Keap1-Nrf2 Pathway Research Articles

Identification of the Nrf2 in the fathead minnow muscle cell line: role for a regulation in response to H2O2 induced the oxidative stress in fish cell.

The Nrf2 (nuclear factor erythroid 2-related factor 2) plays a central role in cell protection against a wide variety of environmental stressors through the Nrf2-Keap1 (Kelch-like ECH-associated protein 1) pathway, but its involvement in modulation of antioxidant system of fish cell is still largely unexplored. The present study focused on the molecular cloning and silencing of the Nrf2 in the fathead minnow muscle cell line (FHM) in response to the oxidative stress induced by H2O2. A full-length cDNA of coding Nrf2 was cloned from FHM cells by RT-PCR and RACE approaches. The obtained cDNA covered 2578bp with an open reading frame (1770bp) of encoding 589 amino acids. Sequence alignment and phylogenetic analysis revealed a high degree of conservation (51-86%) among 16 fishes. Based on the cloned Nrf2 sequence, the siRNA-242 of targeting Nrf2 with the best knocking down efficiency was designed and detected. Then, the mRNA levels of Keap1, Nrf2, Maf (musculoaponeurotic fibrosarcoma oncogene), and HO-1 (haemoxygenase-1); the activities of T-SOD (total superoxide dismutase), CAT (catalase), and GSH-PX (glutathione peroxidase); the levels of GSH (glutathione) and MDA (malonaldehyde); and the cell cycle and apoptosis were analyzed to investigate the molecular responses after H2O2 exposure. These results showed a coordinated transcriptional regulation of Keap1, Maf, and HO-1 and antioxidants (T-SOD, GSH, CAT, and GSH-PX) and MDA levels after H2O2 exposure, leading to oxidative damage and apoptosis. These findings provided an insight to understand the mechanisms of Nrf2 against oxidative stress in fish.

Lapatinib Activates the Kelch-Like ECH-Associated Protein 1-Nuclear Factor Erythroid 2-Related Factor 2 Pathway in HepG2 Cells.

The receptor tyrosine kinase inhibitor lapatinib, indicated to treat patients with HER2-positive breast cancer in combination with capecitabine, can cause severe hepatotoxicity. Lapatinib is further associated with mitochondrial toxicity and accumulation of reactive oxygen species. The effect of lapatinib on the Kelch-like ECH-associated protein 1 (Keap1)-nuclear factor erythroid 2-related factor 2 (Nrf2) pathway, the major cellular defense pathway against oxidative stress, has so far not been studied in detail. In the present study, we show that lapatinib (2–20 µM) activates the Keap1-Nrf2 pathway in HepG2 cells, a hepatocellular carcinoma-derived cell line, in a concentration-dependent manner upon 24 h of treatment. Lapatinib stabilized the transcription factor Nrf2 at concentrations ≥5 µM and caused its nuclear translocation. Well-established Nrf2 regulated genes (Nqo1, Gsta1, Gclc, and Gclm) were upregulated at lapatinib concentrations ≥10 µM. Furthermore, cellular and mitochondrial glutathione (GSH) levels increased starting at 10 µM lapatinib. As a marker of oxidative stress, cellular GSSG significantly increased at 10 and 20 µM lapatinib. Furthermore, the gene expression of mitochondrial Glrx2 and SOD2 were increased upon lapatinib treatment, which was also observed for the mitochondrial SOD2 protein content. In conclusion, lapatinib treatment for 24 h activated the Keap1-Nrf2 pathway in HepG2 cells starting at 10 μM, which is a clinically relevant concentration. As a consequence, treatment with lapatinib increased the mRNA and protein expression of antioxidative and other cytoprotective genes and induced GSH synthesis, but these measures could not completely block the oxidative stress associated with lapatinib.

The Molecular Mechanisms Regulating the KEAP1-NRF2 Pathway.

