Nuclear Nrf2 Protein Levels Research Articles

Cinnamaldehyde alleviates hepatic steatosis correlating with its electrophilic capability

Cinnamaldehyde (CA) is the key component of the essential oil derived from cinnamon bark and recommended as a health factor in foods for the prevention and intervention of metabolic disorders. This research aims to reveal the association between the difference in the electrophilic abilities of functional groups in CA and 3-phenylpropionaldehyde (PA), and their regulation of lipid peroxidation as well as energy metabolism in free fatty acids (FFAs) overloaded HepG2 cells. CA treatment reduced lipid accumulation and synthesis-related factors, increased levels of factors associated with fatty acid β-oxidation, decreased reactive oxygen species (ROS) content, and restored mitochondrial function and was superior to PA. Moreover, CA treatment increased nuclear Nrf2 protein levels and regulated the nuclear/cytoplasmic Nrf2 ratio, which suggested that CA effectively upregulated the Nrf2 signaling pathway. Furthermore, molecular docking showed that CA possessed an electrophilic index of 2.45 covalently bound to Nrf2-associated Keap1 protein with an affinity of −3.6 kcal/mol, which was much stronger than PA, and HPLC-MS/MS analysis further confirmed that CA covalently modified Cys151 on Keap1 protein. In conclusion, CA has the capacity to attenuate hepatic steatosis and cellular oxidative response via Nrf2 signaling pathway, which is closely related to its strong electrophilic ability.

Abstract LB169: Using a global reporter on transcript abundance to identify a selective inhibitor of Nrf2 signaling in KEAP1-mutant non small cell lung cancer

Abstract Loss-of-function mutations in Kelch-like ECH-associated protein 1 (KEAP1) are common in non-small cell lung cancer (NSLC) and result in particularly poor prognosis. Such mutations activate the transcription factor Nrf2, leading to aberrant expression of chemoprotective genes and rendering cells resistant to chemotherapy. We used our chemo-transcriptomic platform, termed GRETA, for Global Reporter on Transcript Abundance, to identify compounds that inhibit Nrf2 transcription factor activity in KEAP1-mutant NSCLC cell lines. After defining the transcriptomic signature imparted by constitutive activation of Nrf2 in a KEAP1 KO NCI-H1975 cell line, we generated over 10,000 transcriptomes to identify compounds that dampen the Nrf2-overactive transcriptional signature. One such compound, ARP-4316, displays selectivity for KEAP1-mutant cell lines, reduces nuclear Nrf2 protein levels, and reduces Nrf2 transcriptional activity in an ARE reporter assay. Furthermore, ARP-4316 synergizes with 5-fluorouracil in two KEAP1-mutant cell models, A549 and NCI-H2122, suggesting that ARP-4316 may sensitize KEAP1 mutant cell lines to chemotherapy by inhibiting Nrf2 transcriptional activity. Efforts to identify the cellular target and mechanism of action of ARP-4316 are underway. Citation Format: Timothy J. Read, Leah Damon, Reo Yoo, Carter Grochala, Cole Liechty, Christy Rhine, Jenna Rimel, David Simpson, Riley Lundblad, Casey Pham, Richard Steel, Joey Azofeifa. Using a global reporter on transcript abundance to identify a selective inhibitor of Nrf2 signaling in KEAP1-mutant non small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB169.

Co-exposure to Environmentally Relevant Levels of Molybdenum and Cadmium Induces Oxidative Stress and Ferroptosis in the Ovary of Ducks.

Excessive doses of molybdenum (Mo) and cadmium (Cd) have toxic effects on animals. Nevertheless, the reproductive toxicity elicited by Mo and Cd co-exposure remains obscure. To evaluate the co-induce toxic impacts of Mo and Cd on ovaries, 8-day-old 40 healthy ducks were stochastically distributed to four groups and were raised a basal diet supplemented with Cd (4 mg/kg Cd) and/or Mo (100 mg/kg Mo). In the 16th week, ovary tissues were gathered. The data revealed that Mo and/or Cd decreased GSH content, CAT, T-SOD, and GSH-Px activities and increased MDA and H2O2 levels. Moreover, there was a significant decrease in nuclear Nrf2 protein level and its related downstream factors, while cytoplasmic Nrf2 protein level showed a substantial increase. Additionally, a marked elevation was observed in ferrous ion content and TFRC, GCLC, SLC7A11, ACSL4, and PTGS2 expression levels, while FTH1, FTL1, FPN1, and GPX4 expression levels were conversely reduced. These indicators exhibited more marked changes in the joint exposure group. In brief, our results announced that Mo and/or Cd resulted in oxidative stress and ferroptosis in duck ovaries. Synchronously, the Cd and Mo mixture intensified the impacts.

