Nrf2 Phosphorylation Research Articles

Structural Characterization and Antioxidant Activity of Polysaccharides from Athyrium multidentatum (Doll.) Ching in d-Galactose-Induced Aging Mice via PI3K/AKT Pathway.

The purpose of this study was to characterize the polysaccharides from Athyrium multidentatum (Doll.) Ching (AMC) rhizome and explore the protective mechanism against d-galactose-induced oxidative stress in aging mice. Methods: A series of experiments, including molecular weight, monosaccharide composition, Fourier transform infrared (FT-IR) spectroscopy, and 1H nuclear magnetic resonance (1H NMR) spectroscopy were carried out to characterize AMC polysaccharides. The mechanism was investigated exploring d-galactose-induced aging mouse model. Quantitative real-time reverse transcription polymerase chain reaction (RT-qPCR) and western blotting assays were performed to assess the gene and protein expression in liver. Key findings: Our results showed that AMC polysaccharides were mainly composed of mannose (Man), rhamnose (Rha), glucuronic acid (Glc A), glucose (Glc), galactose (Gal), arabinose (Ara), and fucose (Fuc) in a molar ratio of 0.077:0.088:0.09:1:0.375:0.354:0.04 with a molecular weight of 33203 Da (Mw). AMC polysaccharides strikingly reversed d-galactose-induced changes in mice, including upregulated phosphatidylinositol 3-kinase (PI3K), Akt, nuclear factor-erythroid 2-related factor 2 (Nrf2), forkhead box O3a (FOXO3a), and hemeoxygenase-1 (HO-1) mRNA expression, raised Bcl-2/Bax ratio, downregulated caspase-3 mRNA expression, enhanced Akt, phosphorylation of Akt (p-Akt), Nrf2 and HO-1 protein expression, decreased caspase-3, and Bax protein expression. Conclusion: AMC polysaccharides attenuated d-galactose-induced oxidative stress and cell apoptosis by activating the PI3K/AKT pathway, which might in part contributed to their anti-aging activity.

Clinically-approved CFTR modulators rescue Nrf2 dysfunction in cystic fibrosis airway epithelia.

Cystic Fibrosis (CF) is a multi-organ progressive genetic disease caused by loss of functional cystic fibrosis transmembrane conductance regulator (CFTR) channel. Previously, we identified a significant dysfunction in CF cells and model mice of the transcription factor nuclear-factor-E2-related factor-2 (Nrf2), a major regulator of redox balance and inflammatory signaling. Here we report that approved F508del CFTR correctors VX809/VX661 recover diminished Nrf2 function and colocalization with CFTR in CF human primary bronchial epithelia by proximity ligation assay, immunoprecipitation, and immunofluorescence, concordant with CFTR correction. F508del CFTR correctors induced Nrf2 nuclear translocation, Nrf2-dependent luciferase activity, and transcriptional activation of target genes. Rescue of Nrf2 function by VX809/VX661 was dependent on significant correction of F508del and was blocked by inhibition of corrected channel function, or high-level shRNA knockdown of CFTR or F508del-CFTR. Mechanistically, F508del-CFTR modulation restored Nrf2 phosphorylation and its interaction with the coactivator CBP. Our findings demonstrate that sufficient modulation of F508del CFTR function corrects Nrf2 dysfunction in CF.

The neuroprotective activities of natural products through the Nrf2 upregulation.

Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is a basic-region leucine zipper transcription factor that regulates the expression of many Phase II detoxifying/antioxidant enzymes. Many natural products act on the Nrf2 pathway and exhibit their preventive and/or therapeutic effects by activating Nrf2 and subsequently antioxidant enzymes. In this article, we reviewed the research literature that described Nrf2 activation as a mechanism for the neuroprotective effect of natural products. Our results showed that the inhibition of Kelch-like ECH-associated protein 1 and the activation of different intracellular kinases that lead to Nrf2 phosphorylation were the main mechanisms of Nrf2 upregulation suggested for these the natural products. Although some natural compounds such as terpenoids and alkaloids were reported to protect the nervous system by Nrf2 activating, flavonoids and phenolic compounds represent the majority of the reported neuroprotective natural substances that activate Nrf2. We also evaluated the physiochemical properties of some of the same substances to predict their oral bioavailability and central nervous system penetration. The results showed that sulforaphane and berberine have good oral bioavailability and would be predicted to cross through blood-brain barrier (BBB) easily, whereas the flavonoids in general would be predicted to be less effective in BBB transition because of their relatively high polar surface area.

