Nrf2 ARE Research Articles

Abstract 2592: Helicobacter pylori-induced FGFR4 mediates nuclear accumulation of NRF2 in gastric tumorigenesis

Abstract Background: Fibroblast growth factor receptor 4 (FGFR4) is a member of the transmembrane tyrosine kinase receptor family and has been linked to a variety of malignancies. NRF2 (Nuclear factor-erythroid 2-related factor) is a cytoprotective factor and a critical regulator in the antioxidant response pathway. In this study, we sought to uncover novel functions of FGFR4 and its role in regulating the antioxidant response in gastric carcinogenesis. Methods and Results: Using western blot and immunofluorescence, H. pylori infection in gastric cancer cell lines demonstrated high levels of reactive oxygen species and induction of both FGFR4 and NRF2. Using Flow cytometry, FGFR4 silencing resulted in decrease of NRF2 with a significant spike in ROS levels and an increase in DNA damage and cell death. FGFR4 knockdown showed a significant decrease in NRF2 transcriptional activity as measured by NRF2 ARE luciferase reporter assay and resulted in reduced mRNA levels of HO-1 (Heme Oxygenase-1), which is a classical target of NRF2. These results were confirmed by immunofluorescence showing a significant increase of nuclear accumulation of NRF2 after H. pylori infection which was abolished after FGFR4 knockdown. Similar results were found using recombinant protein FGF19, a FGFR4 ligand. C57/B6 wild-type mice were infected with the pylori strain (PMSS1). An increase in FGFR4, NRF2, and HO-1 was seen by immunofluorescence, Western blot, and quantitative real-time PCR in H. pylori-infected mice vs control mice. We observed a reduction in NRF2 in our in vitro and in vivo models using H3B-6527, a specific FGFR4 inhibitor. We also discovered an association between an increase in FGFR4 and P62 protein expressions and NRF2 protein stability. We detected a significant increase in FGFR4, NRF2, and HO1 in dysplastic and neoplastic gastric lesions using the TFF1-KO mouse model which was further aggravated by H. pylori infection. In terms of the mechanism, utilizing proximity ligation and immunoprecipitation assays, we found that FGFR4 binds to P62 to inhibit the interaction between NRF2 and KEAP1, allowing NRF2 to avoid degradation facilitating its translocation and accumulation in the nucleus. Conclusion: These findings revealed that FGFR4 has a unique functional role in promoting gastric carcinogenesis by mediating accumulation and activation of the NRF2 antioxidant response. The use of FGFR4 inhibitors is a viable treatment option that warrants further research in patients with gastric cancer. Citation Format: Nadeem S. Bhat, Mohammed Soutto, Xing Zhang, Zheng Chen, Ahmed Gomaa, Marwah Al-Mathkour, Selma Maacha, Heng Lu, Dunfa Peng, Zekuan Xu, Wael El-Rifai. Helicobacter pylori-induced FGFR4 mediates nuclear accumulation of NRF2 in gastric tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2592.

Protective effect of antioxidant peptides from bass (Lateolabrax japonicus) on oxidative stress injury in Caco‐2 cells

AbstractExcessive reactive oxygen species (ROS) can induce oxidative damage to cell membranes and biomolecules, leading to various chronic diseases. Therefore, the exploration of effective natural antioxidants that can replace traditional synthetic antioxidants to reduce excess free radicals without side effects is important. Herein, the antioxidant peptide (Lateolabrax japonicus protein hydrolysates; LPH) was obtained from bass (L. japonicus) by an optimal enzymatic hydrolysis process. The protective effect of LPH on the Caco‐2 cell oxidative stress model and the potential antioxidant signaling pathways were explored. The results showed that LPH was rich in hydrophobic amino acids, and the IC50 for 2,2‐diphenyl‐1‐picrylhydrazyl radical scavenging activity was 0.9667 mg/mL. Furthermore, the LPH treatment reduced cellular ROS levels and lipid peroxidation while increasing antioxidant enzyme activity in a dose‐dependent manner (p < .05). LPH also upregulated the expression of mRNAs related to antioxidant enzymes in the Nrf2 signaling pathway (p < .05), demonstrating the efficacy of LPH on the Nrf2–ARE signaling pathway. These results suggested that LPH could be used as a potential food‐derived antioxidant in the food and healthcare industries.

Quercetin and resveratrol inhibit ferroptosis independently of Nrf2-ARE activation in mouse hippocampal HT22cells.