Effect of miR-27b-3p and Nrf2 in human retinal pigment epithelial cell induced by high-glucose.

To determine whether the microRNA-27b-3p (miR-27b-3p)/NF-E2-related factor 2 (Nrf2) pathway plays a role in human retinal pigment epithelial (hRPE) cell response to high glucose, how miR-27b-3p and Nrf2 expression are regulated, and whether this pathway could be specifically targeted. hRPE cells were cultured in normal glucose or high glucose for 1, 3, or 6d before measuring cellular proliferation rates using cell counting kit-8 and reactive oxygen species (ROS) levels using a dihydroethidium kit. miR-27b-3p, Nrf2, NAD(P)H quinone oxidoreductase 1 (NQO1) and heme oxygenase-1 (HO-1) mRNA and protein levels were analyzed using reverse transcription quantitative polymerase chain reaction (RT-qPCR) and immunocytofluorescence (ICF), respectively. Western blot analyses were performed to determine nuclear and total Nrf2 protein levels. Nrf2, NQO1, and HO-1 expression levels by RT-qPCR, ICF, or Western blot were further tested after miR-27b-3p overexpression or inhibitor lentiviral transfection. Finally, the expression level of those target genes was analyzed after treating hRPE cells with pyridoxamine. Persistent exposure to high glucose gradually suppressed hRPE Nrf2, NQO1, and HO-1 mRNA and protein levels and increased miR-27b-3p mRNA levels. High glucose also promoted ROS release and inhibited cellular proliferation. Nrf2, NQO1, and HO-1 mRNA levels decreased after miR-27b-3p overexpression and, conversely, both mRNA and protein levels increased after expressing a miR-27b-3p inhibitor. After treating hRPE cells exposed to high glucose with pyridoxamine, ROS levels tended to decreased, proliferation rate increased, Nrf2, NQO1, and HO-1 mRNA and protein levels were upregulated, and miR-27b-3p mRNA levels were suppressed. Nrf2 is a downstream target of miR-27b-3p. Furthermore, the miR-27b-3p inhibitor pyridoxamine can alleviate high glucose injury by regulating the miR-27b-3p/Nrf2 axis.

Quercetin Suppresses Ferroptosis in Chondrocytes via Activating the Nrf2/ GPX4 Signaling Pathway

Background: Osteoarthritis is a progressive chronic disease that lacks effective treatment strategies. Ferroptosis features may be involved in the development and progression of osteoarthritis. Quercetin, a widely studied flavonoid compound, is considered a potential candidate for osteoarthritis therapies, although its anti-ferroptosis effect remains uncertain. This research aimed to investigate the regulatory impact of quercetin on ferroptosis and Nrf2 signaling in RSL3-induced C28/I2 cells. Methods: Cell viability was measured using the CCK-8 assay. Cellular lipid reactive oxygen species (ROS) were detected using fluorescence microscopy and flow cytometry combined with C11-BODIPY 581/591 staining. The level of malondialdehyde (MDA) was measured using an MDA assay kit. The level of GPX4 protein was determined by immunofluorescence staining and Western blotting. The protein levels of nuclear-Nrf2 and HO-1 were confirmed using Western blotting. Results: Quercetin improved cell viability, reduced the accumulation of lipid ROS, decreased MDA levels, elevated the protein levels of GPX4 and HO-1, and enhanced nuclear-Nrf2 protein levels in the ferroptosis cell model induced by RSL3. Additionally, the Nrf2 agonist TBHQ reversed ferroptosis in chondrocytes by activating the Nrf2/HO-1 pathway, while the Nrf2 inhibitor ML385 failed to protect against RSL3-induced ferroptosis in chondrocytes. Conclusion: Quercetin suppresses RSL3-induced ferroptosis in chondrocytes by activating the Nrf2/GPX4 signaling pathway, providing a promising therapeutic option for osteoarthritis.

