Induction of the Nrf2-driven antioxidant response by tert-butylhydroquinone prevents ethanol-induced apoptosis in cranial neural crest cells

Previous studies have shown that ethanol exposure causes apoptosis in cranial neural crest cells (NCCs), an ethanol-sensitive cell population implicated in Fetal Alcohol Spectrum Disorders (FASD). Additionally, induction of endogenous antioxidants through activation of nuclear factor-erythroid 2-related factor 2 (Nrf2) has been shown to prevent oxidative stress and apoptosis in ethanol-exposed mouse embryos. The objective of this study was to test whether tert-butylhydroquinone (tBHQ), an Nrf2 inducer, can protect NCCs against ethanol-induced apoptosis. Ethanol exposure was shown to cause a moderate increase in the protein expression of Nrf2 and its downstream antioxidants in the NCCs. Treatment of NCCs with tBHQ alone significantly increased the protein expression of Nrf2 and its downstream antioxidants and also significantly increased the activities of the antioxidant enzymes. In NCCs exposed to ethanol, the tBHQ-mediated antioxidant response prevented oxidative stress and apoptosis. These results clearly demonstrate that the activation of Nrf2 signaling confers protection against ethanol-induced apoptosis in NCCs.     

Role of Nrf2 in preventing ethanol-induced oxidative stress and lipid accumulation

Oxidative stress and lipid accumulation play important roles in alcohol-induced liver injury. Previous reports showed that, in livers of nuclear factor erythroid 2-related factor 2 (Nrf2)-activated mice, genes involved in antioxidant defense are induced, whereas genes involved in lipid biosynthesis are suppressed. To investigate the role of Nrf2 in ethanol-induced hepatic alterations, Nrf2-null mice, wild-type mice, kelch-like ECH-associated protein 1-knockdown (Keap1-KD) mice with enhanced Nrf2, and Keap1-hepatocyte knockout (Keap1-HKO) mice with maximum Nrf2 activation, were treated with ethanol (5 g/kg, po). Blood and liver samples were collected 6 h thereafter. Ethanol increased alanine aminotransferase and lactate dehydrogenase activities as well as thiobarbituric acid reactive substances in serum of Nrf2-null and wild-type mice, but not in Nrf2-enhanced mice. After ethanol administration, mitochondrial glutathione concentrations decreased markedly in Nrf2-null mice but not in Nrf2-enhanced mice. H₂DCFDA staining of primary hepatocytes isolated from the four genotypes of mice indicates that oxidative stress was higher in Nrf2-null cells, and lower in Nrf2-enhanced cells than in wild-type cells. Ethanol increased serum triglycerides and hepatic free fatty acids in Nrf2-null mice, and these increases were blunted in Nrf2-enhanced mice. In addition, the basal mRNA and nuclear protein levels of sterol regulatory element-binding protein 1(Srebp-1) were decreased with graded Nrf2 activation. Ethanol further induced Srebp-1 mRNA in Nrf2-null mice but not in Nrf2-enhanced mice. In conclusion, Nrf2 activation prevented alcohol-induced oxidative stress and accumulation of free fatty acids in liver by increasing genes involved in antioxidant defense and decreasing genes involved in lipogenesis.     

Knockdown of GCN2 inhibits high glucose-induced oxidative stress and apoptosis in retinal pigment epithelial cells

Increasing evidence suggests that general control nonderepressible 2 (GCN2) is a critical regulator of oxidative stress and cell apoptosis in response to various stimuli. However, the role of GCN2 in diabetic retinopathy remains unclear. The aim of the present study was to investigate the effects of GCN2 on oxidative stress and apoptosis in ARPE-19 cells exposed to high glucose. The results showed that GCN2 was highly expressed in high glucose-induced ARPE-19 cells. Moreover, knockdown of GCN2 greatly improved ARPE-19 cell viability in response to high glucose. In addition, GCN2 knockdown significantly suppressed the production of reactive oxygen species (ROS) and malondialdehyde (MDA), as well as increased superoxide dismutase (SOD) activity in high glucose-stimulated ARPE-19 cells. Furthermore, GCN2 knockdown reduced cell apoptosis and enhanced the activation of nuclear factor E2-related factor 2/heme oxygenase-1 (Nrf2/HO-1) pathway in high glucose-stimulated ARPE-19 cells. However, knockdown of Nrf2 reversed the effects of GCN2 on oxidative stress and cell apoptosis. Taken together, our findings suggest that knockdown of GCN2 inhibits high glucose-induced oxidative stress and apoptosis in ARPE-19 cells through activation of the Nrf2/HO-1 pathway.     