The KEAP1-NRF2 pathway is the principal protective response to oxidative and electrophilic stresses. Under homeostatic conditions, KEAP1 forms part of an E3 ubiquitin ligase, which tightly regulates the activity of the transcription factor NRF2 by targeting it for ubiquitination and proteasome-dependent degradation. In response to stress, an intricate molecular mechanism facilitated by sensor cysteines within KEAP1 allows NRF2 to escape ubiquitination, accumulate within the cell, and translocate to the nucleus, where it can promote its antioxidant transcription program. Recent advances have revealed that KEAP1 contains multiple stress sensors and inactivation modalities, which together allow diverse cellular inputs, from oxidative stress and cellular metabolites to dysregulated autophagy, to regulate NRF2 activity. This integration of the KEAP1-NRF2 system into multiple cellular signaling and metabolic pathways places NRF2 activation as a critical regulatory node in many disease phenotypes and suggests that the pharmaceutical modulation of NRF2's cytoprotective activity will be beneficial for human health in a broad range of noncommunicable diseases.

NQO1 Enzyme and its Role in Cellular Protection; an Insight

NAD(P)H:quinone oxidoreductase 1 (NQO1) is considered as one of the most significant enzyme in cellular defense due to its ability to detoxify reactive quinones and quinone imines to their less toxic hydroquinones forms. NQO1 is a xenobiotic metabolizing cytosolic enzyme that catalyzes the reduction of two- or four-electron of numerous exogenous and endogenous quinones by utilizing flavin adenine dinucleotide (FAD) as a cofactor. NQO1 enzyme exists as a homodimer enzyme and is biochemically identified by its noticeable ability to utilize either NADH or NADPH as reducing cofactors and by its inhibition by anticoagulant agents such as dicumarol. NQO1 is a distinctly inducible enzyme and known to be controlled by the Nrf2-Keap1 pathway. The importance of the antioxidant activities exhibited by NQO1 enzyme in suppressing the oxidative stress status is provided by demonstration that induction or reduction of NQO1 levels are linked with increased and reduced susceptibilities to oxidative stress, respectively. The gene coding for NQO1 has two well-recognized polymorphisms at nucleotide site 609(C-T) and 465 (C-T) of the human cDNA. C609T causes complete loss of enzymatic activity due to protein instability, whereas the C465T results in reduction of the enzyme activity. In this Review, we deliberate the protecting activates of NQO1 and discussing its possible transcriptional pathways regulating its induction by Nrf2-Keap1/ARE system.

Effects of Inorganic Arsenic on Human Prostate Stem-Progenitor Cell Transformation, Autophagic Flux Blockade, and NRF2 Pathway Activation.

Background:Inorganic arsenic (iAs) is an environmental toxicant associated with an increased risk of prostate cancer in chronically exposed populations worldwide. However, the biological mechanisms underlying iAs-induced prostate carcinogenesis remain unclear.Objectives:We studied how iAs affects normal human prostate stem-progenitor cells (PrSPCs) and drives transformation and interrogated the molecular mechanisms involved.Methods:PrSPCs were enriched by spheroid culture from normal human primary or immortalized prostate epithelial cells, and their differentiation capability was evaluated by organoid culture. Microarray analysis was conducted to identify iAs-dysregulated genes, and lentiviral infection was used for stable manipulation of identified genes. Soft agar colony growth assays were applied to examine iAs-induced transformation. For in vivo study, PrSPCs mixed with rat urogenital sinus mesenchyme were grafted under the renal capsule of nude mice to generate prostatelike tissues, and mice were exposed to () iAs in drinking water for 3 months.Results:Low-dose iAs () disturbed PrSPC homeostasis in vitro, leading to increased self-renewal and suppressed differentiation. Transcriptomic analysis indicated that iAs activated oncogenic pathways in PrSPCs, including the KEAP1-NRF2 pathway. Further, iAs-exposed proliferative progenitor cells exhibited NRF2 pathway activation that was sustained in their progeny cells. Knockdown of NRF2 inhibited spheroid formation by driving PrSPC differentiation, whereas its activation enhanced spheroid growth. Importantly, iAs-induced transformation was suppressed by NRF2 knockdown. Mechanistically, iAs suppressed Vacuolar ATPase subunit VMA5 expression, impairing lysosome acidification and inhibiting autophagic protein degradation including p62, which further activated NRF2. In vivo, chronic iAs exposure activated NRF2 in both epithelial and stroma cells of chimeric human prostate grafts and induced premalignant events.Conclusions:Low-dose iAs increased self-renewal and decreased differentiation of human PrSPCs by activating the p62-NRF2 axis, resulting in epithelial cell transformation. NRF2 is activated by iAs through specific autophagic flux blockade in progenitor cells, which may have potential therapeutic implications. https://doi.org/10.1289/EHP6471

Saturated and aromatic aldehydes originating from skin and cutaneous bacteria activate the Nrf2-keap1 pathway in human keratinocytes.