The Keap1/Nrf2-ARE signaling pathway is involved in atrazine induced dopaminergic neurons degeneration via microglia activation

ObjectiveTo investigate the mechanisms of ATR-induced dopaminergic toxicity by microglia activation and the response of the Keap1/ Nrf2- ARE signaling pathway. MethodsWistar rats were treated with 50, 100 and 200 mg/kg ATR and BV-2 microglia cells were treated with 50, 100 μM ATR or 100 ng/mL LPS, respectively. Rats behavioral responses and histopathological changes were monitored. Immunohistochemical and immunofluorescence analysis detected Iba-1 and TH+ cells in rats. Keap1/Nrf2-ARE signaling-related proteins and inflammatory factors from BV-2 cells and rats were detected using ELISA, Western blot and Real-time PCR. ResultsAfter ATR treatment, the grip strength of Wistar rats was significantly decreased, and anxiety were clearly observed. TH+ neurons were reduced, however, the number of microglia cells and Iba-1 levels were increased clearly in SN. The release of ROS, TNF-α and IL-Iβ were increased, and levels of SOD and GSH-Px were significantly decreased. Keap1 mRNA expression and protein levels were decreased, while nuclear Nrf2 mRNA expression and protein levels were both increased in vivo and in vitro. ConclusionATR could significantly activate microglia and exacerbate neurotoxicity and neuroinflammation, leading to accelerate dopaminergic neuron cell death by inhibiting Keap1/Nrf2-ARE signaling pathway.

The role of Nrf2 pathway in alleviating fluorine-induced apoptosis by different selenium sources in the chicken duodenum and jejunum

In order to evaluate the alleviative effects and molecular mechanisms of sodium selenite (SS) and selenomethionine (SM) on excessive apoptosis induced by high fluorine (HF) in the duodenum and jejunum of broilers, 720 1 day old Lingnan Yellow broilers were randomly divided into 4 groups (each group assigned 180 chickens with 6 replicates) and offered either a control diet or test diets (800 mg/kg F, HF group; 800 mg/kg F + 0.15 mg selenium (Se)/kg as SS (SS group) or SM (SM group)) for 50 days. High F intake significantly increased (P < 0.05) apoptosis rates of duodenum and jejunum by inducing oxidative stress and leading to mitochondrial damage. Selenomethionine supplementation effectively alleviated mitochondrial damage and severe apoptosis of duodenum and jejunum caused by HF through decreasing oxidative stress parameters. Selenomethionine added group significantly increased (P < 0.05) nuclear factor erythroid 2-related factor 2 (Nrf2) mRNA and nuclear Nrf2 protein levels as well as Nrf2 downstream antioxidant enzymes expressions in the duodenum and jejunum when compared with the HF group. Selenomethionine was superior to SS in activating the Nrf2 pathway and reducing the apoptosis rate of duodenum. It was concluded that dietary SM supplementation could ameliorate F-induced excessive apoptosis by inducing the Nrf2 pathway. Our findings will bring a promising tactics for the utilization of SM as an efficient antioxidant additive for reducing the intestinal damage caused by fluorosis in poultry.

Targeting Nrf2-antioxidant signalling reverses acquired cabazitaxel resistance in prostate cancer cells.

Prostate cancer is known to have a relatively good prognosis, but long-term hormone therapy can lead to castration-resistant prostate cancer (CRPC). Cabazitaxel, a second-generation taxane, has been used for the CRPC treatment, but its tolerance is an urgent problem to be solved. In this study, to elucidate the acquisition mechanism of the cabazitaxel-resistance, we established cabazitaxel-resistant prostate cancer 22Rv1 (Cab-R) cells, which exhibited ∼sevenfold higher LD50 against cabazitaxel than the parental 22Rv1 cells. Cab-R cells showed marked increases in nuclear accumulation of NF-E2 related factor 2 (Nrf2) and expression of Nrf2-inducible antioxidant enzymes compared to 22Rv1 cells, suggesting that Nrf2 signalling is homeostatically activated in Cab-R cells. The cabazitaxel sensitivity of Cab-R cells was enhanced by silencing of Nrf2, and that of 22Rv1 cells was reduced by activation of Nrf2. Halofuginone (HF) has been recently identified as a potent Nrf2 synthetic inhibitor, and its treatment of Cab-R cells not only suppressed the Nrf2 signalling by decreasing both nuclear and cytosolic Nrf2 protein levels, but also significantly augmented the cabazitaxel sensitivity. Thus, inhibition of Nrf2 signalling may be effective in overcoming the cabazitaxel resistance in prostate cancer cells.

Development and optimization of halogenated vinyl sulfones as Nrf2 activators for the treatment of Parkinson’s disease

The Kelch-like ECH-associated protein 1 (Keap1)-Nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway plays a pivotal role in the cellular defense system against oxidative stress by inducing antioxidant and anti-inflammatory effects. We previously developed Nrf2 activators that potentially protect the death of dopaminergic (DAergic) neuronal cells against oxidative stress in Parkinson's disease (PD). In this study, we designed and synthesized a class of halogenated vinyl sulfones by inserting halogens and pyridine to maximize Nrf2 activation efficacy. Among the synthesized compounds, (E)-3-chloro-2-(2-((2-chlorophenyl)sulfonyl)vinyl)pyridine (9d) significantly exhibited potent Nrf2 activating efficacy (9d: EC50=26nM) at least 10-fold compared with the previous developed compounds (1 and 2). Furthermore, treating with 9d remarkably increased Nrf2 nuclear translocation and Nrf2 protein levels in microglial BV-2cells. 9d was shown to induce the expression of antioxidant response genes HO-1, GCLC, GCLM, and SOD-1 at both the mRNA and protein levels and suppress proinflammatory cytokines and enzymes. Also, 9d remarkably protected DAergic neurons and restored the PD-associated motor dysfunction in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced mouse model.

