising and then decreasing. However, the mRNA expression of Kelch-like ECH-associated protein 1 (keap1) was remarkably lower in fish fed the FC diet than that fed diets with tributyrin supplementation (P < 0.05). Overall, fish fed tributyrin supplementation diets can ameliorate the negative effects induced by high proportion of CAP in diets, with an appropriate supplementation of 0.1%.

The KEAP1-NRF2 system and neurodegenerative diseases.

Significance: Central nervous system (CNS) diseases are disorders of the brain and/or spinal cord and include neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson's disease (PD), and multiple sclerosis (MS). NF-E2-related factor 2 (NRF2) is a transcription factor belonging to the cap-n-collar (CNC) family that harbors a unique basic leucine zipper motif and plays as a master regulator of homeostatic responses. Recent Advances: Kelch-like ECH-associated protein 1 (KEAP1) is an adaptor of the Cullin3 (CUL3)-based ubiquitin E3 ligase that enhances the ubiquitylation of NRF2, which promotes the degradation of NRF2 to suppress its transcriptional activity in the absence of stress. Cysteine residues of KEAP1 are modified under stress conditions, and NRF2 degradation is attenuated, allowing it to accumulate and induce the expression of target genes. This regulatory system is referred to as the KEAP1-NRF2 system and plays a central role in protecting cells against various stresses. NRF2 also negatively regulates the expression of inflammatory cytokine and chemokine genes and suppresses pathological inflammation. As oxidative stress, inflammation, and proteostasis are known to contribute to neurodegenerative diseases, the KEAP1-NRF2 system is an attractive target for the treatment of these diseases. Critical Issues: In mouse models of neurodegenerative diseases, Nrf2 depletion exacerbates symptoms and enhances oxidative damage and inflammation in the CNS. In contrast, chemical or genetic NRF2 activation improves these symptoms. Indeed, the NRF2-activating chemical dimethyl fumarate (DMF) is now widely used for the clinical treatment of MS. Future Directions: The KEAP1-NRF2 system is a promising therapeutic target for neurodegenerative diseases.

CRISPR metabolic screen identifies ATM and KEAP1 as targetable genetic vulnerabilities in solid tumors

Cancer treatments targeting DNA repair deficiencies often encounter drug resistance, possibly due to alternative metabolic pathways that counteract the most damaging effects. To identify such alternative pathways, we screened for metabolic pathways exhibiting synthetic lethality with inhibition of the DNA damage response kinase Ataxia-telangiectasia-mutated (ATM) using a metabolism-centered Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 library. Our data revealed Kelch-like ECH-associated protein 1 (KEAP1) as a key factor involved in desensitizing cancer cells to ATM inhibition both invitro and invivo. Cells depleted of KEAP1 exhibited an aberrant overexpression of the cystine transporter SLC7A11, robustly accumulated cystine inducing disulfide stress, and became hypersensitive to ATM inhibition. These hallmarks were reversed in a reducing cellular environment indicating that disulfide stress was a crucial factor. In The Cancer Genome Atlas (TCGA) pan-cancer datasets, we found that ATM levels negatively correlated with KEAP1 levels across multiple solid malignancies. Together, our results unveil ATM and KEAP1 as new targetable vulnerabilities in solid tumors.

FAM117B promotes gastric cancer growth and drug resistance by targeting the KEAP1/NRF2 signaling pathway.

Gastric cancer often shows malignant growth and insensitivity to chemotherapeutic drugs due to the regulation of complex molecular mechanisms, which results in poor prognosis for patients. However, the relevant molecular mechanisms remain unclear. In this study, we reported that family with sequence similarity 117, member B (FAM117B), promoted the growth of gastric cancer cells and reduced the sensitivity of cells to chemotherapeutic drugs. Mechanistically, FAM117B competed with nuclear factor E2-related factor 2 (NRF2) for Kelch-like ECH-associated protein 1 (KEAP1) binding, reduced the ubiquitination degradation of NRF2, and activated the KEAP1/NRF2 signaling pathway. Moreover, FAM117B-induced growth and chemoresistance of gastric cancer cells were NRF2 dependent. We found that FAM117B and NRF2 protein levels were highly expressed in tumor tissues of patients with gastric cancer and their co-overexpression represented an independent factor for poor prognosis. Collectively, our findings reveal that FAM117B is involved in promoting gastric cancer growth and drug resistance, and it could be exploited as a cancer therapeutic target.

Active Nrf2 signaling flexibly regulates HO-1 and NQO-1 in hypoxic Gansu Zokor (Eospalax cansus).