Skin homeostasis is constantly challenged by environmental factors, affecting its delicate redox balance. The skin is also home to a wide variety of bacterial species, including Staphylococci. The cutaneous redox state is governed by the Nrf2-keap1 pathway, which is responsible for the induction of phase II cytoprotective enzymes, thus sustaining a healthy oxidative state. As part of normal metabolism, both bacteria and cutaneous tissue emit copious amounts of volatile organic compounds (VOCs), one subgroup of which are aldehydes. α,β-unsaturated aldehydes are known activators of Nrf2-keap1 pathway by direct oxidation of the keap1 protein. However, we did not encounter reports of Nrf2 activation by saturated or aromatic aldehydes, neither bacteria nor skin-derived. We hypothesized that non-α,β-unsaturated aldehydes derived from skin or cutaneous bacteria may act as Nrf2-keap1 pathway activators and therefore afford protection against environmental insults. The saturated aldehydes nonanal and decanal (known skin metabolites) and the aromatic aldehyde benzaldehyde (known skin and Staphylococcus epidermidis metabolite) were shown to induce the Nrf2-keap1 pathway in human keratinocytes. We also identified a newly described aromatic aldehyde, 3-furaldehyde (3-FA), emitted from S. aureus and S. epidermidis cultures, which also activated the pathway. Moreover, Nrf2-keap1 induction led to a significant protection against UVB-induced apoptosis. The mechanism involved in this activation has been partially elucidated. This work emphasizes the importance of cutaneous bacteria, as well as healthy skin lipid peroxidation processes in the maintenance and regulation of the cellular antioxidant response, namely with regard to coping with environmental stressors.

Keap1-Nrf2 signaling pathway in angiogenesis and vascular diseases.

The transcription factor, nuclear factor E2-related factor 2 (Nrf2), is highly sensitive to oxidative burst products, including reactive oxygen species (ROS) and reactive nitrogen species. In the cell nucleus, Nrf2 activates various antioxidant genes by binding to the antioxidant response elements. As an adapter for cullin 3/ring-box 1, kelch-like ECH-associated protein 1 (Keap1) ubiquitinates and degrades Nrf2 under physiological conditions. Conversely, with the aggravation of oxidative stress, Keap1-Nrf2 interaction could be much more easily dissociated. ROS play a key role in regulating the redox signaling pathway and affect the vasculature in a dose-dependent manner. Long-term production or high concentration of ROS are harmful to the vascular system, while moderate ROS can promote angiogenesis and tissue regeneration. Furthermore, appropriate regulation of oxidative stress mediated via the Keap1-Nrf2 pathway would be beneficial in various diseases associated with abnormal angiogenesis, including diabetes and cancers. Nrf2 deficiency has also been shown to result in significantly impaired survival, proliferation, and angiogenic capacity of endothelial cells in a hind limb ischemia model. Thus, this review will briefly summarize the underlying molecular mechanisms of Keap1-Nrf2 pathway in regulating oxidative stress and also help elucidate the critical role of Keap1-Nrf2 in angiogenesis under physiological and pathological conditions.

Cinnamtannin A2 protects the renal injury by attenuates the altered expression of kidney injury molecule 1 (KIM-1) and neutrophil gelatinase-associated lipocalin (NGAL) expression in 5/6 nephrectomized rat model