Hexavalent chromium induced heart dysfunction via Sesn2-mediated impairment of mitochondrial function and energy supply

Hexavalent chromium (Cr(VI)), the most toxic valence state of chromium, is widely present in industrial effluents and wastes. Although previous study has reported that Cr(VI) can cause cytomembrane structure impairment by aggravating lipid peroxidation in the heart, the detailed mechanism of Cr(VI)-induced heart dysfunction is still unclear. Sesn2, a novel antioxidant and stress-inducible molecule, is evidenced to protect against various cardiometabolic diseases such as atherosclerosis and cardiomyopathy. To define the potential mechanism of heart dysfunction induced by chronic Cr(VI) exposure, Wistar rats were intraperitoneal injected with potassium dichromate (K2Cr2O7) for 35 d in the present study. The data showed that chronic K2Cr2O7 exposure caused dose-dependently hematological variations, oxidative stress, dysfunction, and disorganized structure of heart, cardiomyocyte apoptosis, ATP depletion, and mitochondria impairment in rats. In addition, the expressions of Drp1 and Bax were increased by K2Cr2O7. However, the suppression of Mfn2, PGC-1α, Sesn2, nuclear Nrf2, HO-1, and NQO1 protein levels was observed in K2Cr2O7-treated rat hearts. In conclusion, these results demonstrate that chronic K2Cr2O7 exposure dose-dependently causes heart dysfunction, and the molecular mechanism of this event is associated with the loss of Sesn2 mediated mitochondrial function and energy supply impairment.

Dietary histidine deficiency induced flesh quality loss associated with changes in muscle nutritive composition, antioxidant capacity, Nrf2 and TOR signaling molecules in on-growing grass carp (Ctenopharyngodon idella)

This study was a part of a larger research that involved in determination of the effects of dietary His on fish growth, health and flesh quality, and used one growth trial with our previous study which showed the growth retardation induced by dietary His deficiency. The present study was conducted to assess the effect of dietary His on flesh quality of fish and the possible role of antioxidant capacity in flesh quality in relation to dietary His. A total of 540 on-growing grass carp (Ctenopharyngodon idella) (279.8 ± 1.07 g) were randomly fed graded levels of His at 2.0 (un-supplemented control), 3.7, 5.9, 7.9, 9.8 and 12.2 g/kg diet for 8 weeks. Results showed that compared with the dietary His level of 7.9 g/kg diet, dietary His deficiency (2.0 g/kg diet) significantly decreased nutrients contents, including protein, some essential amino acids (including lysine, methionine, threonine, leucine, histidine and arginine), lipid and unsaturated fatty acid contents in fillet of on-growing grass carp (P < .05). Meanwhile, the His-deficient diet decreased hydroxyproline contents, shear force value and pH (P < .05), whereas increased cooking loss, activities of cathepsin B and L in fish muscle (P < .05). Furthermore, malondialdehyde (MDA) and protein carbonyl contents in muscle were significantly increased by His-deficient diet (P < .05), whereas, total superoxide dismutase (T-SOD), CuZnSOD, catalase (CAT), glutathione peroxidase (GPx) activities and glutathione content in muscle of grass carp were significantly decreased by dietary His deficiency (P < .05). Additionally, the His-deficient diet down-regulated CuZnSOD, CAT, GPx, and signaling molecules Nrf2, TOR and S6K1 genes expression (P < .05), but up-regulated Keap1 gene expression, total and nuclear Nrf2 protein levels, and phosphorylated TOR levels in muscle of grass carp (P < .05). In summary, the present results indicated that dietary His deficiency induced flesh quality loss partly related to the decreased nutritive compositions and antioxidant capacities through Nrf2 signaling in on-growing grass carp.

