Nutritional strategies to modulate inflammation and oxidative stress pathways via activation of the master antioxidant switch Nrf2

The nuclear factor E2-related factor 2 (Nrf2) plays an important role in cellular protection against cancer, renal, pulmonary, cardiovascular and neurodegenerative diseases where oxidative stress and inflammation are common conditions. The Nrf2 regulates the expression of detoxifying enzymes by recognizing the human Antioxidant Response Element (ARE) binding site and it can regulate antioxidant and anti-inflammatory cellular responses, playing an important protective role on the development of the diseases. Studies designed to investigate how effective Nrf2 activators or modulators are need to be initiated. Several recent studies have shown that nutritional compounds can modulate the activation of Nrf2–Keap1 system. This review aims to discuss some of the key nutritional compounds that promote the activation of Nrf2, which may have impact on the human health.     

不同氧浓度下C2C12细胞的Nrf2抗氧化系统对H2O2刺激的反应*

目的:探讨不同氧浓度下小鼠骨骼肌卫星细胞系(C2C12细胞)对H₂O₂刺激反应的变化及其机制。方法:小鼠骨骼肌卫星细胞系(C2C12细胞),经培养复苏后,将细胞分为7组,每组设8个复孔,各组分别加入浓度为0.1 mmol/L、0.25 mmol/L、0.5 mmol/L、0.75 mmol/L、1 mmol/L、2 mmol/L的H₂O₂,分别作用1 h、2 h后测细胞活力,选择细胞H₂O₂刺激的最佳作用时间和浓度;C2C12细胞分为不同氧浓度组:21% O₂、12% O₂、8% O₂、5% O₂每组设8个复孔,12 h后,H₂O₂作用1 h,收集细胞;检测细胞Nrf2蛋白荧光和蛋白表达量,测定Nrf2和抗氧化酶SOD1、SOD2、CAT、NQO-1、HO-1、GPX-1 的mRNA表达量及细胞ROS水平。结果:选择H₂O₂作用时间相对较短的1 h和浓度0.5 mmol/L作为本实验的H₂O₂刺激条件。与21%O₂组相比,12%O₂组细胞Nrf2蛋白荧光增强,Nrf2 的mRNA和蛋白表达以及抗氧化酶SOD1、SOD2、CAT、NQO-1、HO-1、GPX-1的 mRNA表达均显著增加(P＜0.05或P＜0.01),细胞 ROS水平明显降低(P＜0.01);8%O₂组仅GPX-1 mRNA显著增加(P＜0.05),其他指标变化不大;5%O₂组细胞 Nrf2 mRNA和蛋白表达以及抗氧化酶SOD1、SOD2、NQO-1、GPX-1的 mRNA表达均明显降低(P＜0.05或P＜0.01),细胞 ROS水平则明显升高(P＜0.01)。结论:不同氧浓度下C2C12细胞中Nrf2介导的抗氧化系统对H₂O₂刺激反应不同,12 h的12% O₂浓度可促进C2C12细胞Nrf2的抗氧化作用,而5% O₂浓度的严重低氧则作用相反。     

Terrein reduces age‐related inflammation induced by oxidative stress through Nrf2/ERK1/2/HO‐1 signalling in aged HDF cells

This study investigated whether multiple bioactivity of terrein such as anti‐inflammatory and anti‐oxidant inhibits age‐related inflammation by promoting an antioxidant response in aged human diploid fibroblast (HDF) cells. HDF cells were cultured serially for in vitro replicative senescence. To create the ageing cell phenotype, intermediate stage (PD31) HDF cells were brought to stress‐induced premature senescence (SIPS) using hydrogen peroxide (H₂O₂). Terrein increased cell viability even with H₂O₂ stress and reduced inflammatory molecules such as intracellular adhesion molecule‐1 (ICAM‐1), cyclooxygenase‐2 (COX‐2), interleukin‐1beta (IL‐1β) and tumour necrosis factor‐alpha (TNF‐α). Terrein reduced also phospho‐extracellular kinase receptor1/2 (p‐EKR1/2) signalling in aged HDF cells. SIPS cells were attenuated for age‐related biological markers including reactive oxygen species (ROS), senescence associated beta‐galactosidase (SA β‐gal.) and the aforementioned inflammatory molecules. Terrein induced the induction of anti‐oxidant molecules, copper/zinc‐superoxide defence (Cu/ZnSOD), manganese superoxide dismutase (MnSOD) and heme oxygenase‐1 (HO‐1) in SIPS cells. Terrein also alleviated reactive oxygen species formation through the Nrf2/HO‐1/p‐ERK1/2 pathway in aged cells. The results indicate that terrein has an alleviative function of age‐related inflammation characterized as an anti‐oxidant. Terrein might be a useful nutraceutical compound for anti‐ageing. Copyright © 2015 John Wiley & Sons, Ltd.     