Ultraviolet B irradiation induced Nrf2 degradation occurs via activation of TRPV1 channels in human dermal fibroblasts

Ultraviolet (UV) irradiation causes cellular oxidative stress. Under redox imbalance, Keap1-dependent Nrf2 degradation is minimal. In this study, we examined the role of Ca2+ in Nrf2 homeostasis after UVB irradiation using human dermal fibroblasts. UVB irradiation stimulates 12-lipoxygenase and the product 12-hydroxyeicosatetraenoic acid then activates TRPV1 increasing the cell's cytosolic Ca2+ concentration. UVB irradiation induced reactive oxygen species generation and apoptosis are inhibited in the absence of Ca2+ or in the presence of either a 12-lipoxygenase inhibitor or a TRPV1 inhibitor during and after UVB irradiation. Thus, the Ca2+ increase via TRPV1 is a critical factor in UVB irradiation induced oxidative stress. UVB irradiation induces a Ca2+ dependent Nrf2 degradation and thus activation of TRPV1 with 12-hydroxyeicosatetraenoic acid also decreasing Nrf2 levels. UVB irradiation induced Nrf2 degradation is inhibited by co-treatment of cells with W-7, cyclosporin A, SB-216763 or MG-132, which are inhibitors of calmodulin, calcineurin, GSK3β and the proteasome, respectively. Furthermore, UVB irradiation in parallel induces GSK3β dephosphorylation in a Ca2+ dependent manner. Co-immunoprecipitation showed that UVB irradiation induces an increase in Nrf2 phosphorylation, an increase in the binding of β-TrCP and Nrf2, and an increase in Nrf2 ubiquitination; these effects are all Ca2+ dependent. These findings suggest that UVB irradiation induced GSK3β activation in a Ca2+ dependent manner, which then stimulates the phosphorylation and ubiquitination of Nrf2 via β-TrCP. Indeed, silencing of β-TrCP was found to inhibit UVB irradiation-induced oxidative stress, Nrf2 degradation and apoptosis, while it had no effect on the Ca2+ increase. Taken together, our results suggest that a Ca2+ influx via TRPV1 is responsible for UVB irradiation-induced Nrf2 degradation and that modulation of the Ca2+-calmodulin-calcineurin-GSK3β-Nrf2-β-TrCP-Cullin-1 pathway may explain Ca2+ dependent Nrf2 degradation.

Cortisol Excess-Mediated Mitochondrial Damage Induced Hippocampal Neuronal Apoptosis in Mice Following Cold Exposure.

Cold stress can induce neuronal apoptosis in the hippocampus, but the internal mechanism involving neuronal loss induced by cold stress is not clear. In vivo, male and female C57BL/6 mice were exposed to 4 °C, 3 h per day for 1 week. In vitro, HT22 cells were treated with different concentrations of cortisol (CORT) for 3 h. In vivo, CORT levels in the hippocampus were measured using ELISA, western blotting, and immunohistochemistry to assess the neuronal population and oxidation of the hippocampus. In vitro, western blotting, immunofluorescence, flow cytometry, transmission electron microscopy, and other methods were used to characterize the mechanism of mitochondrial damage induced by CORT. The phenomena of excessive CORT-mediated oxidation stress and neuronal apoptosis were shown in mouse hippocampus tissue following cold exposure, involving mitochondrial oxidative stress and endogenous apoptotic pathway activation. These processes were mediated by acetylation of lysine 9 of histone 3, resulting in upregulation involving Adenosine 5‘-monophosphate (AMP)-activated protein kinase (APMK) phosphorylation and translocation of Nrf2 to the nucleus. In addition, oxidation in male mice was more severe. These findings provide a new understanding of the underlying mechanisms of the cold stress response and explain the apoptosis process induced by CORT, which may influence the selection of animal models in future stress-related studies.