Regulation of P-glycoprotein by Bajijiasu in vitro and in vivo by activating the Nrf2-mediated signalling pathway

Context: Bajijiasu (BJJS), a main bioactive compound from Morinda officinalis F.C. How. (Rubiaceae), is widely administered concomitantly with other drugs for treating male impotence, female infertility, fatigue, chronic rheumatism, depression, etc.Objective: This study investigates the regulation of P-glycoprotein (P-gp) by BJJS in vitro and in vivo.Material and methods: HepG2 cells were incubated with BJJS (10, 20 or 40 μM) for 48 h. C57 mice were orally treated with BJJS (25, 50 or 100 mg/kg) for 2 weeks. The protein and mRNA levels of P-gp were measured by using Western blot and real-time PCR, respectively. siNrf2 RNA was used to explore the mediation effects of Nrf2 on the P-gp expression. The efflux activity of P-gp was tested via a flow cytometry.Results: Incubation of HepG2 cells with BJJS at 10, 20, and 40 μM up-regulated the P-gp protein expression by 12.3%, 82.9%, and 134.3%, respectively. Treatment of C57 mice with BJJS at 25, 50 and 100 mg/kg increased the P-gp protein expression by 49.3%, 75.8% and 106.0%, respectively. Incubation of the cells with BJJS at 10, 20 and 40 μM up-regulated the total Nrf2 protein levels by 34.3%, 93.1% and 118.6%, respectively, and also increased the nuclear Nrf2 protein levels by 14.8%, 44.4% and 59.25%, respectively. The total Nrf2 protein levels were increased by 46.3%, 66.5%, and 87.4%, respectively, in the mice exposed to BJJS at 25, 50, and 100 mg/kg. Inhibition of Nrf2 by siRNA diminished the P-gp induction by 25.0%, 33.4%, and 38.7%, respectively, in the cells. In addition, BJJS enhanced the efflux activity of P-gp by 9.6%, 37.1%, and 48.1%, respectively, in the cells.Conclusions: BJJS activates Nrf2 to induce P-gp expression, and enhanced the efflux activity of P-gp. The possibility of potential herb–drug interactions when BJJS is co-administered with other P-gp substrate drugs should be carefully monitored.

Regulation of Nrf2 by X Box-Binding Protein 1 in Retinal Pigment Epithelium.

Normal function of the retinal pigment epithelium (RPE) is essential for maintaining the structural integrity of retinal photoreceptors and the visual process. Sustained oxidative damage of the RPE due to aging and other risk factors contributes to the development of age-related macular degeneration (AMD). The transcription factor NF-E2-related factor 2 (Nrf2) is a central regulator of cellular antioxidant and detoxification responses. Enhancing Nrf2 function protects RPE cells from oxidation-related apoptosis and cell death. Previously, we demonstrated that Nrf2 activation can be induced by endoplasmic reticulum (ER) stress; however, the mechanisms are not fully understood. In the present study, we examined the role of X box-binding protein 1 (XBP1), an ER stress-inducible transcription factor, in regulation of Nrf2 in the RPE. We found that RPE-specific XBP1 conditional knockout (cKO) mice exhibit a significant reduction in Nrf2 mRNA and protein levels, along with decreased expression of major Nrf2 target genes, in the RPE/choroid complex. Using primary RPE cells isolated from XBP1 cKO mice and human ARPE-19 cell line, we confirmed that loss of XBP1 gene or pharmacological inhibition of XBP1 splicing drastically reduces Nrf2 levels in the RPE. Conversely, overexpression of spliced XBP1 results in a modest but significant increase in cytosolic and nuclear Nrf2 protein levels without affecting the transcription of Nrf2 gene. Moreover, induction of ER stress by tunicamycin and thapsigargin markedly increases Nrf2 expression, which is abolished in cells pretreated with XBP1 splicing inhibitors 4μ8C and quinotrierixin. Mechanistic studies indicate that quinotrierixin reduces Nrf2 expression likely through inhibition of protein translation. Finally, we demonstrate that overexpression of Nrf2 protected RPE cells against oxidative injury but appeared to be insufficient to rescue from XBP1 deficiency-induced cell death. Taken together, our results indicate that XBP1 modulates Nrf2 activity in RPE cells and that XBP1 deficiency contributes to oxidative injury of the RPE.