Present investigation determines the protective effect of Cinnamtannin A2 against chronic renal failure (CRF). 5/6 nephrectomized rat model was used to induced CRF by removing the kidneys and rats were treated with Cinnamtannin A2 10 mg/kg, i.p. for the period 30 days. Nephroprotective effect Cinnamtannin A2 was assessed by estimating the biochemical parameters of renal function test and cytokines in the serum of nephractomized rats. Oxidative stress parameters were estimated in the kidney tissue and western blot assay and qRT-PCR assay was performed to determine the expression of protein in renal tissue of nephractomized rats. Moreover histopathology study was done to observe the tubular injury. Data of the report reveals that treatment with Cinnamtannin A2 ameliorates the altered level of creatinine, blood urea nitrogen (BUN), Neutrophil gelatinase-associated lipocalin (NGAL), Kidney Injury Molecule-1 (KIM-1) and cytokines in the serum and microalbuminurea in the urine of 5/6 nephrectomized rat. Oxidative stress level was reduced in Cinnamtannin A2 treated group than CRF group. Moreover treatment with Cinnamtannin A2 attenuates the altered expression of proteins involved in Nrf2-Keap1 pathway in the kidney tissue of 5/6 nephrectomized rat. Result of histopathology reveals that tubular injury score was reduced in the kidney tissue of Cinnamtannin A2 treated group than CRF group. In conclusion, data of the report suggest that treatment with Cinnamtannin A2 ameliorates the level of KIM1 and NAGL in 5/6 nephractomized rats by regulating Nrf2- Keap1 pathway.

Nrf2 suppresses allergic lung inflammation by attenuating the type2-innate lymphoid cell response

Abstract The Keap1-Nrf2 system has an essential role in oxidative-stress responses by inducing a number of cytoprotective genes. Under stress, damaged epithelial cells release cytokines that activate Type 2 Innate lymphoid cells (ILC2s), which mediate the allergic immune response. Here we investigated the role of the Keap1-Nrf2 pathway in ILC2 homeostasis and allergic inflammation, using Nrf-2-knockout mice. ILC2s from Nrf2-deficient mice showed a transient, upregulated IL-33 response, and underwent hyper-proliferation in response to combined stimulation by IL-33 with IL-2, IL-7, or TSLP. This enhanced proliferation was correlated with an increased activation of downstream signals, including JAK1, Akt, and Erk1/2. In contrast, activating Nrf2 with a chemical inducer (CDDO-Im) decreased the viability of wild-type, but not of the Nrf2-deficient ILC2s. The reduced ILC2 viability resembled that exerted by the corticosteroid dexamethasone; however, unlike the latter, the Nrf2-dependent cell death was mediated by neither caspase 3-dependent apoptosis nor necroptosis. Using a mouse intratracheal IL-33-administration allergy model, we found that the activation of Nrf2 by CDDO-Im in vivo decreased the numbers of pulmonary ILC2s and eosinophils. These findings indicated that Nrf2 is an important regulator of the allergic response, by determining the survival and death of ILC2s, and suggest that Nrf2 activation is a potential therapeutic strategy for steroid-resistant allergy alleviation.

RETRACTED ARTICLE: EV71 virus reduces Nrf2 activation to promote production of reactive oxygen species in infected cells

BackgroundEmerging evidence closely links Enterovirus 71 (EV71) infection with the generation of reactive oxygen species (ROS). Excess ROS results in apoptosis and exacerbates inflammatory reactions. The Keap1–Nrf2 axis serves as an essential oxidant counteracting pathway.MethodsThe present study aimed to elucidate the role of the Keap1–Nrf2 pathway in modulating apoptosis and inflammatory reactions triggered by oxidative stress in Vero and RD cells upon EV71 infection.ResultsElevated ROS production was identified in EV71 infected Vero and RD cells. The percentage of dead cells and expression of inflammation-promoting cytokines were increased in these cells. EV71 infected cells also displayed reinforced Keap1 expression and abrogated Nrf2 expression. Keap1 silencing resulted in the downstream aggregation of the Nrf2 protein and heme oxygenase-1 HO-1. Keap1 silencing repressed ubiquitination and reinforced Nrf2 nuclear trafficking. Furthermore, silencing Keap1 expression repressed ROS production, cell death, and inflammatory reactions in EV71 infected RD and Vero cells. In contrast, silencing of both Keap1 and Nrf2 restored ROS production, cell death, and inflammatory reactions. Nrf2 and Keap1 modulated the stimulation of the Akt sensor and extrinsic as well as intrinsic cell death pathways, resulting in EV71-triggered cell death and inflammatory reactions.ConclusionsEV71 infection can trigger ROS production, cell death, and inflammatory reactions by modulating the Nrf2 and Keap1 levels of infected cells.

