CREB1 regulates glucose transport of glioma cell line U87 by targeting GLUT1

Chemical modification of chitosan is a promising method for the improvement of biological activity. In this study, chitosan-caffeic acid (CCA) was prepared and its in vitro hepatoprotective ability against hydrogen peroxide-induced hepatic damage in liver cells was evaluated. Treatment with CCA (50–400 µg/mL) did not show cytotoxicity and also significantly (p < 0.05) recovered cell viability against 650 µM hydrogen peroxide-induced hepatotoxicity. CCA treatment attenuated reactive oxygen species generation and lipid peroxidation in addition to increasing cellular glutathione level in cultured hepatocytes. To validate the underlying mechanism, antioxidant and phase II detoxifying enzyme expressions, which are mediated by NF-E2-related factor 2 (Nrf2) activation, were analyzed and CCA treatment was found to increase the expression of superoxide dismutase-1 (SOD-1), glutathione reductase (GR), heme oxygenase-1 (HO-1), and NAD(P)H:quinine oxidoreductase 1 (NQO1). CCA treatment resulted in increased Nrf2 nuclear translocation. The phosphorylation of extracellular regulated kinase (ERK), c-Jun NH₂-terminal kinase (JNK), and p38 mitogen-activated protein kinase (MAPK) by CCA treatment contributed to Nrf2 activation. Pharmacological blockade of ERK, JNK, and p38 MAPK revealed that SP600125 (JNK inhibitor) and PD98059 (ERK inhibitor) treatment reduced Nrf2 translocation into the nucleus while SB203580 (p38 inhibitor) exhibited weak inhibition. Collectively, CCA protects liver cells against hydrogen peroxide-induced injury and this ability is attributed to the induction of antioxidants and phase II detoxifying enzymes that are mediated by Nrf2 translocation via JNK/ERK signaling.     

Enhanced thioredoxin,glutathione and Nrf2 antioxidant systems by safflower extract and aceglutamide attenuate cerebral ischaemia/reperfusion injury

A large number of reactive oxygen species (ROS) aggravate cerebral damage after ischaemia/reperfusion (I/R). Glutathione (GSH), thioredoxin (Trx) and nuclear factor (erythroid‐derived 2)‐like 2 (Nrf2) represent three major antioxidant systems and play vital roles in affecting each other in eliminating ROS. Identification of drugs targeting triple antioxidant systems simultaneously is vital for inhibiting oxidative damage after cerebral I/R. This study investigated the protective effect of safflower extract and aceglutamide (SAAG) against cerebral I/R injury through modulating multiple antioxidant systems of GSH, Trx and Nrf2 and identified each role of its component acegluatminde (AG) and safflower extract (SA) on these systems. Safflower extract and aceglutamide and its two components decreased neurological deficit scores, infarction rate, apoptosis and oxidative damage after cerebral I/R while enhanced cell viability, decreased reactive oxygen species and nitric oxide level in H₂O₂‐induced PC12 cell model. Importantly, compared to its two components, SAAG demonstrated more effective enhancement of GSH, Nrf2 and Trx systems and a better protection against cerebral I/R injury. The enhanced antioxidant systems prevented ASK1 activation and suppressed subsequent p38 and JNK cascade‐mediated apoptosis. Moreover, inhibition of Trx and Nrf2 systems by auranofin and ML385 abolished SAAG‐mediated protection, respectively. Thus, enhanced triple systems by SAAG played a better protective role than those by SA or AG via inhibition of ASK1 cascades. This research provided evidence for the necessity of combination drugs from the perspective of multiple antioxidant systems. Furthermore, it also offers references for the study of combination drugs and inspires novel treatments for ischaemic stroke.     

DJ-1 plays an important role in caffeic acid-mediated protection of the gastrointestinal mucosa against ketoprofen-induced oxidative damage

Ketoprofen is widely used to alleviate pain and inflammation in clinical medicine; however, this drug may cause oxidative stress and lead to gastrointestinal (GI) ulcers. We previously reported that nuclear factor erythroid 2-related factor 2 (Nrf2) plays a crucial role in protecting cells against reactive oxygen species, and it facilitates the prevention of ketoprofen-induced GI mucosal ulcers. Recent reports suggested that Nrf2 becomes unstable in the absence of DJ-1/PARK7, attenuating the activity of Nrf2-regulated downstream antioxidant enzymes. Thus, increasing Nrf2 translocation by DJ-1 may represent a novel means for GI protection. In vitro, caffeic acid increases the nuclear/cytosolic Nrf2 ratio and the mRNA expression of the downstream antioxidant enzymes, _ϒ-glutamyl cysteine synthetase, glutathione peroxidase, glutathione reductase, and heme oxygenase-1, by activating the JNK/p38 pathway in Int-407 cells. Moreover, knockdown of DJ-1 also reversed caffeic acid-induced nuclear Nrf2 protein expression in a JNK/p38-dependent manner. Our results also indicated that treatment of Sprague–Dawley rats with caffeic acid prior to the administration of ketoprofen inhibited oxidative damage and reversed the inhibitory effects of ketoprofen on the antioxidant system and DJ-1 protein expression in the GI mucosa. Our observations suggest that DJ-1 plays an important role in caffeic acid-mediated protection against ketoprofen-induced oxidative damage in the GI mucosa.     