Gansu zokor (Eospalax cansus) is a typical subterranean rodent species with resistance to ambient hypoxia. The nuclear factor erythroid 2-related factor 2 (Nrf2) signaling plays a key role in regulating redox homeostasis. However, little is known about the regulation of Nrf2 signaling in Gansu zokor. We exposed Gansu zokors and SD rats to chronic hypoxia (44h at 10.5% O2) or acute hypoxia (6h at 6.5% O2) andmeasured the activities of heme oxygenase-1 (HO-1) and NAD(P)H quinone oxidoreductase-1 (NQO-1)，gene expression of HO-1, NQO-1, Nrf2, Kelch-like ECH-associated protein-1 (KEAP1), and β-transducin repeat-containing protein (β-TRCP) in the brain and liver. We found that Gansu zokor increased the NQO-1 protein content and activity, HO-1 protein content in the brain, and increased HO-1 activity and mRNA level, NQO-1 activity and protein content in the liver by up regulating Nrf2 gene expression under chronic hypoxia. Although acute hypoxia enhanced the expression of Nrf2 gene, only the level of HO-1 mRNA in the liver increased. Besides, the HO-1 and NQO-1 genes in the brain, HO-1 genes and NQO-1 mRNA in the Gansu zokor liver were significantly higher than those in SD rats under normoxia. Negative regulators of Nrf2 signaling were tissue specific: KEAP1 protein decreased in the brain, and β-TRCP decreased in the liver. The Nrf2 signaling and expression of downstream antioxidant enzymes were different under different oxygen concentrations, reflecting the flexible characteristics of Gansu zokor to deal with the hypoxic environment.

Activation of the Keap1/Nrf2 Pathway as an Adaptive Response to an Electrophilic Metabolite of Morphine.

Morphinone (MO) is an electrophilic metabolite of morphine that covalently binds to protein thiols via its α,β-unsaturated carbonyl group, resulting in toxicity in vitro and in vivo. Our previous studies identified a variety of redox signaling pathways that are activated during electrophilic stress. Here, we examined in vitro activation of a signaling pathway involving Kelch-like ECH-associated protein 1 (Keap1) and nuclear factor erythroid 2-related factor 2 (Nrf2) in response to MO. Exposure of HepG2 cells to MO caused covalent modification of Keap1 thiols (evaluated using biotin-PEAC5-maleimide labeling) and nuclear translocation of Nrf2, thereby up-regulating downstream genes encoding ATP binding cassette subfamily C member 2, solute carrier family 7 member 11, glutamate-cysteine ligase catalytic subunit, glutamate-cysteine ligase modifier subunit, glutathione S-transferase alpha 1, and heme oxygenase 1. However, dihydromorphinone, a metabolite of morphine lacking the reactive C7-C8 double bond, had little effect on Nrf2 activation. These results suggest that covalent modification is crucial in the Keap1/Nrf2 pathway activation and that this pathway is a redox signaling-associated adaptive response to MO metabolism.

Benzo[a]pyrene treatment modulates Nrf2/Keap1 axis and changes the metabolic profile in rat lung cancer

Lung cancer is an aggressive malignancy and the leading cause of cancer-related deaths. Benzo[a]pyrene (B[a]P), a polycyclic hydrocarbon, plays a pivotal role in lung carcinogenesis. Uncovering the molecular mechanism underlying the pathophysiology of B[a]P induced malignancy is crucial. Male Sprague Dawley rats were induced with B[a]P to generate a lung cancer model. The B[a]P administered rats show increased body and lung weight, loss of normal pulmonary architecture, and decreased survival. This study demonstrated that B[a]P upregulates activating transcription factor-6 (ATF6) and C/EBP Homologous Protein (CHOP) and induces endoplasmic reticulum (ER) stress. B[a]P also dysregulated mitochondrial homeostasis by upregulating, PTEN-induced putative kinase-1 (PINK1) and Parkin. B[a]P affected the levels of superoxide dismutase (SOD), reduced glutathione (GSH), malondialdehyde (MDA), and increased oxidative stress. B[a]P exposure downregulated Kelch-like ECH-associated protein 1 (Keap1) and upregulated nuclear factor erythroid 2–related factor 2 (Nrf2) and Heme oxygenase-1(HO1). The metabolomic study identified that biosynthesis of nucleotide, amino acids, pentose phosphate pathway (PPP), tricarboxylic acid cycle (TCA), and glutathione metabolism were up-accumulated. On the other hand, lower accumulation of fatty acids e.g., palmitic acid, stearic acid, and oleic acid were reported in the B[a]P induced group. Overall, the results of this study indicate that B[a]P treatment affects several signaling and metabolic pathways, whose dysregulation might be involved in lung cancer induction.

17β-estradiol plays the anti-osteoporosis role via a novel ESR1-Keap1-Nrf2 axis-mediated stress response activation and Tmem119 upregulation.