The role of NOX enzymes in ethanol-induced oxidative stress and apoptosis in mouse embryos

Reactive oxygen species (ROS) play an important role in ethanol-induced apoptosis and teratogenesis. However, the major sources of ROS in ethanol-exposed embryos have remained undefined. This study was conducted to determine the role of NADPH oxidase (NOX) in ethanol-induced oxidative stress and apoptosis in mouse embryos. Analyses of mRNA expression indicated that ethanol treatment resulted in a significant increase in mRNA expression of NOX catalytic subunit Duox-1 in gestational day 9 (GD 9:0) mouse embryos. Ethanol exposure also resulted in significant increases in mRNA expression of NOX regulatory subunits, p22phox, p67phox, NOXA1 and NOXO1. In addition, a significant increase in NOX enzyme activity was found in the ethanol-exposed embryos as compared to controls. Co-treatment with the NOX inhibitor, diphenyleneiodonium (DPI), significantly prevented ethanol-induced increases in NOX enzyme activity, ROS generation and oxidative DNA damage in ethanol-exposed embryos. DPI treatment also resulted in a reduction in caspase-3 activation, decreased caspase-3 activity and diminished prevalence of apoptosis in ethanol-exposed embryos. These results support the hypothesis that NOX is a critical source of ROS in ethanol-exposed embryos and that it plays an important role in ethanol-induced oxidative stress and pathogenesis.     

Nrf2-ARE信号通路功能与临床疾病及相关药物的研究新进展

Nrf2（NF-E2-related factor 2）核因子E2相关因子是一种机体抵抗内界和外界氧化或化学等刺激的中枢调节者。Nrf2-ARE则是近年来新发现的细胞氧化应激反应的关键传导通路,当其在体内被有毒有害物质激活后转位进入细胞核能与抗氧化反应元件（antioxidant response element,ARE）结合形成Nrf2-ARE信号通路,从而调控下游抗氧化蛋白、氧化酶和Ⅱ相解毒酶等。研究发现该通路在抗衰老、抗肿瘤、抗炎症、神经损伤、眼科等多方面均有重要作用。以Nrf2为靶点的药物有望用于肿瘤、糖尿病、神经退行性疾病等。本文综述了Nrf2-ARE信号通路功能及以其为靶点的药物研究的进展。     

Resveratrol protects human keratinocytes HaCaT cells from UVA-induced oxidative stress damage by downregulating Keap1 expression

Ultraviolet radiation A (UVA)-induced oxidative stress is recognized as an important factor in the development of skin carcinogenesis. Resveratrol is demonstrated to possess remarkable antioxidant activity in the organism. The aim of this study was to investigate the protective role of resveratrol in human keratinocytes (HaCaT) against UVA-induced oxidative damage and the possible mechanism of the translocation of NF-E2-related factor-2 (Nrf2) into the nucleus. The HaCaT cells were UVA-irradiated and the effects of resveratrol on cell viability, reactive oxygen species generation and membrane-lipid peroxidation were measured. The proteins and mRNA of Nrf2 and Kelch-like-ECH-associated protein 1 (Keap1) were determined by immunofluorescence staining, Western blot and quantitative PCR, respectively. UVA exposure led to a decrease in viability and an increase in reactive oxygen species generation in HaCaT cells. Resveratrol could effectively increase the viability of HaCaT cells after UVA exposure and protect them from UVA-induced oxidative stress. Moreover, resveratrol increased the level of Nrf2 protein and facilitated Nrf2 accumulation in the nucleus; as a result, the activity of antioxidant enzymes was also upregulated. The main finding was that Keap1 protein, a repressor of Nrf2 in the cytoplasm, was clearly decreased by resveratrol treatment 12 h and beyond though the level of Keap1 mRNA still increased. Our results suggest that resveratrol can degrade Keap1 protein and facilitate Nrf2 accumulation in the nucleus, thereby protecting HaCaT cells from UVA-induced oxidative stress. Resveratrol could be a more useful natural medicine for the protection of epidermal cells from UVA-induced damage.     