341 - MnTE-2-PyP protects the prostate from radiation-induced fibrosis (RIF) by activating Nrf2/Nqo1/SIRT signaling axis

Prostate cancer treated with radiation therapy is accompanied by damage to healthy tissues, resulting in radiation induced-fibrosis (RIF) and a reduction in quality of life. Radiation exposure increases reactive oxygen species (ROS) that leads to molecular remodeling of fibroblast cells to induce RIF. Our work demonstrates that MnTE-2-PyP, a scavenger of superoxide molecules, prevents acute and chronic changes to prostate fibroblast cells that are responsible for the development of RIF. Fibroblast cells exposed to radiation (≥ 3Gy) show a decrease in the redox-sensitive nuclear Nrf2 protein levels without any change in Nrf2 mRNA expression. This correlates with a decrease in mRNA and protein levels of a Nrf2 downstream target, Nqo1, which is a cytoprotective protein. Therefore, we hypothesize that MnTE-2-PyP can prevent ROS mediated molecular remodeling of fibroblast cells in RIF by increasing Nrf2 activity and Nqo1 expression. We have confirmed that MnTE-2-PyP increases nuclear Nrf2 levels and causes an upregulation of Nqo1 expression (2.23-fold ± 0.41). Upregulation of Nqo1 expression correlates with a decrease in Keap1 levels that control Nrf2 degradation and translocation to the nucleus (p=0.0161). Inhibition of Nqo1 activity using Dicoumarol (50μM) or siRNA (10nM) significantly decrease cell viability (40-50%). Interestingly, Nqo1 upregulation and its cytoprotective function correlate with an increase in NAD+ levels (2.24-fold ± 0.60). NAD+ is an essential cofactor for sirtuin (SIRT) proteins that can deacetylate other proteins involved epigenetic remodeling of fibroblast cells. We have observed a significant increase in sirtuin activity in irradiated cells treated with MnTE-2-PyP (1.88-fold) as compared to no treatment. Therefore, we believe that activating the Nrf2 signaling pathway that causes an increase in Nqo1 expression, followed by an increase in NAD+ levels and sirtuin activity by MnTE-2-PyP treatment, is responsible for prevention of ROS mediated molecular alterations in RIF.

Isoniazid induces apoptosis: Role of oxidative stress and inhibition of nuclear translocation of nuclear factor (erythroid-derived 2)-like 2 (Nrf2)

AimsLong-term treatment of Isoniazid (INH) in tuberculosis (TB) patients can lead to anti-tuberculosis drug-induced hepatotoxicity. To understand the mechanism of hepatotoxicity, an attempt has been made to elucidate the role of Nrf2, a transcription factor induced by oxidative stress, in INH induced apoptosis liver cancer cell lines. Materials and methodsCytotoxicity was evaluated by MTT assay. Apoptosis and reactive oxygen species (ROS) generation was performed by flow cytometry. mRNA and protein expression of various genes involves in INH induced toxicity was evaluated via Real-time PCR and western blot analysis respectively. Differential protein expression was performed by two-dimensional gel electrophoresis followed by identification using MALDI TOF/TOF. Key findingsINH induced ROS and apoptosis in HepG2 as well as THLE-2 cells. Nuclear damage was also observed by INH treatment in HepG2 cells. Expression of apoptotic (Cytochrome C and Caspase 9) and antioxidative (Keap1 and Nrf2) genes were observed to increase. INH induced PKCδ phosphorylation and released Nrf2 from its inhibitor Keap1 in the cytoplasm of HepG2 cells. However, over-expression of Nrf2 did not affect nuclear Nrf2 protein level as well as its downstream target NQO1. Nrf2 importer, Karyopherin β1 level was observed to decrease in HepG2 as well as THLE-2 cells following INH treatment. SignificanceThese findings suggest that INH prevented Nrf2 translocation into the nucleus by inhibiting its importer Karyopherin β1. Therefore Nrf2 might not able to bind ARE sequences from inducing antioxidative response for protecting the cells undergoing apoptosis.

Niga-ichigoside F1 ameliorates high-fat diet-induced hepatic steatosis in male mice by Nrf2 activation.

Hepatic lipid accumulation and oxidative stress (OS) lead to non-alcoholic fatty liver disease (NAFLD). Thus, we hypothesized that antihyperlipidemic and antioxidant activities of niga-ichigoside F1 (NI) would ameliorate events leading to NAFLD. Lanbuzheng (Geum japonicum Thunb. var. chinense), a type of wild vegetable found in Southwest China, was used to extract NI. Male C57BL/6J mice were fed a standard diet (Con) or a high-fat diet (HFD) (denoted as diet) with or without 40 mg kg-1 NI (defined as treatment) for 12 weeks. Diet-treatment interactions were observed in the final body weight, fat pad mass, respiratory exchange ratio (RER) in the daytime, and energy expenditure during the whole day. Moreover, NI alleviated hepatic steatosis, possibly by significantly interacting with HFD to regulate lipid metabolism genes (including Srebp1c, Acc1, Fasn, Scd1, Cpt1a and Fabp5). We also found significant diet-treatment interactions on superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase (CAT) activities, and thiobarbituric acid reactive substance (TBARS) levels, as well as the nuclear and cellular Nrf2 protein levels. Significant free fatty acid (FFA)-treatment interactions on Nrf2 nuclear translocation, antioxidant enzymes activities, genes in lipogenesis (Srebp1c, Acc1, Fasn, and Scd1), and fatty acid oxidation (Pparα) and transport (Fabp5 and Cd36) were also detected in 1 mM FFA-treated HepG2 cells with or without 20 μM NI. These beneficial effects of NI on oxidative stress and lipid accumulation were abolished by Nrf2 siRNA. Our data revealed that dietary NI could prevent HFD-induced hepatic steatosis, possibly via interacting with HFD to activate Nrf2 nuclear translocation to maintain a redox status, thus regulating lipid metabolism genes expressions.