2-Sulfonylpyridines as Tunable, Cysteine-Reactive Electrophiles.

The emerging use of covalent ligands as chemical probes and drugs would benefit from an expanded repertoire of cysteine-reactive electrophiles for efficient and diverse targeting of the proteome. Here we use the endogenous electrophile sensor of mammalian cells, the KEAP1-NRF2 pathway, to discover cysteine-reactive electrophilic fragments from a reporter-based screen for NRF2 activation. This strategy identified a series of 2-sulfonylpyridines that selectively react with biological thiols via nucleophilic aromatic substitution (SNAr). By tuning the electrophilicity and appended recognition elements, we demonstrate the potential of the 2-sulfonylpyridine reactive group with the discovery of a selective covalent modifier of adenosine deaminase (ADA). Targeting a cysteine distal to the active site, this molecule attenuates the enzymatic activity of ADA and inhibits proliferation of lymphocytic cells. This study introduces a modular and tunable SNAr-based reactive group for targeting reactive cysteines in the human proteome and illustrates the pharmacological utility of this electrophilic series.

Clinical Manifestations of Hereditary Leiomyomatosis and Renal Cell Carcinoma: a Case Report

Introduction: Hereditary leiomyomatosis and renal cell carcinoma (HLRCC) is a rare genetic disorder etiologically stemming from mutations in the FH gene. This disorder has been reported in 300 families worldwide. We were presented with the following case as part of the Rare Disease Interdepartmental Science Case Competition (RISCC) in order to successfully diagnose the patient and examine the pathophysiology, socioeconomics, and treatment strategy of this disorder.Case Presentation: A 27-year-old woman presented to the emergency room with a two-week history of hematuria. Her medical history included a single benign skin lesion. She did not report a family history of cancer but stated that her mother underwent a hysterectomy for unknown reasons.Clinical Investigations: An abdominal ultrasound revealed an isoechoic nodular formation on the right kidney, and CT scans confirmed the presence of a 7-cm renal tumour and extensive leiomyomas in the patient’s uterus. Genetic testing revealed a mutation in the fumarate hydratase (FH) gene, leading to the diagnosis of hereditary leiomyomatosis and renal cell carcinoma.Pathophysiology: HLRCC is a condition caused by an autosomal dominant germline mutation in the FH gene, characterized by skin lesions, uterine leiomyomas, and aggressive renal cell carcinoma. FH acts as a tumour suppressor gene and encodes the fumarase protein. HLRCC arises from a variety of missense mutations in the FH gene that prevent the formation of a full fumarase tetramer. The disease is likely related to the dosage of the gene, rather than the site of mutation. Consequently, there is no known genotype/phenotype correlation. Fumarate inhibits HIF-α hydroxylases, so loss of the FH gene causes fumarate accumulation within cells and prevents the degradation of HIF-α. This induces a state of pseudohypoxia and leads to upregulation of genes involved in cell growth and tumorigenesis. Fumarate accumulation also modulates the Keap1-Nrf2 pathway by inducing activation of Nrf2 and allows for the protection of cancer cells from oxidative and electrophilic stressors. Furthermore, deficiency of fumarase prevents oxidative phosphorylation, thus promoting aerobic glycolysis, the most common method of metabolism used by cancer cells.Socioeconomics: The risk of infertility and developing an aggressive cancer makes it essential to have frequent and thorough screening procedures early in life for children with a family history of HLRCC. Medical costs, infertility risk, and frequent hospital visits breed anxiety and stress for patients and family members. Fortunately, organizations like HLRCC Family Alliance exist to provide accessible information and represent a welcoming support community.Treatment Strategy: To treat the patient’s renal tumour, we suggest a radical nephrectomy with wide margins due to the aggressive nature of renal cell carcinoma. Additional imaging should be performed to identify and excise any metastatic tumours. The patient should also consult a gynecologist regarding her extensive uterine leiomyomas and will likely undergo a hysterectomy or myomectomy. A full dermatological exam should be performed to locate additional cutaneous leiomyomas. We also strongly suggest that family members undergo genetic testing. Lastly, we recommend the patient connect with support groups such as HLRCC Family Alliance to receive continued social support following her diagnosis and throughout her life.