Increased oxidative stress and decreased osteoblastic bone formation contribute to estrogen deficiency-induced osteoporosis. However, the role and mechanism of estrogen-deficiency in regulating oxidative stress and osteoblastic activity remain unclear. Here, we showed that estrogen-deficient bone marrow stromal/stem cells (BMSCs) exhibited impaired capacity to combat stress, characterized by increased oxidative stress, shortened cell survival and reduced osteogenic differentiation and bone formation, which were due to a decrease of nuclear factor erythroid 2-related factor 2 (Nrf2). Nrf2 re-activation induced by the pyrazinyl dithiolethione oltipraz significantly rescued the cell phenotype of estrogen-deficient BMSCs in vitro and ex vivo. Mechanistically, we found that 17β-estradiol/ESR1 (Estrogen Receptor 1) facilitated Nrf2 accumulation, and activated its target genes by competing with Nrf2 for binding to Kelch-like ECH-associated protein 1 (Keap1) via ESR1 containing a highly conserved DLL motif. Of note, oltipraz, an Nrf2 activator, rescued ovariectomy-induced osteoporosis partly by inhibiting oxidative stress and promoting osteoblastic bone formation via Nrf2-induced antioxidant signaling activation and Tmem119 (transmembrane protein 119) upregulation. Conversely, Nrf2 knockout largely blocked the bone anabolic effect of 17β-estradiol in vivo and ex vivo. This study provides insight into the mechanisms whereby estrogen prevents osteoporosis through promoting osteoblastic bone formation via Nrf2-mediated activation of antioxidant signaling and upregulation of Tmem119, and thus provides evidence for Nrf2 as a potential target for clinical prevention and treatment of menopause-related osteoporosis.

Efficacy of Artemisia annua Linné in improving cognitive impairment in a chronic cerebral hypoperfusion-induced vascular dementia animal model

BackgroundVascular dementia (VaD) is the second most common type of dementia after Alzheimer's disease. Currently, no FDA-approved drugs are available for the treatment of VaD. Artemisia annua Linné (AA) is known to have antioxidant properties, but its effects and mechanisms of action on cognitive impairment are still unknown. PurposeIn this study, the improvement in cognitive impairment by AA in terms of protection against oxidative stress, neuroinflammation, and preservation of the integrity of the neurovascular unit (NVU) was assessed in an animal model of VaD with bilateral common carotid artery occlusion (BCCAO). MethodsEight-week-old male Wistar rats were allowed to adapt for four weeks, and BCCAO was induced at 12 weeks of age. The rats were randomly assigned into four groups, with seven rats in each group: sham group without BCCAO, VaD group that received oral administration of distilled water after BCCAO surgery, and two AA groups that received oral administration of 150 mg/kg or 750 mg/kg AA after BCCAO surgery for 8 weeks. Nine weeks after BCCAO surgery, the cognitive function of the rats was evaluated and accumulated oxidative stress was assessed by immunohistochemistry, immunofluorescence, and western blotting. Damage to the components of the NVU was evaluated, and sirtuin (Sirt) 1 and 2 expression and nuclear factor-erythrocyte 2-associated factor 2 (Nrf2)/Kelch-like ECH-associated protein1 (Keap1) activation were investigated to assess the reduction in cell signaling and antioxidant pathways. ResultsBCCAO-induced cerebral perfusion decreased memory function and induced neuroinflammation and oxidative stress. But AA treatment mitigated cognitive impairment and reduced neuroinflammation and oxidative stress caused by chronic cerebral hypoperfusion. AA extracts activated the Nrf2/Keap1/activating antioxidant response elements pathway and maintained Sirt 1 and 2, subsequently leading to the maintenance of neurons, improved construct of microvessels, increased platelet-derived growth factor receptor beta, and platelet-endothelial cell adhesion molecule-1 associated with the blood-brain barrier integrity. ConclusionAA is effective in alleviating BCCAO-induced cognitive decline and its administration may be a useful therapeutic approach for VaD.

Kynureninase Upregulation Is a Prominent Feature of NFR2-Activated Cancers and Is Associated with Tumor Immunosuppression and Poor Prognosis.