2141-P: Nrf2 Mediates Glucose-Stimulated Beta-Cell Proliferation

Glucose catabolism drives adaptive beta cell mass expansion by promoting proliferation under conditions of increased insulin demand. Previously, we showed that induction of the antioxidant regulator, Nrf2, is required for glucose-stimulated β-cell proliferation, and that overexpression of Nrf2 stimulates human beta cell proliferation. Here we manipulate the Nrf2 pathway using pharmacological and genetic approaches to explore the link between glucose metabolism, Nrf2 activation and beta cell proliferation. Treatment of isolated mouse islets with either a Nrf2 inhibitor (brusatol) or Cre-recombinase deleted Nrf2, resulted in complete inhibition of glucose-stimulated proliferation (both n=3, p < 0.01). Conversely, stimulation of Nrf2 by CDDO-me or genetic gain of function (Cre-mediated depletion of Nrf2 inhibitor, Keap1) increased beta cell proliferation of beta cells from isolated mouse islets cultured in 5 mM glucose (4.9 ± 0.2 fold and 6.9 ± 1.4 fold, respectively; n=3, p < 0.05). Inhibition or depletion of Nrf2 was associated with a remarkable decrease in insulin content, suggesting that beta cell identity requires basal levels of Nrf2 expression. Importantly, isolated human beta cells displayed similar responses to Nrf2 loss and gain of function. Activation and nuclear translocation of Nrf2 requires dissociation from its inhibitor, Keap1, and phosphorylation of Ser-40 on Nrf2. Five minutes of 20 mM glucose treatment profoundly increased Ser-40 phosphorylation and translocation of Nrf2, while the same effect by CDDO-Me treatment took 50 minutes in INS1 cells. The glucose-mediated activation and translocation of Nrf2 was blocked by the antioxidant N-acetylcysteine (NAC), which also attenuated glucose-stimulated proliferation of isolated mouse beta cells. In addition, the activation and phosphorylation of Nrf2 was prevented by the pan-PKC inhibitor, calphostin C. We conclude that manipulation of the Nrf2 pathway is an exciting novel target for regulation of beta cell proliferation. Disclosure S. Baumel-Alterzon: None. L.S. Katz: None. G. Brill: None. A. Garcia-Ocaña: None. D. Scott: None. Funding American Diabetes Association (1-17-IBS-116 to D.S.); National Institutes of Health

A novel compound AB-38b improves diabetes-associated cognitive decline in mice via activation of Nrf2/ARE pathway

ObjectiveDiabetes-associated cognitive decline (DACD) is increasingly being concerned, and oxidative stress plays a vital role in the pathological process. AB-38b is a novel synthetic compound with two specific active groups of biphenyl dicarboxylate and α, β unsaturated ketone, showing good antioxidant activity. The aim of this study was to investigate the ameliorative effects of AB-38b on DACD in mice, and to explore the possible mechanisms from glyoxylase 1 (Glo-1) enhancement and NF-E2-related factor-2 (Nrf2) activation. MethodsExperimental type 2 mouse model of diabetes with C57BL/6 mice was made through high-fat diet combining with intraperitoneal streptozotocin. Diabetic mice were treated by gavage with AB-38b (0, 10, 20 and 40 mg/kg) or resveratrol (40 mg/kg), a typical inducer of Nrf2, for 8 weeks. Cognitive performances were evaluated by the novel object recognition task. Then brain tissues were collected to assess hippocampal damages, protein glycation, Glo-1 functions and protein expression, and the classic Nrf2/ARE pathway. ResultAB-38b markedly increased the preference index to novel object and the number of neurons in hippocampal CA1 area of diabetic mice. AB-38b significantly elevated the activity and protein of Glo-1, while reduced the levels of advanced glycation end products (AGEs) and protein expression of its receptor RAGE. Moreover, AB-38b raised Nrf2 expression and phosphorylation, as well as the protein expression and enzymatic activity of γ-glutamylcysteine synthetase, a well-known gene of Nrf2/ARE pathway, in hippocampus of the diabetic mice. ConclusionAB-38b improved the cognitive performances of diabetic mice, which was achieved via up-regulation of Glo-1 and activation of Nrf2/ARE pathway.

PMA-triggered PKCε activity enhances Nrf2-mediated antiviral response on fish rhabdovirus infection

Viral infection is often accompanied with alteration of intracellular redox state, especially an imbalance between reactive oxygen species (ROS) production and antioxidant cellular defenses. The previous studies showed that an antioxidant cellular defense system, the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2), played an important role against spring viraemia of carp virus (SVCV) infection in fish. To further reveal the mediated mechanism that Nrf2 active state was affected by protein kinase C (PKC), here we evaluated SVCV replication in host cells by treated with a strong activator of PKC phorbol-12-myristate-13-acetate (PMA) and an inhibitor staurosporine. Our results showed that PMA significantly repressed SVCV replication and viral-induced apoptosis in Epithelioma papulosum cyprini (EPC) cell, suggesting that PKC may exhibit an anti-SVCV effect. Likewise, PMA resulted in a higher phosphorylation levels of PKCε rather than PKCα/β to participate in the activation of Nrf2, mainly involved in the activation of Nrf2 phosphorylation of Ser40 to favor Nrf2 translocation to nucleus. Furthermore, the data revealed that PMA up-regulated an antiviral response heme oxygenase-1 (HO1) gene expression that was confirmed as the key player against SVCV infection by HO1 specific siRNA. Overall, this study provided a new therapeutic target for the treatment of SVCV infection, and modulating PKC activity could be used for the prevention and treatment of SVCV.