Dysfunction of Nrf2-ARE Signaling Pathway: Potential Pathogenesis in the Development of Neurocognitive Impairment in Patients with Moderate to Severe Obstructive Sleep Apnea-Hypopnea Syndrome.

The present study investigated the nuclear factor erythroid 2-related factor 2- (Nrf2-) antioxidant response element (ARE) signaling pathway in patients with moderate to severe obstructive sleep apnea-hypopnea syndrome (OSAHS). Their correlation with neurocognitive impairment metrics was investigated to explore potential pathogenesis in OSAHS. Forty-eight patients with OSAHS and 28 controls underwent testing with the Epworth Sleep Scale (ESS), MATRICS Consensus Cognitive Battery (MCCB), Stroop Color and Word Test, polysomnography (PSG), and measurements of the concentration of plasma superoxide dismutase (SOD) and thioredoxin (Trx). Further, 20 pairs of matched patients with OSAHS and controls were selected for measurement of the expression (protein and mRNA) of Nrf2 and of its downstream antioxidase, heme oxygenase-1 (HO-1), in peripheral mononuclear cells (PBMCs). Finally, correlations between neurocognitive impairment and the above metrics were analyzed. Expression of Nrf2 and HO-1 mRNA and protein in the PBMCs, as well as plasma SOD and Trx levels, were significantly reduced in patients with OSAHS. After adjusting for education, sex, age, and smoking index, the expression of Nrf2-ARE signaling pathway proteins (or mRNA) was closely correlated with sleep respiratory parameters. An inverse relationship was demonstrated between the expression of nuclear Nrf2 in PBMCs, concentration of plasma SOD and Trx, and apnea-hypopnea index (AHI) in patients with OSAHS. Trx, nuclear Nrf2 protein, and HO-1 protein were also negatively correlated with the percent of time that SaO2 was less than 90% (TSat90). Total Nrf2 protein level was positively correlated with AHI and TSat90 and negatively correlated with minimum SaO2 (LSaO2), while nuclear Nrf2 protein and HO-1 protein were positively correlated with LSaO2. Moreover, significant positive correlations were found between maze scores and expression of nuclear Nrf2 protein, HO-1 protein, and SOD and Trx levels. Furthermore, inverse relationships between total Nrf2 protein in PBMCs and HVLT-R and maze scores were found. Multiple linear regression showed plasma Trx concentration as a potential predictor of maze and BVMT-R scores. In conclusion, the expression of Nrf2-ARE molecules and related antioxidases is significantly decreased in patients with OSAHS and is correlated with neurocognitive dysfunction. The Nrf2-ARE signaling pathway may play a crucial role in neurocognitive impairment in patients with moderate to severe OSAHS. Further studies are needed to explore the exact mechanisms and potential treatment interventions.

P21Waf1/Cip1 depletion promotes dexamethasone-induced apoptosis in osteoblastic MC3T3-E1 cells by inhibiting the Nrf2/HO-1 pathway.

Dexamethasone (Dex), a glucocorticoid with strong anti-inflammatory and immunosuppressive activities, has been shown to exhibit marked cytotoxicity and apoptosis in osteoblasts, but the underlying mechanisms have not yet been comprehensively investigated. P21Waf1/Cip1 (p21) plays a critical role in the regulation of cell cycle progression and apoptosis. The present study aims to investigate the role of p21 in Dex-induced apoptosis in osteoblastic MC3T3-E1 cells, and to explore its mechanisms. Results demonstrated that Dex-induced apoptosis decreased the phosphorylation of Akt in a concentration-dependent manner. Moreover, LY294002, an inhibitor of the PI3K/Akt pathway enhanced the Dex-induced apoptosis of osteoblasts. On the contrary, insulin-like growth factor-1 (IGF-1), an activator of PI3K/Akt, attenuated the apoptosis of Dex in MC3T3-E1 cells. The protein level of p21 was downregulated by shortening its half-life, which was associated with inhibition of the PI3K/Akt pathway by Dex. Furthermore, depletion of p21 by siRNA enhanced Dex-induced caspase-3 activation and ROS generation, and promoted apoptosis of MC3T3-E1 cells. In addition, suppression of p21 led to a reduction of Dex-induced upregulation of nuclear Nrf2 and heme oxygenase-1 (HO-1) protein levels. These findings demonstrate that p21 depletion promotes Dex-induced apoptosis of MC3T3-E1 cells by inhibiting the antioxidant Nrf2/HO-1 pathway, which highlights the anti-apoptotic effect of p21 in MC3T3-E1 cells.