Oxidative stress is the central pathomechanism in multiple cell death pathways, including ferroptosis, a form of iron-dependent programmed cell death. Various phytochemicals, which include the inducers of the nuclear factor erythroid-2-related factor 2-antioxidant response element (Nrf2-ARE) transcription pathway, prevent ferroptosis. We recently reported that several compounds, such as the potent Nrf2-ARE inducer curcumin, protect mouse hippocampus-derived HT22cells against ferroptosis independently of Nrf2-ARE activity. The present study characterized the anti-ferroptotic mechanisms of two additional Nrf2-ARE inducers, quercetin and resveratrol. Both compounds prevented erastin- and RSL3-induced ferroptosis of wild-type HT22cells, and also blocked the exacerbated erastin- and RSL3-induced ferroptosis of Nrf2-knockdown HT22cells. In both HT22cells, quercetin and resveratrol blocked erastin- and RSL3-induced elevation in reactive oxygen species. These results suggest that the Nrf2-ARE pathway does protect against ferroptosis, but quercetin and resveratrol act by reducing oxidative stress independently of Nrf2-ARE induction. Quercetin and resveratrol also reduced Fe2+ concentrations in HT22cells and in cell-free reactions. Thus, quercetin and resveratrol likely protect against erastin- and RSL3-induced ferroptosis by inhibiting the iron-catalyzed generation of hydroxyl radicals. Unlike quercetin, resveratrol cannot form a chelate structure with Fe2+ but the density functional theory computation demonstrates that resveratrol can form stable monodentate complexes with the alkene moiety and the electron-rich A ring.

Hesperetin, a Promising Dietary Supplement for Preventing the Development of Calcific Aortic Valve Disease.

No effective therapeutic agents for calcific aortic valve disease (CAVD) are available currently. Dietary supplementation has been proposed as a novel treatment modality for various diseases. As a flavanone, hesperetin is widely abundant in citrus fruits and has been proven to exert protective effects in multiple diseases. However, the role of hesperetin in CAVD remains unclear. Human aortic valve interstitial cells (VICs) were isolated from aortic valve leaflets. A mouse model of aortic valve stenosis was constructed by direct wire injury (DWI). Immunoblotting, immunofluorescence staining, and flow cytometry were used to investigate the roles of sirtuin 7 (Sirt7) and nuclear factor erythroid 2-related factor 2 (Nrf2) in hesperetin-mediated protective effects in VICs. Hesperetin supplementation protected the mice from wire-injury-induced aortic valve stenosis; in vitro, hesperetin inhibited the lipopolysaccharide (LPS)-induced activation of NF-κB inflammatory cytokine secretion and osteogenic factors expression, reduced ROS production and apoptosis, and abrogated LPS-mediated injury to the mitochondrial membrane potential and the decline in the antioxidant levels in VICs. These benefits of hesperetin may have been obtained by activating Nrf2-ARE signaling, which corrected the dysfunctional mitochondria. Furthermore, we found that hesperetin could directly bind to Sirt7 and that the silencing of Sirt7 decreased the effects of hesperetin in VICs and potently abolished the ability of hesperetin to increase Nrf2 transcriptional activation. Our work demonstrates that hesperetin plays protective roles in the aortic valve through the Sirt7-Nrf2-ARE axis; thus, hesperetin might be a potential dietary supplement that could prevent the development of CAVD.

Dynamics of Metabolic Pathways and Stress Response Patterns during Human Neural Stem Cell Proliferation and Differentiation.

Understanding early nervous system stress response mechanisms is crucial for studying developmental neurotoxicity and devising neuroprotective treatments. We used hiPSC-derived long-term self-renewing neuroepithelial stem (lt-NES) cells differentiated for up to 12 weeks as an in vitro model of human neural development. Following a transcriptome analysis to identify pathway alterations, we induced acute oxidative stress (OS) using tert-butyl hydroperoxide (TBHP) and assessed cell viability at different stages of neural differentiation. We studied NRF2 activation, autophagy, and proteasomal function to explore the contribution and interplay of these pathways in the acute stress response. With increasing differentiation, lt-NES cells showed changes in the expression of metabolic pathway-associated genes with engagement of the pentose phosphate pathway after 6 weeks, this was accompanied by a decreased susceptibility to TBHP-induced stress. Microarray analysis revealed upregulation of target genes of the antioxidant response KEAP1–NRF2–ARE pathway after 6 weeks of differentiation. Pharmacological inhibition of NRF2 confirmed its vital role in the increased resistance to stress. While autophagy was upregulated alongside differentiation, it was not further increased upon oxidative stress and had no effect on stress-induced cell loss and the activation of NRF2 downstream genes. In contrast, proteasome inhibition led to the aggravation of the stress response resulting in decreased cell viability, derangement of NRF2 and KEAP1 protein levels, and lacking NRF2-pathway activation. Our data provide detailed insight into the dynamic regulation and interaction of pathways involved in modulating stress responses across defined time points of neural differentiation.