Intervention of curcumin and its analogue J7 on oxidative stress injury in testis of type 2 diabetic rats

To investigate the intervention of curcumin and its analogue J7 on oxidative stress injury in testis of type 2 diabetic rats. Sixty male SD rats, 10 rats were chosen as normal control group (NC), the other 50 rats were assigned to experiment group. Experiment diabetic rats were induced by high-fat food and intraperitoneal injection of steptozotocin (STZ). After the model was established successfully, diabetic rats were divided into four groups randomly: diabetes mellitus group (DM, n=12), curcumin treatment group (CUR, n=10), high dose treatment group of J7 (J+, n=10), low dose treatment group of J7 (J-, n=10). The CUR group were intragastrically administered with curcumin 20 mg/kg daily, in addition, the J+ group and the J- group were intragastrically administered with J7 20 mg/kg and 10 mg/kg daily respectively. After 8 weeks, the fast blood glucose was detected biochemically. The activity of superoxide dismutase (SOD) and the level of malondialdehyde (MDA) were detected by hydroxylamine method and thiobarbituric acid method respectively. The protein expressions of the nuclear factor-erythroid 2-related factor 2 (tNrf2), phosphorylation of Nrf2 (pNrf2), catalase (CAT), NAD(P)H quinine oxidoreductase 1 (NQO1) were measured by Western blot. The mRNA expressions of CAT, NQO1, hemeoxygenase-1 (HO1) were measured by quantitative real-time PCR (qRT-PCR). Morphological structure of testis was observed by hematoxylin-eosin (HE) staining. The expressions of Nrf2 and CAT were also detected by immunohistochemical method. The levels of fast blood glucose and MDA in DM group were increased significantly(P＜0.05), while the body weight, the activity of SOD, the protein expressions of pNrf2/tNrf2, CAT, NQO1 and the mRNA expressions of CAT, NQO1, HO1 were decreased (P＜0.05). Under light microscope, the DM group showed disrupted histological appearance. Immunohistochemistry showed that the protein expressions of Nrf2 around the nucleus and CAT were decreased. With the treatment of curcumin and J7, the MDA levels in the three treatment groups were decreased (P＜0.05). The activity of SOD, the protein expressions of pNrf2/tNrf2, CAT, NQO1 and the mRNA expressions of NQO1, HO1 were increased (P＜0.05). the levels of fast blood glucose were decreased in the J+ and J- group (P＜0.05), and the mRNA expression of CAT was increased in the J+ group (P＜0.05). The ratio of pNrf2/tNrf2 in the J+ group was significantly higher than that in CUR and J- group (P＜0.05). The protein level of CAT in the J+ group was also significantly higher than that in J- group (P＜0.05). There were no significant differences in other indexes among the three treatment groups. Under light microscope, the morphology was obviously improved in the three treatment groups. Immunohistochemistry showed that the protein expressions of Nrf2 around the nucleus and CAT were increased in the three treatment groups. It was suggested that high dose J7 had better antioxidant stress ability in testis of diabetic rats. Curcumin and J7 could inhibit the oxidative stress damage of testicular tissue in diabetic rats, which might be related with the activation of the Nrf2-ARE signaling pathway.

Cell-specific regulation of Nrf2 during ROS-Dependent cell death caused by 2,3,5-tris(glutathion-S-yl)hydroquinone (TGHQ)

2,3,5-tris(Glutathion-S-yl)hydroquinone (TGHQ), a potent nephrotoxic and nephroncarcinogenic metabolite of benzene and hydroquinone, retains the ability to redox cycle and create oxidative stress. We have previously detected that TGHQ induces ROS-dependent necrotic or apoptotic cell death in renal epithelial HK-2 and human leukemic HL-60 cells respectively. Herein, we sought to determine the nature of the Nrf2 regulation in HK-2 and HL-60 cells undergoing TGHQ-mediated ROS-dependent cell death, due to the key role of Nrf2 in oxidative stress. Intriguingly, Nrf2 was upregulated in HK-2, but not in HL-60 cells, despite the ROS-dependent nature of cell death in both cell types. The possibility that TGHQ targeted the GSK3β-dependent Nrf2 stabilization pathway in HL-60 cells was discounted, whereas TGHQ-induced decreases in Nrf2 phosphorylation at Ser40 site appears to partially underlie the inability of TGHQ to up-regulate Nrf2 expression in HL-60 cells. Moreover, whereas the TGHQ-induced post-translational stabilization of Nrf2 in HK-2 cells resulted in the expected upregulation of HO1 and NQO1 mRNA, TGHQ actually decreased Nrf2 mRNA in HL-60 cells, with a concomitant decrease in NQO1, but not HO1 mRNA. In summary, we define differences between the two cell types that might contribute to the engagement of the Nrf2 signaling pathways. By extension, these data provide evidence that Nrf2 is not necessarily activated in ROS-dependent cell death, and further delve into the knowledge that Nrf2 regulation sensing by cells might be achieved at solely transcriptional level, not related to its degradation.