The Bcl-2 inhibitor venetoclax inhibits Nrf2 antioxidant pathway activation induced by hypomethylating agents in AML

Induction of reactive oxygen species (ROS), an important process for the cytotoxicity of various acute myeloid leukemia (AML) therapies including hypomethylating agents (HMAs), concurrently activates the NF-E2-related factor 2 (Nrf2) antioxidant response pathway which in turn results in induction of antioxidant enzymes that neutralize ROS. In this study, we demonstrated that Nrf2 inhibition is an additional mechanism responsible for the marked antileukemic activity in AML seen with the combination of HMAs and venetoclax (ABT-199). HMA and venetoclax combined treatment augmented mitochondrial ROS induction and apoptosis compared with treatment HMA alone. Treatment of AML cell lines as well as primary AML cells with venetoclax disrupted HMA decitabine-increased nuclear translocation of Nrf2 and induction of downstream antioxidant enzymes including heme oxygenase-1 and NADP-quinone oxidoreductase-1. Venetoclax treatment also leads to dissociation of B-cell lymphoma 2 from the Nrf2/Keap-1 complex and targets Nrf2 to ubiquitination and proteasomal degradation. Thus, our results here demonstrated an undiscovered mechanism underlying synergistic effect of decitabine and venetoclax in AML cells, elucidating for impressive results in antileukemic activity against AML in preclinical and early clinical studies by combined treatment of these drugs.     

Involvement of Nrf2 and Keap1 in the activation of antioxidant responsive element (ARE) by chemopreventive agent peptides from soft-shelled turtle

The antioxidant response element (ARE) is a cis-acting enhancer sequence located in the region containing genes related to antioxidant and detoxification. Under oxidative stress, the induction of nuclear factor-E2-related factor 2 (Nrf2)/ARE is considered as a fundamental process involved in defending reactive oxygen species (ROS) and providing protection against toxic xenobiotics. In this study, we obtained seven antioxidant peptides from soft-shelled turtle and concluded that Glu-Asp-Tyr-Gly-Ala (EDYGA) is the most potent ARE-luciferase inducer. To gain fundamental insights into the role of EDYGA in oxidative stress, we evaluated the effects of EDYGA on the Nrf2/Keap1 system in HepG₂ cells. The results revealed that EDYGA modulated the Nrf2/ARE pathway by enhancing Nrf2 level through the stabilization of Nrf2, which was accomplished by a decrease in the level of Keap1. These actions eventually led to an increase in nuclear Nrf2 accumulation and ARE-binding activity. Moreover, silencing Nrf2 markedly reduced ARE-driven activity induced by EDYGA. Docking results proved that glutamate residues of peptide EDYGA directly bind to Arg 415 of Kelch domain receptor pocke. The results were helpful in understanding the antioxidant activity of peptides from soft-shelled turtle which have potential to be used in foods and drugs as functional ingredients.     

Genipin up-regulates heme oxygenase-1 via PI3-kinase-JNK1/2-Nrf2 signaling pathway to enhance the anti-inflammatory capacity in RAW264.7 macrophages

Genipin, an aglycon of geniposide, has been reported to exhibit diverse pharmacological functions such as antitumor and anti-inflammatory effects. This study aimed to elucidate the anti-inflammatory mechanism of genipin, focusing particularly on the role of heme oxygenase-1 (HO-1), a potent anti-inflammatory enzyme. In RAW264.7 cells, genipin increased HO-1 expression and its enzyme activity via a NF-E2-related factor 2 (Nrf2)–antioxidant response element (ARE) pathway. These effects were significantly inhibited by exposure to the phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor, LY294002, or by expression of a dominant negative mutant of PI 3-kinase. Additional experiments showed that the activation of c-Jun NH₂-terminal kinase 1/2 (JNK1/2) is required for genipin-induced phosphorylation and nuclear translocation of Nrf2 and antioxidant response element (ARE)-driven induction of HO-1, and acts as a downstream effector of PI 3-kinase. Furthermore, functional significance of HO-1 induction was revealed by genipin-mediated inhibition of lipopolysaccharide-stimulated inducible nitric oxide synthase expression or cyclooxygenase-2 promoter activity, the response was reversed by the blocking of HO-1 protein synthesis or HO-1 enzyme activity. Therefore, identification of PI 3-kinase-JNK1/2-Nrf2-linked signaling cascade in genipin-mediated HO-1 expression defines the signaling event that could participate in genipin-mediated anti-inflammatory response.     