核因子E2相关因子2在运动改善非酒精性脂肪性肝病中的作用

核因子E2相关因子2 (nuclear factor erythroid 2-related factor 2,Nrf2)信号通路在维持心血管疾病、神经系统退行性疾病以及慢性代谢性疾病中的细胞稳态方面起关键作用。研究表明,以氧化应激、炎症和线粒体功能失调为特征的慢性疾病可通过增加Nrf2表达来恢复机体氧化还原状态,治疗或预防疾病。非酒精性脂肪性肝病（nonalcoholic fatty liver disease,NAFLD）是一种由除酒精以外的其他多种因素导致的以肝脏脂肪变性为特征的慢性代谢性肝脏疾病,其患病率近年来在全球范围内逐渐增加。运动是防治NAFLD的有效手段,可通过运动方式、运动强度、运动环境和运动疲劳等因素影响Nrf2信号通路。本文通过阐述Nrf2信号通路的激活、其调控抗氧化的相关机制以及运动对Nrf2信号通路的影响,以NAFLD的发病机制为基础,探讨运动、Nrf2和NAFLD之间的关系,综述Nrf2在运动改善NAFLD中的作用及相关机制。为运动改善NAFLD的分子机制研究提供理论参考依据。     

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.     

Trehalose targets Nrf2 signal to alleviate d-galactose induced aging and improve behavioral ability

As an important factor leading to aging and chronic diseases, oxidative stress has become a hot research topic. Trehalose is a natural sugar widely found in many edible plants, animals and natural microorganisms, and recent studies have suggested that trehalose is an antioxidant, although its underlying molecular mechanism is unclear. Therefore, we evaluated the protective mechanism of trehalose against oxidative stress-induced senescence. In the mouse model of d-galactose (D-gal) induced aging, we found that trehalose significantly reversed the learning and memory impairment caused by D-gal and improved the ability to explore unknown things, which was associated with a significant reduction in brain tissue damage. Further studies have shown that trehalose activates the expressions of downstream target genes HO-1, NQO1, SOD, GSH and CAT by promoting the nuclear translocation of Nrf2 in the liver. The detoxification ability of organs is increased, antioxidant enzyme activity is enhanced, lipid peroxidation is reduced, and the secretion of inflammatory factors TNF-α, IL-1β, il-6 is decreased. In conclusion, trehalose play an anti-aging role by activating genes related to Nrf2 pathway.     

Electronegative LDL induction of apoptosis in macrophages: Involvement of Nrf2

The aim of this study was to determine the apoptotic pathways and mechanisms involved in electronegative LDL [LDL(−)]-induced apoptosis in RAW 264.7 macrophages and the role of Nrf2 in this process. Incubation of RAW 264.7 macrophages with LDL(−) for 24 h resulted in dose-dependent cell death. Activated caspases were shown to be involved in the apoptosis induced by LDL(−); incubation with the broad caspase inhibitor z-VAD prevented apoptosis in LDL(−)-treated cells. CD95 (Fas), CD95 ligand (FasL), CD36 and the tumor necrosis factor (TNF) ligand Tnfsf10 were overexpressed in LDL(−)-treated cells. However, Bax, Bcl-2 and Mcl-1 protein levels remained unchanged after LDL(−) treatment. LDL(−) promoted hyperpolarization of the mitochondrial membrane, elevated reactive oxygen species (ROS) production and translocation of Nrf2 to the nucleus, a process absent in cells treated with native LDL. Elicited peritoneal macrophages from Nrf2-deficient mice exhibited an elevated apoptotic response after challenge with LDL(−), together with an increase in the production of ROS in the absence of alterations in CD36 expression. These results provide evidence that CD36 expression induced by LDL(−) is Nrf2-dependent. Also, it was demonstrated that Nrf2 acts as a compensatory mechanism of LDL(−)-induced apoptosis in macrophages.     