NOX4‐ and Nrf2‐mediated oxidative stress induced by silver nanoparticles in vascular endothelial cells

It has been widely reported that silver nanoparticles (AgNPs) induce oxidative stress in various cell lines. However, the mechanism for this effect and its consequences for cellular signaling are poorly understood. In this study, human umbilical vein endothelial cells (HUVECs) were used to assess the toxicity and investigate the associated molecular mechanisms caused by exposure to AgNPs. We demonstrated that AgNP exposure significantly and dose‐dependently decreased the cell viability, induced reactive oxygen species (ROS) generation and led to early apoptosis in HUVECs. Our findings showed that AgNPs induced excess ROS production that affected the signaling pathways by a mechanism that depended on activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity through upregulation of NADPH oxidase 4 (NOX4) protein expressions. Moreover, AgNPs could disrupt the inactivation of the nuclear factor erythroid 2‐related factor 2 (Nrf2)‐mediated antioxidant response, which is considered another important element for oxidative stress caused by AgNPs in HUVECs. The redox imbalance between NOX4 and Nrf2 was an important cause for the ROS overproduction that led to cell injury in HUVECs. The results provided insight into the mechanisms of oxidative stress induced by AgNPs in vascular endothelial cells.     

Ginkgolide B attenuates ethanol-induced neurotoxicity through regulating NADPH oxidases

Ethanol has long been demonstrated to trigger cell apoptosis in the central nervous system. The over-production of reactive oxygen species (ROS) is considered as one of the most important mechanisms involving in the apoptosis caused by ethanol. Ginkgolide B (GB), which was widely used as a monomer of traditional Chinese medicine, was reported to scavenge free radicals in endothelial cells and smooth muscle cells. But whether GB can prevent ethanol-induced neurotoxicity is still unknown. The aim of this study was to investigate effects of GB on ethanol-induced cytotoxicity, oxidative stress and apoptosis and explore potential protective molecular mechanism of GB. It was found that GB inhibited cell injury and apoptosis in a dose-dependent manner in ethanol-treated PC12 cells by MTT and LDH assays. It was also found that activities of caspase-3 increased by ethanol were mostly abrogated by GB. Further, GB decreased the production of ROS and subsequent over-production of lipid peroxides. A significant increase of alcohol dehydrogenase (ADH) and CYP2E1 enzyme activity was found in the ethanol-exposed PC12 cells as compared to controls. However, GB pretreatment did not significantly affect ethanol-induced ADH and CYP2E1 activities. Quantitative real-time PCR and Western blot analysis demonstrated that ethanol treatment resulted in a significant increase in mRNA and protein expression of NADPH oxidases, which are main oxidases producing ROS in neurons. Moreover, expression and activities of NADPH oxidases were down-regulated by GB. These results indicate that ethanol-induced neurotoxicity is ameliorated by GB mainly through regulating expression and activity of NADPH oxidases.     

Alpha-lipoic acid regulates heme oxygenase gene expression and nuclear Nrf2 activation as a mechanism of protection against arsenic exposure in HepG2 cells

Oxidative stress is a known mechanism induced, among other things, by arsenic toxicity. As a response, the cell triggers the synthesis of antioxidant and stress response elements like glutathione and heme oxygenase. Alpha-lipoic acid (ALA) is a well-known antioxidant that confers protection to oxidative stress conditions. We analyzed the effect of ALA pretreatment on Nrf2-responsive gene expression of HepG2 cells exposed to As(3+). Cells were treated with 5mM ALA and 8h later exposed to 50μM As(3+) for 24h, analyzing MTT-activity, glutathione content, Nrf2 induction and antioxidant gene expression. As(3+) increased glutathione (154%), heme oxygenase, glutamate cystein ligase, modifier subunit and metallothionein (35-fold, 10-fold and 9-fold, respectively). ALA prevented the strong expression of heme oxygenase by As(3+) exposure (from 35- to 5-times of control cells), which correlated with the reduction of Nrf2 observed in As(3+) group. ALA pretreatment can down-modulate the response mediated by Nrf2 and provide protection to As(3+) exposed HepG2 cells.