Sulforaphane induces autophagy through ERK activation in neuronal cells

Sulforaphane (SFN), an activator of nuclear factor E2-related factor 2 (Nrf2), has been reported to induce autophagy in several cells. However, little is known about its signaling mechanism of autophagic induction. Here, we provide evidence that SFN induces autophagy with increased levels of LC3-II through extracellular signal-regulated kinase (ERK) activation in neuronal cells. Pretreatment with NAC (N-acetyl-l-cysteine), a well-known antioxidant, completely blocked the SFN-induced increase in LC3-II levels and activation of ERK. Knockdown or overexpression of Nrf2 did not affect autophagy. Together, the results suggest that SFN-mediated generation of reactive oxygen species (ROS) induces autophagy via ERK activation, independent of Nrf2 activity in neuronal cells.     

Neuroprotective effects of 1‐O‐hexyl‐2,3,5‐trimethylhydroquinone on ischaemia/reperfusion‐induced neuronal injury by activating the Nrf2/HO‐1 pathway

1‐O‐Hexyl‐2,3,5‐trimethylhydroquinone (HTHQ), a lipophilic phenolic agent, has an antioxidant activity and reactive oxygen species (ROS) scavenging property. However, the role of HTHQ on cerebral ischaemic/reperfusion (I/R) injury and the underlying mechanisms remain poorly understood. In the present study, we demonstrated that HTHQ treatment ameliorated cerebral I/R injury in vivo, as demonstrated by the decreased infarct volume ration, neurological deficits, oxidative stress and neuronal apoptosis. HTHQ treatment increased the levels of nuclear factor erythroid 2–related factor 2 (Nrf2) and its downstream antioxidant protein, haeme oxygenase‐1 (HO‐1). In addition, HTHQ treatment decreases oxidative stress and neuronal apoptosis of PC12 cells following hypoxia and reperfusion (H/R) in vitro. Moreover, we provided evidence that PC12 cells were more vulnerable to H/R‐induced oxidative stress after si‐Nrf2 transfection, and the HTHQ‐mediated protection was lost in PC12 cells transfected with siNrf2. In conclusion, these results suggested that HTHQ possesses neuroprotective effects against oxidative stress and apoptosis after cerebral I/R injury via activation of the Nrf2/HO‐1 pathway.     

Gualou Guizhi Granule Protects Against Oxidative Injury by Activating Nrf2/ARE Pathway in Rats and PC12 Cells

Stroke involves numerous pathophysiological processes and oxidative stress is considered as a main cellular event in its pathogenesis. The nuclear factor erythroid-2-related factor 2 (Nrf2)/antioxidant response element (ARE) pathway plays a key role in inducing phase II detoxifying enzymes and antioxidant proteins and is now considered as a interesting therapeutic target for the treatment of stroke. The objective of this study is to investigate the protective effect of Gualou Guizhi granule (GLGZG) against oxidative stress and explore the protective mechanism of the Nrf2/ARE pathway. In vivo, administration of GLGZG in a rat model of focal cerebral ischemia significantly suppressed oxidative injury by increasing the activity of superoxide dismutase and glutathione level and decreasing reactive oxygen species and malondialdehyde levels. Western blot analysis showed that GLGZG induced nuclear translocation of Nrf2, and combined with real-time PCR results, which indicated that GLGZG up-regulated the Nrf2/ARE pathway. In addition, in cultured PC12 cells, GLGZG protected against H₂O₂ induced oxidative injury and activated the Nrf2/ARE pathway. All the results demonstrated that GLGZG in the management of cerebral ischemia and H₂O₂ induced oxidative injury may be associated with activation of Nrf2/ARE signaling pathway.     