Protocatechuic acid induces antioxidant/detoxifying enzyme expression through JNK-mediated Nrf2 activation in murine macrophages

Protocatechuic acid (PCA) is a main metabolite of anthocyanins, whose daily intake is much higher than that of other polyphenols. PCA has biological effects, e.g., it induces the antioxidant/detoxifying enzyme gene expression. This study was aimed at defining the molecular mechanism responsible for PCA-induced over-expression of glutathione (GSH) peroxidase (GPx) and GSH reductase (GR) in J774 A.1 macrophages. New evidence is provided that PCA increases GPx and GR expression by inducing C-JUN NH₂-terminal kinase (JNK)-mediated phosphorylation of Nuclear factor erythroid 2 (NF-E2)-related factor 2 (Nrf2). RNA and proteins were extracted from cells treated with PCA (25 μM) for different time points. Quantitative real-time polymerase chain reaction and immunoblotting analyses showed a rapid increase in mRNA (>60%) and protein (>50%) for both the enzymes. This was preceded by the up-regulation of Nrf2, in terms of mRNA and protein, and by its significant activation as assessed by increased Nrf2 phosphorylation and nuclear translocation (+60%). By using specific kinase inhibitors and detecting the activated form, we showed that JNK was the main upstream kinase responsible for Nrf2 activation. Convincing evidence is provided of a causal link between PCA-induced Nrf2 activation and increased enzyme expression. By silencing Nrf2 and using a JNK inhibitor, enzyme enhancement was counteracted. Finally, with the ChIP assay, we demonstrated that PCA-activated Nrf2 specifically bound ARE sequences in enzyme gene promoters. Our study demonstrates for the first time that PCA improves the macrophage endogenous antioxidant potential by a mechanism in which JNK-mediated Nrf2 activation plays an essential role. This knowledge could contribute to novel diet-based approaches aimed at counteracting oxidative injury by reinforcing endogenous defences.     

基于模式生物斑马鱼Nrf2的相关研究进展

Nrf2/ARE信号通路是大多数生物体内抗氧化应激反应、抵抗内外界刺激的关键通路,在抗炎症、免疫、抗肿瘤、抗凋亡、神经保护等方面起着重要的作用。斑马鱼作为一种常见的模式动物,广泛地应用于发育生物学、遗传学和毒理学等研究领域。研究表明转录因子NF_E2相关因子2(NF-E2-related factor 2,Nrf2)不仅在哺乳动物体内存在,也在斑马鱼体内存在并且高度保守,并在抗氧化应激反应中发挥着重要作用。本文通过对斑马鱼Nrf2的结构、生物学功能及其信号通路等方面的最新研究进行阐述,以期为Nrf2及其信号通路引发的相关疾病的预防和治疗提供新的思路。     

Nrf2 protects human alveolar epithelial cells against injury induced by influenza A virus

Background

Influenza A virus (IAV) infection primarily targets respiratory epithelial cells and produces clinical outcomes ranging from mild upper respiratory infection to severe pneumonia. Recent studies have shown the importance of lung antioxidant defense systems against injury by IAV. Nuclear factor-erythroid 2 related factor 2 (Nrf2) activates the majority of antioxidant genes.

Methods

Alveolar type II (ATII) cells and alveolar macrophages (AM) were isolated from human lungs not suitable for transplantation and donated for medical research. In some studies ATII cells were transdifferentiated to alveolar type I-like (ATI-like) cells. Alveolar epithelial cells were infected with A/PR/8/34 (PR8) virus. We analyzed PR8 virus production, influenza A nucleoprotein levels, ROS generation and expression of antiviral genes. Immunocytofluorescence was used to determine Nrf2 translocation and western blotting to detect Nrf2, HO-1 and caspase 1 and 3 cleavage. We also analyzed ingestion of PR8 virus infected apoptotic ATII cells by AM, cytokine levels by ELISA, glutathione levels, necrosis and apoptosis by TUNEL assay. Moreover, we determined the critical importance of Nrf2 using adenovirus Nrf2 (AdNrf2) or Nrf2 siRNA to overexpress or knockdown Nrf2, respectively.