335 - Frataxin-Deficiency in the Heart Results in an Impaired Nrf2 Response: A Dual Mechanism Mediated via Up-Regulation of Keap1 and GSK3β Axis

Friedreich's ataxia (FA) is a neuro- and cardio-degenerative disease due to frataxin-deficiency and leads to marked mitochondrial iron loading. The ensuing oxidative stress is likely a key contributor to the pathology. Recent studies in neuronal FA models have reported defective expression of the transcription factor, nuclear factor E2-related factor 2 (Nrf2), which regulates the cellular antioxidant response. However, the mechanism has not been elucidated and no molecular examination of the anti-oxidant response has been conducted in the FA heart where fatal cardiomyopathy develops. Using the MCK conditional frataxin knockout (KO) mouse, which exhibits a cardiomyopathy that mimics the human disease, we examined the Nrf2 antioxidant response pathway in the frataxin-deficient heart. Our studies demonstrated protein and GSH oxidation in the KO relative to wild-type (WT) littermates. Despite this, we found decreased total and nuclear Nrf2 protein levels and increased Keap1-mediated Nrf2 degradation in the heart. Moreover, we also demonstrated the involvement of Gsk3β-dependent export and degradation of nuclear Nrf2 in the heart, as evident by: (i) increased Gsk3β activation; (ii) increased Fyn phosphorylation, mediating nuclear export; and (iii) increased expression of β-TrCP, a substrate recognition subunit of the E3 ubiqitin ligase complex that is involved in Nrf2 degradation. Furthermore, electrophoretic mobility shift assays demonstrated decreased Nrf2-DNA-binding activity and a general decrease in target gene expression in frataxin KO hearts. These results indicate the blunted anti-oxidant response in the frataxin-deficient heart is, at least in part, due to reduced Nrf2 activity caused by its increased degradation via the Keap1 and Gsk3β pathways.

Activation of the Keap1/Nrf2 stress response pathway in autophagic vacuolar myopathies.

Nrf2 (nuclear factor [erythroid-derived 2]-like 2; the transcriptional master regulator of the antioxidant stress response) is regulated through interaction with its cytoplasmic inhibitor Keap1 (Kelch-like ECH-associated protein 1), which under basal conditions targets Nrf2 for proteasomal degradation. Sequestosome 1 (SQSTM1)/p62–a multifunctional adapter protein that accumulates following autophagy inhibition and can serve as a diagnostic marker for human autophagic vacuolar myopathies (AVMs)–was recently shown to compete with Nrf2 for Keap1 binding, resulting in activation of the Nrf2 pathway. In this study, we used 55 human muscle biopsies divided into five groups [normal control, hydroxychloroquine- or colchicine-treated non-AVM control, hydroxychloroquine- or colchicine-induced toxic AVM, polymyositis, and inclusion body myositis (IBM)] to evaluate whether Keap1-SQSTM1 interaction led to increased Nrf2 signaling in human AVMs. In toxic AVMs and IBM, but not in control muscle groups or polymyositis, Keap1 antibody labeled sarcoplasmic protein aggregates that can be used as an alternate diagnostic marker for both AVM types; these Keap1-positive aggregates were co-labeled with the antibody against SQSTM1 but not with the antibody against autophagosome marker LC3 (microtubule-associated protein 1 light chain 3). In human AVM muscle, sequestration of Keap1 into the SQSTM1-positive protein aggregates was accompanied by an increase in mRNA and protein levels of Nrf2 target genes; similarly, treatment of differentiated C2C12 myotubes with autophagy inhibitor chloroquine led to an increase in the nuclear Nrf2 protein level and an increase in expression of the Nrf2-regulated genes. Taken together, our findings demonstrate that Nrf2 signaling is upregulated in autophagic muscle disorders and raise the possibility that autophagy disruption in skeletal muscle leads to dysregulation of cellular redox homeostasis.Electronic supplementary materialThe online version of this article (doi:10.1186/s40478-016-0384-6) contains supplementary material, which is available to authorized users.