Protective Effect of GSK-3β/Nrf2 Mediated by Dimethyl Fumarate in Middle Cerebral Artery Embolization Reperfusion Rat Model.

This study investigated the protective effect of dimethyl fumarate (DMF) in rats by mediating glycogen synthase kinase 3β (GSK-3β)/Nrf2 using the middle cerebral artery embolization reperfusion (MCAO/R) rat model. After an acute ischemic stroke (AIS), oxidative stress occurs. Dimethyl fumarate (DMF), a nuclear factor-E2-related factor 2 (Nrf2) activator, approved by the US Food and Drug Administration (FDA), was observed to regulate the Nrf2 pathway by acting as an anti-oxidative stress agent; however, whether this agent is involved in inhibiting GSK-3β remains to be established. DMF model was used to explore the effects of GSK-3β on Nrf2 expression level, Nrf2- ARE binding activity and Nrf2/ARE downstream expression level of anti-oxidant stress protein in Cerebral ischemia-reperfusion injury (CIRI). 60 rats were randomly divided into Sham group, MCAO/R group, solvent control group (DMSO group) and DMF treatment group, with 15 rats in each group. The MCAO/R, DMSO and DMF groups were considered in the MCAO/R model using the modified thread embolization method. In contrast, the Sham group was only anaesthetized and disinfected, and tissue muscle was dissected without inserting suture emboli. DMF group was gavaged with 45mg/kg per day of DMF, DMSO control group was gavaged with DMSO of equal volume, while MCAO/R group was only modeled without any intragastric treatment. The rats were treated seven days after the operation, and a neurological function Longa score was estimated. The rats were sacrificed seven days later, and the infarct volume was assessed by TTC staining. Hematoxylin- eosin (HE) staining was used to observe the pathological changes in rat brain tissue. Nissl staining was used to observe the expression of neurons in the infarcted cortex. Western blotting (WB) was used to observe the protein expression levels of GSK-3β, Nrf2, downstream heme oxygenase 1 (HO1) and NADPH quinone oxidoreductase 1 (NQO1) in four groups. The expression levels of GSK-3β and Nrf2 in the four groups were observed by immunohistochemistry and immunofluorescence. (1) The Longa score of the MCAO/R, DMSO and DMF groups was found to be higher compared to the Sham group, indicating successful operation. The Longa score of the DMF group was lower than that of the other three groups 4-7 days after surgery (P<0.05). (2) HE and Nissl staining showed that the DMF group had lower neuron necrosis and higher gliosis compared to the control groups. (3) TTC staining results showed that the infarct volume of the DMF group was significantly smaller than the MCAO/R and DMSO groups. (4) Protein results showed that the GSK-3β expression in the DMF group was lower than that in all groups, while the expression of Nrf2, HO1 and NQO1 was higher compared to other groups. DMF can reduce neurological deficits and infarct size in the MCAO/R model. The protective effect may be related to decreased GSK-3β expression and increased Nrf2 expression, which may play a role in anti-oxidative stress.

A Characterization of the Effects of Minocycline Treatment During Adolescence on Structural, Metabolic, and Oxidative Stress Parameters in a Maternal Immune Stimulation Model of Neurodevelopmental Brain Disorders.

BackgroundMinocycline (MIN) is a tetracycline with antioxidant, anti-inflammatory, and neuroprotective properties. Given the likely involvement of inflammation and oxidative stress (IOS) in schizophrenia, MIN has been proposed as a potential adjuvant treatment in this pathology. We tested an early therapeutic window, during adolescence, as prevention of the schizophrenia-related deficits in the maternal immune stimulation (MIS) animal model.MethodsOn gestational day 15, Poly I:C or vehicle was injected in pregnant Wistar rats. A total 93 male offspring received MIN (30 mg/kg) or saline from postnatal day (PND) 35–49. At PND70, rats were submitted to the prepulse inhibition test. FDG-PET and T2-weighted MRI brain studies were performed at adulthood. IOS markers were evaluated in frozen brain tissue.ResultsMIN treatment did not prevent prepulse inhibition test behavioral deficits in MIS offspring. However, MIN prevented morphometric abnormalities in the third ventricle but not in the hippocampus. Additionally, MIN reduced brain metabolism in cerebellum and increased it in nucleus accumbens. Finally, MIN reduced the expression of iNOS (prefrontal cortex, caudate-putamen) and increased the levels of KEAP1 (prefrontal cortex), HO1 and NQO1 (amygdala, hippocampus), and HO1 (caudate-putamen).ConclusionsMIN treatment during adolescence partially counteracts volumetric abnormalities and IOS deficits in the MIS model, likely via iNOS and Nrf2–ARE pathways, also increasing the expression of cytoprotective enzymes. However, MIN treatment during this peripubertal stage does not prevent sensorimotor gating deficits. Therefore, even though it does not prevent all the MIS-derived abnormalities evaluated, our results suggest the potential utility of early treatment with MIN in other schizophrenia domains.