Dietary nanoencapsulated quercetin homeostated transcription of redox-status orchestrating genes in zebrafish (Danio rerio) exposed to silver nanoparticles

The present study assessed the protective effect of chitosan-nanoencapsulated quercetin (Qu-ChiNPs) against oxidative stress caused by silver nanoparticles (AgNPs). To this end, the transcription of prime genes regulating hepatic Keap1-Nrf2 pathway as well as downstream antioxidant enzymes were monitored prior to and after oxidative stress by AgNPs. Zebrafish (Danio rerio; n = 225) was assigned into five experimental groups based on feeding with diets supplemented with different additives as follows: negative and positive control groups, without additive; ChiNPs, 400 mg nanochitosan per kg diet; Quercetin, 400 mg free quercetin per kg diet; and Qu-ChiNPs, 400 mg Qu-ChiNPs per kg diet. At the end of the feeding trial (40 days), the experimental groups, except the negative control, were exposed to sublethal concentration of AgNPs (0.15 mg L−1) for 96h. Before exposure to AgNPs, free quercetin-treated diet significantly upregulated Keap1, Nrf2, Cat, SOD, GPx, and GST genes in the liver tissue when compared with the control diet, whereas Qu-Chi.NPs downregulated their transcription to the lowest levels. After exposure to AgNPs, all genes exhibited different responses in the AgNPs-exposed groups. The highest transcription of Nrf2, Cat, SOD, GPx, and GST was observed in the positive group, with being upregulated about 8, 10, 8, 8, and 7 times, respectively, when compared to the respective ones in the negative control. However, Keap1 showed a reverse response with being transcripted 12 times lower. The quercetin treatments, especially Qu-Chi.NPs, significantly reduced the transcription of Nrf2, Cat, SOD, GPx, and GST genes, yet enhanced Keap1 expression. Qu-Chi.NPs reduced the expression of Nrf2, SOD, Cat, GPx, and GST about 11, 10, 15, 10, and 10 times, respectively, yet increased that of Keap1 about 12 times. Taken together, nanoencapsulation can improve the antioxidant efficacy of quercetin against AgNPs toxicity and might reduce involvement of the cellular antioxidant system through tuning redox status. More broadly, it would be interesting to assess the effects of Qu-Chi.NPs against other metallic and organic oxidative stressors or pollutants.

Potential Applications of NRF2 Modulators in Cancer Therapy.

The nuclear factor erythroid 2-related factor 2 (NRF2)–Kelch-like ECH-associated protein 1 (KEAP1) regulatory pathway plays an essential role in protecting cells and tissues from oxidative, electrophilic, and xenobiotic stress. By controlling the transactivation of over 500 cytoprotective genes, the NRF2 transcription factor has been implicated in the physiopathology of several human diseases, including cancer. In this respect, accumulating evidence indicates that NRF2 can act as a double-edged sword, being able to mediate tumor suppressive or pro-oncogenic functions, depending on the specific biological context of its activation. Thus, a better understanding of the mechanisms that control NRF2 functions and the most appropriate context of its activation is a prerequisite for the development of effective therapeutic strategies based on NRF2 modulation. In line of principle, the controlled activation of NRF2 might reduce the risk of cancer initiation and development in normal cells by scavenging reactive-oxygen species (ROS) and by preventing genomic instability through decreased DNA damage. In contrast however, already transformed cells with constitutive or prolonged activation of NRF2 signaling might represent a major clinical hurdle and exhibit an aggressive phenotype characterized by therapy resistance and unfavorable prognosis, requiring the use of NRF2 inhibitors. In this review, we will focus on the dual roles of the NRF2-KEAP1 pathway in cancer promotion and inhibition, describing the mechanisms of its activation and potential therapeutic strategies based on the use of context-specific modulation of NRF2.