C-Src regulates Nrf2 activity through PKCδ after oxidant stimulus

Nrf2 is the main transcription factor involved in expression of cell defense enzymes, which is altered in several oxidant-related disorders. Cytosolic Nrf2 activation is modulated through phosphorylation by PKCδ, an enzyme controlled by Src tyrosine kinases. Of relevance, Src family members are involved in numerous cellular processes and regulated by hydrogen peroxide (H2O2). In this study we analysed the activation of cell survival-related signaling proteins, c-Src and Nrf2, and the influence of c-Src kinase on Nrf2 regulation after exposure to H2O2. Acute exposure of HT22 mouse hippocampal neural cells to H2O2 increased c-Src and Nrf2 phosphorylation/activation at Tyr416 and Ser40, respectively. Nrf2 phosphorylation at Ser40, its nuclear accumulation and transcriptional activity involving heme oxygenase-1 (HO-1) expression were dependent on c-Src kinase activation. Moreover, modulation of Nrf2 activity by c-Src occurred through PKCδ phosphorylation at Tyr311. We demonstrate, for the first time, c-Src-mediated regulation of Nrf2 transcriptional activity, via PKCδ activation, following an acute H2O2 stimulus. This work supports that the c-Src/PKCδ/Nrf2 pathway may constitute a novel signaling pathway stimulated by H2O2 and a potential target for the treatment of diseases involving redox deregulation.

Catalpol and panax notoginseng saponins synergistically alleviate triptolide-induced hepatotoxicity through Nrf2/ARE pathway

Triptolide (TP), as the main component of Tripterygium wilfordii (TW), could induce significant hepatic damage when exerting the therapeutic effect. However, The previous studies have shown that catalpol (CAT) and panax notoginseng saponins (PNS) have synergistic protecting effect against hepatotoxicity induced by TP. This study aims to address the role of the nuclear factor erythroid-2-related factor-2 (Nrf2)/antioxidant response element (ARE) pathway in their protecting effect, and to explore their synergistic mechanisms. For this purpose, the human hepatocyte cell line L-02 was selected, and the synergistic antioxidative effect of CAT and PNS was confirmed. Then the effects of CAT and PNS on different aspects of the Nrf2/ARE pathway were analyzed. The results showed that CAT significantly reduced TP-induced inhibition of Nrf2 transcription and made it increased, PNS significantly increased Nrf2 phosphorylation for relieving TP-induced potential inhibition of Nrf2/ARE binding activity, while the combination of the two further enhanced their activation by synergistically inducing Nrf2 transcriptional expression and phosphorylated Nrf2 (p-Nrf2) expression, followed with NAD(P)H:quinine oxidoreductase 1 (NQO1) expression and glutathione (GSH) activity increasing, which further enhanced their antioxidative effects. Subsequently, the gene silencing techniques were used, and that the Nrf2/ARE pathway is necessary in this effect was confirmed. In conclusion, the synergistic protecting effect of CAT and PNS is dependent on Nrf2/ARE pathway, while the natural mutual promotion between the regulatory links of CAT and PNS may be the main source of the synergistic effect.

A physiological concentration of luteolin induces phase II drug-metabolizing enzymes through the ERK1/2 signaling pathway in HepG2 cells

The flavon luteolin has various health-promoting activities including cardiovascular protection, anti-inflammatory activity and anticancer activity. A serum concentration of about 100 nM luteolin is reached by dietary habit. However, little is known about the function of luteolin over its physiological concentration range. In this study, we investigated whether a physiological concentration of luteolin could activate nuclear factor-erythroid-2-related factor 2 (Nrf2)-mediated expression of phase II drug-metabolizing enzymes in human hepatoma HepG2 cells. Interestingly, less than 1 nM of luteolin could induce phase II drug-metabolizing enzymes, such as GSTs, HO-1, and NQO1. Both 1 and 100 nM luteolin increased expression and activity of ALDH2, which metabolized toxic acetaldehyde into nontoxic acetic acid. Luteolin increased nuclear accumulation of Nrf2 and enhanced the ARE-binding complex through increasing the stability of the Nrf2 protein. Luteolin increased phosphorylation of Nrf2 at Ser40, and MEK inhibitors (U0126 and PD98059) canceled luteolin-induced phosphorylation of Nrf2. Furthermore, luteolin increased modified Keap1. In conclusion, a physiological concentration of luteolin induces the expression of phase II drug-metabolizing enzymes by enhancement of Nrf2 nuclear accumulation through MEK1/2-ERK1/2-mediated phosphorylation of Nrf2, increasing Nrf2 stability and inducing a conformational change of Keap1.