Omega-3 fatty acids during adolescence prevent schizophrenia-related behavioural deficits: Neurophysiological evidences from the prenatal viral infection with PolyI:C

The likely involvement of inflammation and oxidative stress (IOS) in mental disease has led to advocate anti-oxidant and anti-inflammatory drugs as therapeutic strategies in the treatment of schizophrenia. Since omega-3 fatty acids (ω-3) show anti-inflammatory/neuroprotective properties, we aim to evaluate whether ω-3 treatment during adolescence in the maternal immune stimulation (MIS) animal model of schizophrenia could prevent the brain and behavioural deficits described in adulthood. At gestational day 15, PolyI:C (4 mg/kg) or saline (VH) were injected to pregnant Wistar rats. Male offspring received ω-3 (800 mg/kg) or saline (Sal) daily from postnatal day (PND) 35–49, defining 4 groups: MIS-ω-3; MIS-Sal; VH-ω-3 and VH-Sal. At PND70, rats were submitted to prepulse inhibition test (PPI). FDG-PET and T2-weighted MRI brain studies were performed in adulthood and analyzed by means of SPM12. IOS markers were measured in selected brain areas. MIS-offspring showed a PPI deficit compared with VH-offspring and ω-3 treatment prevented this deficit. Also, ω-3 reduced the brain metabolism in the deep mesencephalic area and prevented the volumetric abnormalities in the hippocampus but not in the ventricles in MIS-offspring. Besides, ω-3 reduced the expression of iNOS and Keap1 and increased the activity/concentration of HO1, NQO1 and GPX. Our study demonstrates that administration of ω-3 during adolescence prevents PPI behavioural deficits and hippocampal volumetric abnormalities, and partially counteracts IOS deficits via iNOS and Nrf2–ARE pathways in the MIS model. This study highlights the need for novel strategies based on anti-inflammatory/anti-oxidant compounds to alter the disease course in high-risk populations at early stages.

Nitrosative Redox Homeostasis and Antioxidant Response Defense in Disused Vastus lateralis Muscle in Long-Term Bedrest (Toulouse Cocktail Study).

Increased oxidative stress by reactive oxygen species (ROS) and reactive nitrogen species (RNS) is a major determinant of disuse-induced muscle atrophy. Muscle biopsies (thigh vastus lateralis, VL) obtained from healthy male subjects enrolled in the Toulouse Cocktail bedrest (BR) study were used to assess efficacy of an antioxidant cocktail (polyphenols, omega-3, vitamin E, and selenium) to counteract the increased redox homeostasis and enhance the antioxidant defense response by using label-free LC–MS/MS and NITRO-DIGE (nitrosated proteins), qPCR, and laser confocal microscopy. Label-free LC–MS/MS indicated that treatment prevented the redox homeostasis dysregulation and promoted structural remodeling (TPM3, MYH7, MYBPC, MYH1, MYL1, HRC, and LUM), increment of RyR1, myogenesis (CSRP3), and skeletal muscle development (MUSTN1, LMNA, AHNAK). These changes were absent in the Placebo group. Glycolysis, tricarboxylic acid cycle (TCA), oxidative phosphorylation, fatty acid beta-oxidation, and mitochondrial transmembrane transport were normalized in treated subjects. Proteins involved in protein folding were also normalized, whereas protein entailed in ion homeostasis decreased. NITRO-DIGE analysis showed significant protein nitrosylation changes for CAT, CA3, SDHA, and VDAC2 in Treatment vs. Placebo. Similarly, the nuclear factor erythroid 2-related factor 2 (Nrf-2) antioxidant response element (Nrf-2 ARE) signaling pathway showed an enhanced response in the Treatment group. Increased nitrosative redox homeostasis and decreased antioxidant defense response were found in post-BR control (Placebo, n = 10) vs. the antioxidant cocktail treated group (Treatment, n = 10). Taken together, increased nitrosative redox homeostasis and muscle deterioration during BR-driven physical inactivity were prevented, whereas decreased antioxidant nitrosative stress defense response was attenuated by Treatment suggesting positive effects of the nutritional intervention protocol in bedrest.

Protective effects of ginsenoside-Rg2 and -Rh1 on liver function through inhibiting TAK1 and STAT3-mediated inflammatory activity and Nrf2/ARE-mediated antioxidant signaling pathway.