The nuclear factor erythroid 2-related factor 2 (NRF2) pathway is frequently activated in various cancer types. Aberrant activation of NRF2 in cancer is attributed to gain-of-function mutations in the NRF2-encoding gene NFE2L2 or a loss of function of its suppressor, Kelch-like ECH-associated protein 1 (KEAP1). NRF2 activation exerts pro-tumoral effects in part by altering cancer cell metabolism. Previously, we reported a novel mechanism of NRF2 tumoral immune suppression through the selective upregulation of the tryptophan-metabolizing enzyme kynureninase (KYNU) in lung adenocarcinoma. In the current study, we explored the relevance of NRF2-mediated KYNU upregulation across multiple cancer types. Specifically, using a gene expression dataset for 9801 tumors representing 32 cancer types from The Cancer Genome Atlas (TCGA), we demonstrated that elevated KYNU parallels increased gene-based signatures of NRF2-activation and that elevated tumoral KYNU mRNA expression is strongly associated with an immunosuppressive tumor microenvironment, marked by high expression of gene-based signatures of Tregs as well as the immune checkpoint blockade-related genes CD274 (PDL-1), PDCD1 (PD-1), and CTLA4, regardless of the cancer type. Cox proportional hazard models further revealed that increased tumoral KYNU gene expression was prognostic for poor overall survival in several cancer types, including thymoma, acute myeloid leukemia, low-grade glioma, kidney renal papillary cell carcinoma, stomach adenocarcinoma, and pancreatic ductal adenocarcinoma (PDAC). Using PDAC as a model system, we confirmed that siRNA-mediated knockdown of NRF2 reduced KYNU mRNA expression, whereas activation of NFE2L2 (the coding gene for NRF2) through either small-molecule agonists or siRNA-mediated knockdown of KEAP1 upregulated KYNU in PDAC cells. Metabolomic analyses of the conditioned medium from PDAC cell lines revealed elevated levels of KYNU-derived anthranilate, confirming that KYNU was enzymatically functional. Collectively, our study highlights the activation of the NRF2-KYNU axis as a multi-cancer phenomenon and supports the relevance of tumoral KYNU as a marker of tumor immunosuppression and as a prognostic marker for poor overall survival.

Optineurin regulates NRF2-mediated antioxidant response in a mouse model of Paget's disease of bone.

Degenerative diseases affecting the nervous and skeletal systems affect the health of millions of elderly people. Optineurin (OPTN) has been associated with numerous neurodegenerative diseases and Paget's disease of bone (PDB), a degenerative bone disease initiated by hyperactive osteoclastogenesis. In this study, we found age-related increase in OPTN and nuclear factor E2-related factor 2 (NRF2) in vivo. At the molecular level, OPTN could directly interact with both NRF2 and its negative regulator Kelch-like ECH-associated protein 1 (KEAP1) for up-regulating antioxidant response. At the cellular level, deletion of OPTN resulted in increased intracellular reactive oxygen species and increased osteoclastogenic potential. At the tissue level, deletion of OPTN resulted in substantially increased oxidative stress derived from leukocytes that further stimulate osteoclastogenesis. Last, curcumin attenuated hyperactive osteoclastogenesis induced by OPTN deficiency in aged mice. Collectively, our findings reveal an OPTN-NRF2 axis maintaining bone homeostasis and suggest that antioxidants have therapeutic potential for PDB.

Moschus exerted protective activity against H2O2-induced cell injury in PC12 cells through regulating Nrf-2/ARE signaling pathways

The pivotal characteristics of Alzheimer's disease (AD) are irreversible memory loss and progressive cognitive decline, eventually causing death from brain failure. In the various proposed hypotheses of AD, oxidative stress is also regarded as a symbolic pathophysiologic cascade contributing to brain diseases. Using Chinese herbal medicine may be beneficial for treating and preventing AD. As a rare and valuable animal medicine, Moschus possesses antioxidant and antiapoptotic efficacy and is extensively applied for treating unconsciousness, stroke, coma, and cerebrovascular diseases. We aim to evaluate whether Moschus protects PC12 cells from hydrogen peroxide (H2O2)-induced cellular injury. The chemical constituents of Moschus are analyzed by GC-MS assay. The cell viability, reactive oxygen species (ROS) levels, mitochondrial membrane potential (MMP) levels, oxidative stress-related indicators, and apoptotic proteins are determined. Through GC-MS analysis, nineteen active contents were identified. The cell viability loss, lactate dehydrogenase releases, MMP levels, ROS productions, and Malondialdehyde (MDA) activities decreased, and BAX, Caspase-3, and Kelch-like ECH-associated protein 1 expression also significantly down-regulated and heme oxygenase 1, nuclear factor erythroid-2-related factor 2 (Nrf-2), and quinine oxidoreductase 1 expression upregulated after pretreatment of Moschus. The result indicated Moschus has neuroprotective activity in relieving H2O2-induced cellular damage, and the potential mechanism might be associated with regulating the Nrf-2/ARE signaling pathway. A more in-depth and comprehensive understanding of Moschus in the pathogenesis of AD will provide a fundamental basis for in vivo AD animal model research, which may be able to provide further insights and new targets for AD therapy.