Bamboo Stems (Phyllostachys nigra variety henosis) Containing Polyphenol Mixtures Activate Nrf2 and Attenuate Phenylhydrazine-Induced Oxidative Stress and Liver Injury.

This study was designed to investigate the hepatoprotective effect of bamboo stems using in vitro and in vivo experimental liver damage models. Ethyl acetate fraction of 80% ethanol extract of Phyllostachys nigra stem (PN3) containing polyphenols had a higher NQO1-ARE reporter gene activity as monitored by the activity of the NF-E2-related factor (Nrf2) antioxidant pathway in cells in comparison to extracts from other species and under other conditions. The Nrf2 was translocated from the cytosol to the nucleus in response to PN3, followed by induction of the Nrf2 target gene expression, including HO-1, GCL, and NQO-1 in HepG2 cells. Phosphorylation of Nrf2 in HepG2 cells was enhanced in PN3, which was mediated by PKCδ, ERK, and p38 MAPK. Consequently, PN3 inhibited arachidonic acid (AA) + iron-induced reactive oxygen species generation and glutathione depletion, and, thus, highlighted their role in cytotoxicity. Treatment with major polyphenols of PN3, including catechin, chlorogenic acid, caffeic acid, and p-coumaric acid, also improved AA + iron-mediated oxidative stress and, thus, improved cell viability. Treatment with phenylhydrazine in mice, i.e., the iron overload liver injury model, increased plasma alanine aminotransferase and aspartate aminotransferase levels and changed histological features in mice—a response that was almost completely blocked by PN3 administration. Moreover, PN3 extract mitigated phenylhydrazine-induced oxidative stress and inflammatory responses. Conclusively, PN3 can exert a hepatoprotective effect against iron overload-induced acute liver damage due to its antioxidant properties.

TNFα Induces Multidrug Resistance-Associated Protein 4 Expression through p38-E2F1-Nrf2 Signaling in Obstructive Cholestasis.

PurposeTo explore the molecular mechanism of the upregulation of multidrug resistance-associated protein 4 (MRP4) in cholestasis.Materials and MethodsThe mRNA and protein levels of MRP4 in liver samples from cholestatic patients were determined by quantitative real-time PCR and Western blot. In human hepatoma HepG2 cells, electrophoretic mobility shift assay (EMSA) was used to determine the affinity of nuclear factor-E2-related factor (Nrf2) binding to MRP4 promoter. Dual-luciferase reporter assay was used to detect the binding of tumor necrosis factor α (TNFα) to the promotor of E2F1. The bile duct ligation mouse models were established using male C57BL/6 mice.ResultsThe mRNA and protein levels of MRP4 were significantly increased in cholestatic patients. TNFα treatment induced the expression of MRP4 and Nrf2 and enhanced cell nuclear extract binding activity to MRP4 promoter, as demonstrated by EMSA. Nrf2 knockdown reduced MRP4 mRNA levels in both HepG2 and Hep-3B cells. In addition, TNFα increased Rb phosphorylation and expression of MRP4 and Nrf2 and activated E2F1 and phosphorylated p38 in HepG2 and Hep-3B cells. These effects were markedly inhibited by pretreatment with E2F1 siRNA. Dual-luciferase reporter assay validated that TNFα induces the transcription of E2F1. Furthermore, the expression of MRP4, Nrf2, E2F1, and p-p38 proteins was improved with treatment of TNFα in a mouse model of cholestasis. E2F1 siRNA lentivirus or SB 203580 (p38 inhibitor) inhibited these positive effects.ConclusionOur findings indicated that TNFα induces hepatic MRP4 expression through activation of the p38-E2F1-Nrf2 signaling pathway in human obstructive cholestasis.