Systemic or hepatic inflammation is caused by intraperitoneal application of lipopolysaccharide (LPS). In this study, we investigated anti-inflammatory and antioxidant properties of combination of ginsenoside-Rg2 (G-Rg2) and -Rh1 (G-Rh1) on liver function under LPS challenging. We first confirmed that G-Rg2 and -Rh1 at 100μg/ml did not show cytotoxicity in HepG2 cells. G-Rg2 and -Rh1 treatment significantly inhibited activation of STAT3 and TAK1, and inflammatory factors including iNOS, TNF-α, and IL-1β in peritoneal macrophages. In HepG2 cells, G-Rg2 and -Rh1 treatment inhibited activation of STAT3 and TAK1/c-Jun N-terminal kinase, and down-regulated nuclear translocation of NF-κB transcription factor. In addition, LPS-induced mitochondrial dysfunction was restored by treatment with G-Rg2 and -Rh1. Interestingly, pretreatment with G-Rg2 and -Rh1 effectively inhibited mitochondrial damage-mediated ROS production induced by LPS stimulation, and alterations of Nrf2 nuclear translocation and ARE promotor activity were involved in G-Rg2 and -Rh1 effects on balancing ROS levels. In liver tissues of LPS-treated mice, G-Rg2 and -Rh1 treatment protected liver damages and increased Nrf2 expression while reducing CD45 expression. Taken together, G-Rg2 and -Rh1 exerts a protective effect on liver function by increasing antioxidant through Nrf2 and anti-inflammatory activities through STAT3/TAK1 and NF-κB signaling pathways in liver cells and macrophages.

Discovery of disubstituted xylylene derivatives as small molecule direct inhibitors of Keap1-Nrf2 protein-protein interaction

The Keap1–Nrf2–ARE system represents a crucial antioxidant defense mechanism that protects cells against reactive oxygen species. Targeting Keap1–Nrf2 protein–protein interaction (PPI) has become a promising drug target for several oxidative stress-related and inflammatory diseases including pulmonary fibrosis, chronic obstructive pulmonary disorder (COPD) and cancer chemoprevention. For the development of a potential therapeutic agent, drug-like properties and potency are important considerations. In this work, we focused on the modification of 4 as a lead through a molecular dissection strategy in an effort to improve its metabolic stability, leading to the discovery of a series of new disubstituted xylylene derivatives. The preliminary SAR of 9a indicated that compound 21a containing S-methylated acetate moieties exhibited comparable potency to the lead compound 4 in a fluorescent polarization assay but with improved metabolic stability in the presence of human liver microsomes.

Sevoflurane suppresses oxidation-induced stress and inflammatory responses, via promotion of Nrf2-induced antioxidant signaling

This study aimed to investigate the role of sevoflurane on anti-oxidant effects in sepsis-induced multiple organ failure. Western blot and quantitative RT–PCR were used to measure the expression of Nrf2 and its downstream genes. The DCFH-DA fluorescence probe assay was performed to assess the intracellular levels in HUVECs and LMVECs. Electrophoretic mobility shift assay was used to assess the DNA binding activities of Nrf2. Better survival, reduced expression of inflammatory cytokines, and improved pathological infiltration in the lungs and kidneys were observed in septic rats treated with sevoflurane. SOD activity increased and Nrf2–ARE signaling pathway was activated following sevoflurane treatment. The protective effect was lost in sepsis-induced acute lung injury (ALI) following inhibition of Nrf2 by intratracheal delivery of siRNA-Nrf2. In vitro experiments, we found the sevoflurane could suppress ROS production after stimulation with LPS and the application of sevoflurane augments the DNA-binding activity of Nrf2 in HUVECs and LMVECs. However, knockdown of Nrf2 by siRNA-Nrf2, the Protein kinase C (PKC) inhibitor, and the Nrf2 inhibitor ML385 could partially recover ROS production, inhibit the activity of SOD, and repress the expression of anti-oxidant genes following sevoflurane treatment. Sevoflurane could have protective effects in ALI and septic multiple organ disorders (MODs).