TRIM25 promotes the cell survival and growth of hepatocellular carcinoma through targeting Keap1-Nrf2 pathway

Tumor cells often exhibit augmented capacity to maintain endoplasmic reticulum (ER) homeostasis under adverse conditions, yet the underlying mechanisms are not well defined. Here, through the evaluation of all human TRIM proteins, we find that TRIM25 is significantly induced upon ER stress. Upregulation of TRIM25 ameliorates oxidative stress, promotes ER-associated degradation (ERAD), and reduces IRE1 signaling in the UPR pathway. In contrast, depletion of TRIM25 leads to ER stress and attenuates tumor cell growth in vitro and in vivo. Mechanistically, TRIM25 directly targets Keap1 by ubiquitination and degradation. This leads to Nrf2 activation, which bolsters anti-oxidant defense and cell survival. TRIM25 expression is positively associated with Nrf2 expression and negatively with Keap1 expression in hepatocellular carcinoma (HCC) xenografts and specimens. Moreover, high TRIM25 expression correlates with poor patient survival in HCC. These findings reveal TRIM25 as a regulator of ER homeostasis and a potential target for tumor therapy.

NBR1-mediated p62-liquid droplets enhance the Keap1-Nrf2 system.

p62/SQSTM1 is a multivalent protein that has the ability to cause liquid-liquid phase separation and serves as a receptor protein that participates in cargo isolation during selective autophagy. This protein is also involved in the non-canonical activation of the Keap1-Nrf2 system, a major oxidative stress response pathway. Here, we show a role of neighbor of BRCA1 gene 1 (NBR1), an autophagy receptor structurally similar to p62/SQSTM1, in p62-liquid droplet formation and Keap1-Nrf2 pathway activation. Overexpression of NBR1 blocks selective degradation of p62/SQSTM1 through autophagy and promotes the accumulation and phosphorylation of p62/SQSTM1 in liquid-like bodies, which is required for the activation of Nrf2. NBR1 is induced in response to oxidative stress, which triggers p62-mediated Nrf2 activation. Conversely, loss of Nbr1 suppresses not only the formation of p62/SQSTM1-liquid droplets, but also of p62-dependent Nrf2 activation during oxidative stress. Taken together, our results show that NBR1 mediates p62/SQSTM1-liquid droplet formation to activate the Keap1-Nrf2 pathway.

Biomonitoring of biomarkers among pesticide sprayers and nonsprayers across cropping seasons in Punjab, India

Introduction: Pesticide exposure causes acute and chronic adverse health effects on humans affecting the Kelch ECH-associated protein (Keap1)- nuclear factor erythroid 2-related factor 2 (Nrf2) and other oxidative pathways. We assessed the Keap1-Nrf2 pathway and other oxidative stress biomarker levels among a cohort of agricultural pesticide sprayers (SP) and nonspraying (NSP) farmers of a rural community during the cotton and wheat cropping seasons in India. Methodology: We randomly selected eight villages from the four blocks of Bathinda district, Punjab. Using a cohort study design, we collected the socio-demographic characteristics and biological samples (blood and urine) from 68 SP and 71 NSP at baseline, during the flowering seasons of cotton and wheat crops at scheduled time points. The United States Environment Protection Agency methods and standards were used to detect pesticide residues. Standard and validated enzyme-linked immunosorbent assay-based kits and colorimetric method-based kits were used to detect the biomarkers of Keap1-Nrf2 and oxidative stress pathways. Results: In Keap1-Nrf2 pathway, cotton season glutathione peroxidase (P = 0.004) and baseline heme oxygenase-1 (P Conclusion: The Keap1-Nrf2 and other oxidative stress biomarker levels between the group were not consistently different all times across the seasons. The biomarker levels of SP and NSP need to be compared with the nonagricultural population.

The Nrf2-Keap1 pathway: A secret weapon against pesticide persecution in Drosophila Kc cells

Nrf2-Keap1 pathway defends organisms against the detrimental effects of oxidative stress, and play pivotal roles in preventing xenobiotic-related toxicity. We designed experiments to explore and verify its role and function under deltamethrin (DM) stress. In experiments, DM was selected as the inducer, and Drosophila Kc cells were treated as the objects. The result showed the oxidative stress of cells proliferated in a very short time after DM treatment, reaching the maximum after one hour of treatment. The experimental data showed Nrf2 could be up-regulated and activated by DM which were manifested by the increase of Nrf2 mRNA, Nrf2 protein in the nucleus and the expression of detoxification enzyme genes. We further tested the activity of all groups, and found the survival rate of cells was basically proportional to the expression of Nrf2. Based on the above experimental results, Keap1 overexpression (K+), Nrf2 overexpression (N+) or interference (N-) cells were used to verified the relationship between Nrf2, cell survival and detoxification enzymes expression. We found the cell survival rate of N+ group was significantly higher than that of other groups and the expression of detoxification enzymes were increased compared to the control group. These results demonstrated that Nrf2 is related to cell detoxification and associated with the tolerance to DM. Our evidence suggested Nrf2 is a potential therapeutic target for oxidative stress and a potential molecular target gene of resistance control.