Neuroprotection of quercetin on central neurons against chronic high glucose through enhancement of Nrf2/ARE/glyoxalase-1 pathway mediated by phosphorylation regulation

Although dietary flavonoid quercetin alleviates diabetes-associated cognitive decline in rodents, the mechanisms are not clearly clarified. This study was designed to investigate whether quercetin showed neuroprotection on central neurons against chronic high glucose through the enhancement of Nrf2/ARE/glyoxalase 1 (Glo-1) pathway. SH-SY5Y cells were divided into 8 groups: normal glucose, high glucose (HG), osmotic pressure control, solvent control, HG plus low, middle, high concentrations of quercetin, or Nrf2 activator (sulforaphane). After treatment for 72 h, the associated parameters were measured. We found quercetin and sulforaphane increased cell viability, and enhanced Glo-1 functions (Glo-1 activity, the reduced glutathione and advanced glycation end-products levels) as well as Glo-1 protein and mRNA levels in SH-SY5Y cells cultured with HG. Meanwhile, quercetin and sulforaphane activated Nrf2/ARE pathway, reflected by the raised Nrf2 and p-Nrf2 levels, and the elevated protein and mRNA levels of γ-glutamycysteine synthase (γ-GCS), a known target gene of Nrf2/ARE signaling. Moreover, Nrf2/ARE pathway was activated after pretreatment with a PKC activator, p38 MAPK inhibitor, or GSK-3β inhibitor under the condition of HG, and quercetin addition further strengthened this pathway; however, PKC inhibition or GSK-3β activation pretreatment reversed the effects of quercetin on the protein expression of γ-GCS in the HG condition. In summary, quercetin exerts the neuroprotection by enhancing Glo-1 functions in central neurons under chronic HG condition, which may be mediated by activation of Nrf2/ARE pathway; furthermore, the increased Nrf2 phosphorylation mediated by PKC activation and/or GSK-3β inhibition may involve in the activation of Nrf2/ARE pathway.

Ginsenoside Rb3 protects cardiomyocytes against hypoxia/reoxygenation injury via activating the antioxidation signaling pathway of PERK/Nrf2/HMOX1

ObjectivesThis study aimed to investigate the pharmacological function and underlying regulation mechanisms of Ginsenoside-Rb3 (G-Rb3) in cardioprotection. MethodsCultured H9C2 cells were pre-treated with gradient concentrations of G-Rb3, and subsequently challenged with hypoxia/reoxygenation (H/R) treatment. The generation of intracellular reactive oxygen species (ROS) and cellular antioxidatant capacity were quantified. Cell apoptosis was measured by flow cytometry. Myocardial ischemia reperfusion injury (MIRI) rat models constructed by coronary artery ligation surgery were orally administrated with G-Rb3 for 5 consecutive days, and then infarction area, apoptosis ratio and total antioxidant capacity (T-AOC) of myocardial tissues were measured. PERK phosphorylation inhibitor GSK2656157 and Nrf2 translocation inhibitor ML385 were co-treated with G-Rb3 to further verify the signaling pathway mediated by G-Rb3. ResultsH/R treatment induced prominent ROS deposition and elevated cell apoptosis ratio in H9C2 cells. G-Rb3 pretreatment suppressed intracellular ROS accumulation and enhanced T-AOC, partially rescuing cardiomyocytes from oxidative stress and apoptosis induced by H/R. In vivo, the cardiac infarction area of MIRI model rats was reduced by G-Rb3 treatment via improved total antioxidant levels. In the further functional and mechanistic studies, G-Rb3 was found to induce PERK phosphorylation and nuclear translocation of transcriptional factor Nrf2, promoting the expression of antioxidative genes such as HMOX1. Inhibitors GSK2656157 and ML385 reversed the effects of G-Rb3. ConclusionOur studies revealed a novel mechanism of G-Rb3 to attenuates oxidative stress via activating the antioxidation signaling pathway of PERK/Nrf2/HMOX1 in vivo and in vitro, which may help us to enrich the theoretical knewledge of Ginsenoside-Rb3 in cardiopretection

Mini-GAGR, an intranasally applied polysaccharide, activates the neuronal Nrf2-mediated antioxidant defense system

Oxidative stress triggers and exacerbates neurodegeneration in Alzheimer's disease (AD). Various antioxidants reduce oxidative stress, but these agents have little efficacy due to poor blood–brain barrier (BBB) permeability. Additionally, single-modal antioxidants are easily overwhelmed by global oxidative stress. Activating nuclear factor erythroid 2 (NF-E2)-related factor 2 (Nrf2) and its downstream antioxidant system are considered very effective for reducing global oxidative stress. Thus far, only a few BBB-permeable agents activate the Nrf2-dependent antioxidant system. Here, we discovered a BBB-bypassing Nrf2-activating polysaccharide that may attenuate AD pathogenesis. Mini-GAGR, a 0.7-kDa cleavage product of low-acyl gellan gum, increased the levels and activities of Nrf2-dependent antioxidant enzymes, decreased reactive oxygen species (ROS) under oxidative stress in mouse cortical neurons, and robustly protected mitochondria from oxidative insults. Moreover, mini-GAGR increased the nuclear localization and transcriptional activity of Nrf2 similarly to known Nrf2 activators. Mechanistically, mini-GAGR increased the dissociation of Nrf2 from its inhibitor, Kelch-like ECH-associated protein 1 (Keap1), and induced phosphorylation and nuclear translocation of Nrf2 in a protein kinase C (PKC)- and fibroblast growth factor receptor (FGFR1)-dependent manner. Finally, 20-day intranasal treatment of 3xTg-AD mice with 100 nmol of mini-GAGR increased nuclear p-Nrf2 and growth-associated protein 43 (GAP43) levels in hippocampal neurons, reduced p-tau and β-amyloid (Aβ) peptide–stained neurons, and improved memory. The BBB-bypassing Nrf2-activating polysaccharide reported here may be effective in reducing oxidative stress and neurodegeneration in AD.