Protective effect of Shaoyao Decoction against colorectal cancer via the Keap1–Nrf2–ARE signaling pathway

Ethnopharmacological relevanceUlcerative colitis is one of the three high risk factors for colorectal cancer. Studies have found that about 20% of cancers are caused by repeated chronic inflammatory stimuli over a long period of time. Ulcer-related colorectal cancer is one of the main causes of death in patients with ulcerative colitis. At present, surgery is the first choice for the treatment of colorectal cancer, combined with radiotherapy and chemotherapy, which have serious side effects. However, reportedly, a compound prescription of Chinese traditional medicine Shaoyao Decoction (SYD) commonly used to treat damp-heat dysentery has anti-colorectal cancer effect. Thus this study described the effect of SYD to AOM/DSS-induced colon cancer model. Aim of the studyIn this study, modern biomedical approaches were employed for investigating the protective/preventive effects of SYD in mice with azoxymethane (AOM)/DSS-induced CRC. Materials and methodsThe mice pretreated with AOM/DSS were randomly allocated to SYDL, SYDM, SYDH group and SASP (sulfasalazine) group. Mice without AOM/DSS treatment were randomly divided into PBS control group and SYD control group. ResultsIt was found that SYD inhibited the production of inflammatory cytokines, TNF-α, IL-1β, superoxide dismutase (SOD), and malonaldehyde (MDA), and increased the antioxidant indices, as measured by the mRNA expression of GR, TR, HO-1, γ-GCSc, γ-GCSm, NQO-1, UGT1A1, and UGT1A10 in AOM-treated mice. Particularly, the expressions rates of NF-κB and Ki-67 in the SYD-treated experimental groups were significantly lower than those in the model group, indicating that the proliferative ability of the CRC tissues was weaker in the SYD-treated experimental groups. Moreover, the positive levels of Nrf2 in the SYD-treated experimental groups were slightly higher than those in the model group, suggesting that SYD exhibited antioxidant activity. ConclusionsTo sum up, our results suggest that SYD inhibits the development of acute/chronic colitis and prevents colitis-associated CRC by suppressing inflammation and preventing oxidative stress-induced cellular damage.

β-carotene provides neuro protection after experimental traumatic brain injury via the Nrf2-ARE pathway.

We investigate whether β-carotene, a known natural antioxidant, can reduce oxidative stress induced by traumatic brain injury. In addition, we investigated the underlying mechanism of traumatic brain injury focusing on the NF-E2-related factor (Nrf2) pathway. A controlled cortical impact model was used to mimic traumatic brain injury. Using this model, we evaluated brain edema, lesion volume, neurologic deficits, reactive oxygen species, and the expression of Nrf2-related protein markers. The results of our study demonstrated that cognitive performance and neural functions were improved with β-carotene administration. In addition, β-carotene reduced brain edema and reactive oxygen species levels after traumatic brain injury. Nrf2 nuclear accumulation was increased and was accompanied by decreased Keap1 expression. The expression of quinone oxidoreductase 1, a target gene of the Nrf2 signaling pathway was increased. However, lesion volume was not significantly reduced after β-carotene treatment. Taken together, our data demonstrated that β-carotene administration was neuroprotective and alleviated oxidative stress by modulating the Nrf2/Keap1- mediated antioxidant pathway in the traumatic brain injury model.

Catalpol Exerts an Anti-Epilepticus Effect, Possibly by Regulating the Nrf2-Keap1-ARE Signaling Pathway.

BackgroundStatus epilepticus (SE) is a refractory neurological disease with high mortality and morbidity rates. SE can be induced by numerous factors, including oxidative stress. Catalpol has several biological activities, including regulating the oxidative stress response. However, the role of catapol in SE has not been fully elucidated.Material/MethodsThirty Wistar rats were randomly and equally divided into 3 groups: a control group, an SE group established by LiCl-pilocarpine intraperitoneal injection, and an SE+catalpol group established administering catalpol to SE rats. Epileptic seizure level and after-discharge duration (ADD) were analyzed. Cognitive function was assessed by Morris water maze. Myeloperoxidase (MPO) and superoxide dismutase (SOD) activities were tested. Keap1 and ARE mRNA expressions were detected by real-time PCR. Nrf2 protein expression was determined by Western blot.ResultsCatalpol significantly decreased epileptic seizure level, extended ADD, and improved cognitive function compared with the SE group (P<0.05). MPO was increased, SOD was reduced, Keap1 mRNA was upregulated, and Nrf2 protein and ARE mRNA were reduced in the SE group compared with the control group (P<0.05). Catalpol markedly decreased MPO, enhanced SOD activity, decreased Keap1 mRNA level, and elevated Nrf2 protein and ARE mRNA expressions compared with the SE group (P<0.05).ConclusionsCatalpol plays an anti-epileptic role and improves cognitive function by regulating the Nrf2-Keap1-ARE signaling pathway to inhibit oxidative stress response.