Glutamine improves heat stress–induced oxidative damage in the broiler thigh muscle by activating the nuclear factor erythroid 2–related 2/Kelch-like ECH-associated protein 1 signaling pathway

The aim of the present study was to evaluate the effect of glutamine (Gln) on modulating heat stress–induced oxidative damage in the broiler thigh muscle through nuclear factor erythroid 2–related 2/Kelch-like ECH-associated protein 1 (Nrf2-Keap1) pathway. Three-hundred 22-day-old Arbor Acres broilers were reallocated into 5 groups: a control group (24 °C) fed with basal diet and 4 heat stress (HS) groups (34 °C for 8 h/D) fed with basal diet containing 0, 0.5, 1.0, and 1.5% Gln. This experiment lasted 21 D. Heat stress decreased (P < 0.05) pH, redness, and Gln levels, and increased (P < 0.05) luminance, water loss rate, and cooking loss (CL) values of the thigh meat. Compared with the HS group, supplementation with 1.5% Gln increased (P < 0.05) pH, redness, and Gln levels, but decreased (P < 0.05) luminance and CL values in the thigh meat. There were significant decreases (P < 0.05) in glutathione (GSH), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), total antioxidant capacity (T-AOC), and Nrf2 levels, but significant increases (P < 0.05) in the malondialdehyde (MDA) and Keap1 levels of the thigh muscle after HS treatment. Compared with the HS group, supplementation with 1.0, and 1.5% Gln decreased (P < 0.05) MDA and Keap1 levels; supplementation with 1.5% Gln increased (P < 0.05) GSH, GSH-Px, T-AOC, CAT, SOD, and Nrf2 levels in the thigh muscle of heat-stressed broilers. Furthermore, HS decreased (P < 0.05) Nrf2, SOD, CAT, and GSH-Px mRNA expression levels, but increased (P < 0.05) Keap1 mRNA level in the thigh muscle of broiler. Dietary supplementation with 1.5% Gln increased (P < 0.05) Nrf2, GSH-Px, CAT, and SOD mRNA expression levels, but decreased (P < 0.05) Keap1 mRNA level in the thigh muscle of heat-stressed broilers. In conclusion, dietary Gln improved the resistance of heat-stressed broiler muscles to oxidative damage possibly through reversing the muscle Gln level and inducing the expression of the Nrf2-Keap1 pathway.

Flavonoids from Apios americana Medikus Leaves Protect RAW264.7 Cells against Inflammation via Inhibition of MAPKs, Akt-mTOR Pathways, and Nfr2 Activation.

Apios americana Medikus was once widely accepted as staple food in India for a long time, and the tuber of which possesses high nutrients. During the past decades, most of the research has focused on the biological activity in the tubers of Apios americana Medikus whereas the leaves were ignored. In this study, the Apios americana Medikus leaf extract (ALE) was obtained and seven compounds were identified. LPS-induced RAW264.7 cells were used to study the anti-inflammation activity of ALE. As expected, ALE reduced the secretion of nitric oxide (NO) and inflammatory cytokines via inhibition of NF-κB and MAPK signaling together with activation of Nrf2-Keap1 and FOXO pathways, as well as alleviating the oxidative stress and mitochondrial dysfunction. In addition, ALE could activate HMGB1-Beclin1 and Sirt1-FoxO1 pathways and inhibit the Akt-mTOR signaling pathway to activate autophagy, protecting RAW264.7 cells from inflammation. In summary, our results suggested that ALE might help activate the anti-inflammation system, resulting in the prevention of LPS-induced damage in RAW264.7 cells.