CB1 receptor blockade ameliorates hepatic fat infiltration and inflammation and increases Nrf2-AMPK pathway in a rat model of severely uncontrolled diabetes.

Previous studies have shown that the CB1 receptor antagonist reverses steatohepatitis and its related features of metabolic syndrome, such as obesity and type 2 diabetes. However, the beneficial effects of CB1 receptor blockade on hepatic steatosis and inflammation have not been investigated independently of its effects on body weight and glycemic control. At 32 weeks of age, OLETF rats were administered with rimonabant (10 mg·kg−1·day−1) by oral gavage for 6 weeks. No significant changes in body weight, OGTT, and serum glucose were observed in spite of rimonabant-decreased food intake. Moreover, there was a significant difference between initial and final body weight, regardless of rimonabant administration, indicating that OLETF rats were severely diabetic rats. Rimonabant administration significantly decreased serum liver enzyme levels such as ALT and AST, hepatic fat accumulation, lipid peroxidation, and cell death as demonstrated by the number of TUNEL-positive cells in severely uncontrolled diabetic OLETF rats. Significant decreases in hepatic gene expression of proinflammatory cytokines (CD11b, F4/80, MCP1, and TNFα), negative inflammatory mediators (SOCS1 and SOCS3), and fibrosis-related proteins (TGFβ, collagen 1, and TIMP1) were found in rimonabant-treated OLETF rats. Six-week administration of rimonabant significantly upregulated mRNA levels of CPT1α and PPARα related to β-oxidation. Moreover, significant increases in Nrf2 gene expression and its downstream genes, NQO1, GSAT, HO-1, and TXNRD1 along with increased AMPK phosphorylation were noted in uncontrolled diabetic rats treated with rimonabant. The observed potent inhibitory effects of CB1 receptor blockade on hepatic fat infiltration and cellular death in severely uncontrolled diabetic rats indicate that CB1 receptor is a possible therapeutic target. Increased Nrf2 and AMPK phosphorylation may play a role in the mechanism of rimonabant action.

Berberine ameliorates lipopolysaccharide-induced acute lung injury via the PERK-mediated Nrf2/HO-1 signaling axis.

A fundamental element of acute lung injury (ALI) is the inflammatory response, which can affect the entire respiratory system, including the respiratory tract and alveoli. Berberine has gained attention because of its anti-inflammatory effects. Nuclear factor-erythroid 2-related factor 2 (Nrf2) and endoplasmic reticulum (ER) stress are involved in lung injury. Nrf2 also acts as a protein kinase-like ER kinase (PERK) substrate in heart disease. Therefore, this study investigated the effect of berberine against lipopolysaccharide (LPS)-induced ALI and the role of the PERK-mediated Nrf2/HO-1 signaling axis. Berberine promoted Nrf2 nuclear translocation and phosphorylation in vitro. After LPS stimulation, this effect was further enhanced, whereas inflammatory factor (IL-6 and IL-8) release and reactive oxygen species generation were significantly decreased. Berberine effectively alleviated lung injury by reducing lung edema and neutrophil infiltration. Berberine also significantly reduced histopathological inflammatory changes via inhibition of ER stress and activation of Nrf2 signaling. Thapsigargin-induced ER stress and small interference RNA (siRNA)-mediated Nrf2 inhibition abrogated the protective effects of berberine in vitro, whereas siRNA-mediated suppression of ER stress and sulforaphane-induced Nrf2 activation further improved those effects. Importantly, ER stress induction led to Nrf2 activation, whereas PERK depletion partly reduced the level of Nrf2 phosphorylation and translocation in LPS-induced cells. Therefore, berberine inhibits LPS-induced ALI through the PERK-mediated Nrf2/HO-1 signaling axis.