Nrf2 inhibition reverses resistance to GPX4 inhibitor-induced ferroptosis in head and neck cancer

Glutathione peroxidase 4 (GPX4) is a regulator of ferroptosis (iron-dependent, non-apoptotic cell death); its inhibition can render therapy-resistant cancer cells susceptible to ferroptosis. However, some cancer cells develop mechanisms protective against ferroptosis; understanding these mechanisms could help overcome chemoresistance. In this study, we investigated the molecular mechanisms underlying resistance to ferroptosis induced by GPX4 inhibition in head and neck cancer (HNC). The effects of two GPX4 inhibitors, (1S, 3R)-RSL3 and ML-162, and of trigonelline were tested in HNC cell lines, including cisplatin-resistant (HN3R) and acquired RSL3-resistant (HN3-rslR) cells. The effects of the inhibitors and trigonelline, as well as of inhibition of the p62, Keap1, or Nrf2 genes, were assessed by cell viability, cell death, lipid ROS production, and protein expression, and in mouse tumor xenograft models. Treatment with RSL3 or ML-162 induced the ferroptosis of HNC cells to varying degrees. RSL3 or ML-162 treatment increased the expression of p62 and Nrf2 in chemoresistant HN3R and HN3-rslR cells, inactivated Keap1, and increased expression of the phospho-PERK–ATF4–SESN2 pathway. Transcriptional activation of Nrf2 was associated with resistance to ferroptosis. Overexpression of Nrf2 by inhibiting Keap1 or Nrf2 gene transfection rendered chemosensitive HN3 cells resistant to RSL3. However, Nrf2 inhibition or p62 silencing sensitized HN3R cells to RSL3. Trigonelline sensitized chemoresistant HNC cells to RSL3 treatment in a mouse model transplanted with HN3R. Thus, activation of the Nrf2–ARE pathway contributed to the resistance of HNC cells to GPX4 inhibition, and inhibition of this pathway reversed the resistance to ferroptosis in HNC.

Tannins Enriched Fraction of Emblica officinalis Fruits Alleviates High-Salt and Cholesterol Diet-Induced Cognitive Impairment in Rats via Nrf2-ARE Pathway.

Modern diets containing high quantities of salt and cholesterol have exhibited to cause a considerable effect on our health. Such diets, when consumed in the long term, have also shown to be a precursor to several disorders such as the metabolic disorder and consequently, various other diseases, including cognitive deficits. In the present study, we used a high salt and cholesterol diet (HSCD) to induce cognitive impairment in rats and also investigated the pharmacological action of tannins enriched fractions of Emblica officinalis (EOT) – a fruit that has been traditionally used for the treatment of numerous disorders for centuries. Significant alterations in MDA, GSH, TBARS, GPx, mitochondrial ATP, and mitochondrial membrane potential levels were observed in rats fed HSCD, which indicated presence of oxidative stress. Moreover, classic signs of cognitive impairment and deficits in spatial learning and memory were observed in the neurobehavioral tests. E. officinalis tannins exhibited good affinity to Nrf2 receptors in in silico studies, significantly reversed the changes in the aforementioned biomarkers of oxidative stress which were altered in the model group, as well as improved the performance of rats in Morris water maze task. Our results also reflected that EOT supplementation significantly increased the expression of Nrf2 in the CA1 region of hippocampus and cortex. Additionally, TUNEL assay indicated that EOT supplementation led to reversal of DNA fragmentation and apoptosis caused by HSCD. Immunohistochemical analysis and western blot further revealed a surge in the nuclear location of Nrf2. Through our study, we have demonstrated that cognitive impairment can be caused in rats via HSCD as a result of the oxidative stress induced by the same. Additionally, we have investigated a novel mechanism of action for EOT (which strongly suggests to be via the Nrf2–ARE pathway) and demonstrated that it has the potential to be used in the treatment of cognitive impairment.

Rosuvastatin alleviates high-salt and cholesterol diet-induced cognitive impairment in rats via Nrf2–ARE pathway

ABSTRACTObjective: The objectives of our study were to investigate the possible effect of rosuvastatin in ameliorating high salt and cholesterol diet (HSCD)-induced cognitive impairment and to also investigate its possible action via the Nrf2-ARE pathway.Methods: In silico studies were performed to check the theoretical binding of rosuvastatin to the Nrf2 target. HSCD was used to induce cognitive impairment in rats and neurobehavioral studies were performed to evaluate the efficacy of rosuvastatin in enhancing cognition. Biochemical analyses were used to estimate changes in oxidative markers. Western blot and immunohistochemical analyses were done to check Nrf2 translocation. TUNEL and caspase 3 tests were performed to evaluate reversal of apoptosis by rosuvastatin.Results: Rosuvastatin showed good theoretical affinity to Nrf2, significantly reversed changes in oxidative biomarkers which were induced by HSCD, and also improved the performance of rats in the neurobehavioral test. A rise in nuclear translocation of Nrf2 was revealed through immunohistochemical analysis and western blot. TUNEL staining and caspase 3 activity showed attenuation of apoptosis.Discussion: We have investigated a novel mechanism of action for rosuvastatin (via the Nrf2–ARE pathway) and demonstrated that it has the potential to be used in the treatment of cognitive impairment.