Rg1 protects H9C2 cells from high glucose‐/palmitate‐induced injury via activation of AKT/GSK‐3β/Nrf2 pathway

Our previous studies have assessed ginsenoside Rg1 (Rg1)‐mediated protection in a type 1 diabetes rat model. To uncover the mechanism through which Rg1 protects against cardiac injury induced by diabetes, we mimicked diabetic conditions by culturing H9C2 cells in high glucose/palmitate. Rg1 had no toxic effect, and it alleviated the high glucose/palmitate damage in a dose‐dependent manner, as indicated by 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl tetrazolium bromide assay and lactate dehydrogenase release to the culture medium. Rg1 prevented high glucose/palmitate‐induced cell apoptosis, assessed using cleaved caspase‐3 and terminal deoxynucleotidyl transferase dUTP nick end labelling staining. Rg1 also reduced high glucose‐/palmitate‐induced reactive oxygen species formation and increased intracellular antioxidant enzyme activity. We found that Rg1 activates protein kinase B (AKT)/glycogen synthase kinase‐3 (GSK‐3β) pathway and antioxidant nuclear factor erythroid 2‐related factor 2 (Nrf2) pathway, indicated by increased phosphorylation of AKT and GSK‐3β, and nuclear translocation of Nrf2. We used phosphatidylinositol‐3‐kinase inhibitor Ly294002 to block the activation of the AKT/GSK‐3β pathway and found that it partially reversed the protection by Rg1 and decreased Nrf2 pathway activation. The results suggest that Rg1 exerts a protective effect against high glucose and palmitate damage that is partially AKT/GSK‐3β/Nrf2‐mediated. Further studies are required to validate these findings using primary cardiomyocytes and animal models of diabetes.     

Nuclear Nrf2 Induction by Protein Transduction Attenuates Osteoclastogenesis

It has been reported that reactive oxygen species (ROS) play a role as intracellular signaling molecules in RANKL stimulation. Previously we demonstrated that induction of cytoprotective enzyme expression by Nrf2-gene transfer successfully ameliorated RANKL-dependent osteoclastogenesis. In the present study, we hypothesized that Nrf2 activation by inhibiting ubiquitination and degradation of Nrf2 by ETGE-peptide would induce Nrf2-dependent cytoprotective enzyme expression, attenuate ROS signaling, and thereby inhibit RANKL-dependent osteoclastogenesis. ETGE-peptide containing a cell-permeable sequence (seven consecutive arginine; 7R-ETGE) was applied to a mouse macrophage cell-line RAW 264.7 cell or a primary macrophage culture. ETGE-peptide prevents Keap1 from binding to Nrf2. Nrf2 nuclear translocation and Nrf2-dependent cytoprotective enzyme induction was observed. The effects of 7R-ETGE on RANKL-dependent induction of intracellular ROS levels and osteoclastogenesis were examined. Finally, the protective effect of 7R-ETGE on RANKL-mediated bone destruction was investigated in mice. 7R-ETGE dose-dependently induced nuclear Nrf2, followed by the induction of cytoprotective enzyme expression at both the gene and protein level. 7R-ETGE inhibited upregulation of intracellular ROS levels by RANKL stimulation, and osteoclastogenesis was attenuated. Of particular interest was that local injection of 7R-ETGE ameliorated RANKL-mediated bone destruction. Local induction of nuclear Nrf2 by protein transduction is a potential novel therapeutic target for bone destruction diseases such as periodontitis and rheumatoid arthritis.     

Schisandrin B elicits a glutathione antioxidant response and protects against apoptosis via the redox-sensitive ERK/Nrf2 pathway in AML12 hepatocytes

Abstract This study examined the effects of (-)schisandrin B [(-)Sch B] on MAPK and Nrf2 activation and the subsequent induction of glutathione antioxidant response and cytoprotection against apoptosis in AML12 hepatocytes. Pharmacological tools, such as cytochrome P-450 (CYP) inhibitor, antioxidant, MAPK inhibitors and Nrf2 RNAi, were used to delineate the signalling pathway. (-)Sch B caused a time-dependent activation of MAPK in AML12 cells, particularly the ERK1/2. The MAPK activation was followed by an enhancement in Nrf2 nuclear translocation and the eliciting of a glutathione antioxidant response. Reactive oxygen species arising from a CYP-catalysed reaction with (-)Sch B seemed to be causally related to the activation of MAPK and Nrf2. ERK inhibition by U0126 or Nrf2 suppression by Nrf2 RNAi transfection almost completely abrogated the cytoprotection against menadione-induced apoptosis in (-)Sch B-pre-treated cells. (-)Sch B pre-treatment potentiated the menadione-induced ERK activation, whereas both p38 and JNK activations were suppressed. Under the condition of ERK inhibition, Sch B treatment did not protect against carbon tetrachloride-hepatotoxicity in an in vivo mouse model. In conclusion, (-)Sch B triggers a redox-sensitive ERK/Nrf2 signalling, which then elicits a cellular glutathione antioxidant response and protects against oxidant-induced apoptosis in AML12 cells.