Protective effect of acupuncture preconditioning on oxidative stress injury induced by myocardial ischemia-reperfusion injury in rats

To observe the protective effect of acupuncture preconditioning at "Jiaji" (EX-B 2) on acute myocardial ischemia-reperfusion injury (MIRI) in the rats and explore the mechanism. Fifty Wistar rats were randomly divided into a control group, a model group, a Jiaji group, a Neiguan group and a Quchi group, 10 rats in each one. In the Jiaji group, the Neiguan group and the Quchi group, electroacupuncture was given for preconditioning at "Jiaji" T4~T5 (EX-B 2), "Neiguan" (PC 6) and "Quchi" (LI 11) for 7 days before modeling. In the control group and the model group, the regular feeding was given, without any acupuncture. At the end of acupuncture, except the control group, ligating the left anterior descending coronary artery (LAD) was adopted to duplicate MIRI models in the rest groups. Electrocardio-gram (ECG) was monitored and ST-segment shift was analyzed. HE staining method was adopted to observe the morphology of cardiac tissue in the rats of the groups. The transmission electron microscope was used to observe myocardial cell ultrastructure. WST-1 method was used to determine the activity of serum superoxide dismutase (SOD), TBA method was used to determine the content of serum malondialehyde (MDA) and the real-time fluorescent quantitative PCR method to determine the expressions of Nrf 2 in ischemic myocardial tissue and downstream HO-1 gene. Compared with the control group, after LAD ligation, ST-segment was elevated and depressed in ECG apparently after reperfusion in the rest groups (all P<0.05). The ST-segment elevation in the Jiaji group and the Neiguan group was less than that in the model group (both P<0.05). Compared with the model group, SOD activity was increased apparently in the Jiaji group and the Neiguan group (both P<0.05), and MDA content was reduced apparently (both P<0.05). The effects in the Jiaji group were better than those in the Neiguan group (both P<0.05). Pathologically, "Jiajia" (EX-B 2) and "Neiguan" (PC 6) all improved the morphology of cardiac tissue and cell ultrastructure. The effects in the Jiaji group were much more significant and the improvements in the Quchi group were not apparent. Compared with the control group, the expressions of Nrf 2 and HO-1 gene in myocardial tissue were down-regulated in the model group (both P<0.05). Those were up-regulated apparently in the Jiaji group and the Neiguan group as compared with the model group (P<0.05, P<0.01). The up-regulation times of the expressions of Nrf 2 and HO-1 gene in the Jiajia group were the highest in comparison. Acupuncture preconditioning at "Jiaji" (EX-B 2) has the protective effect on cardiac ischemia and reperfusion damage, which is probably relevant with the up-regulation of Nrf 2-ARE pathway expression, the activation of endogenous anti-oxidative pathway, the improvement of oxygen free radical scavenging capacity and the alleviation of lipid peroxide damage.

Hesperetin protects against H2O2-triggered oxidative damage via upregulation of the Keap1-Nrf2/HO-1 signal pathway in ARPE-19 cells.

Age-related macular degeneration (AMD) is an irreversible vision loss disease that primarily results from oxidative stress that causes oxidative damage to the retinal pigment epithelial (RPE) cells. Hesperetin (Hesp) is a common flavanone glycoside compound that has been demonstrated to exhibit a variety of biological and pharmacological properties that include anti-inflammatory and antioxidant properties. The aim of this study is to explore the ability of Hesp to attenuate oxidative damage in hydrogen peroxide (H2O2)-stimulated ARPE-19 cells. The results indicated that Hesp treatment not only increased cell survival but also decreased reactive oxygen species (ROS) generation, whereas these roles were effectively enhanced the superoxide dismutase (SOD) and glutathione (GSH) levels, and reduced malondialdehyde (MDA) formation. Importantly, the level of heme oxygenase-1 (HO-1) expression was increased by Hesp exposure, which resulted in a decrease after the transfection of cells with Nrf2-siRNA. Additionally, further results revealed that Hesp treatment significantly elevated Keap-1 protein expression, Nrf2 nuclear translocation and ARE activities. These observations indicated that Hesp treatment effectively protected against H2O2-induced oxidative damage in ARPE-19 cells by inhibiting cell apoptosis, ROS overproduction and MDA formation as well as enhancing the SOD and GSH levels. The underlying mechanisms may be related to the activation of the Keap1-Nrf2/HO-1 signal pathway, which may provide biological evidence to further encourage the investigation of the protective effect of Hesp in AMD disease.