Results

We found that IAV induced oxidative stress, cytotoxicity and apoptosis in ATI-like and ATII cells. We also found that AM can ingest PR8 virus-induced apoptotic ATII cells (efferocytosis) but not viable cells, whereas ATII cells did not ingest these apoptotic cells. PR8 virus increased ROS production, Nrf2, HO-1, Mx1 and OAS1 expression and Nrf2 translocation to the nucleus. Nrf2 knockdown with siRNA sensitized ATI-like cells and ATII cells to injury induced by IAV and overexpression of Nrf2 with AdNrf2 protected these cells. Furthermore, Nrf2 overexpression followed by infection with PR8 virus decreased virus replication, influenza A nucleoprotein expression, antiviral response and oxidative stress. However, AdNrf2 did not increase IFN-λ1 (IL-29) levels.

Conclusions

Our results indicate that IAV induces alveolar epithelial injury and that Nrf2 protects these cells from the cytopathic effects of IAV likely by increasing the expression of antioxidant genes. Identifying the pathways involved in protecting cells from injury during influenza infection may be particularly important for developing new therapeutic strategies.     

A novel small molecule inhibitor targeted at Bcl-2

Recently, the heterocyclic compound 8-oxo-3-thiomorpholino-8H-acenaphtho[1,2-b]pyrrole-9-carboni-trile (S1) was synthesized and shown to induce apoptosis in both (H22) hematoma and (MCF-7) ade-nocarcinoma cells. The IC50 values of S1 against the two cell lines were 0.17 and 0.09 μmol/L, respec-tively. Furthermore, the apoptosis-inducing activity of this compound was highlighted both in vivo and in vitro. Subsequent experiments identified Bcl-2 as the primary target of S1, as a significant reduc-tion in Bcl-2 protein levels was observed in H22 cells following a two-hour treatment with 10 μmol/L S1. While rapid depolarization of mitochondrial membranes led immediately to caspase 9 activation, no changes were identified in either caspase 8 levels or levels in Bcl-2 mRNA. These data were consistent with the results of circular dichroism (CD) spectra analysis, revealing that S1 inactivated the Bcl-2 protein by destroying its critical alpha helices. Taken together, these results suggest the potential of S1 in the development of new therapeutic agents.     

A novel small molecule inhibitor targeted at Bcl-2

Recently, the heterocyclic compound 8-oxo-3-thiomorpholino-8H-acenaphtho[1,2-b]pyrrole-9-carbonitrile (S1) was synthesized and shown to induce apoptosis in both (H22) hematoma and (MCF-7) adenocarcinoma cells. The IC₅₀ values of S1 against the two cell lines were 0.17 and 0.09 μmol/L, respectively. Furthermore, the apoptosis-inducing activity of this compound was highlighted both in vivo and in vitro. Subsequent experiments identified Bcl-2 as the primary target of S1, as a significant reduction in Bcl-2 protein levels was observed in H22 cells following a two-hour treatment with 10 μmol/L S1. While rapid depolarization of mitochondrial membranes led immediately to caspase 9 activation, no changes were identified in either caspase 8 levels or levels in Bcl-2 mRNA. These data were consistent with the results of circular dichroism (CD) spectra analysis, revealing that S1 inactivated the Bcl-2 protein by destroying its critical alpha helices. Taken together, these results suggest the potential of S1 in the development of new therapeutic agents.     

Administration of vitamin E attenuates airway inflammation through restoration of Nrf2 in a mouse model of asthma

Accumulating evidence reveals that ROS is one of the key mediators that contribute to the development of asthma. Studies on antioxidants have shown to have beneficial effects on asthma management. However, we still do not know the precise mechanism, and the effects depend on age. This study was conducted to assess the levels of ROS and the effect of antioxidants in younger and older mice using an eosinophilic asthma model. We analyzed airway hyperresponsiveness (AHR), cytokines in bronchoalveolar lavage fluid (BALF), inflammatory cell counts, and the expression levels of NFκB, Nrf2, EPx, and EDN in the lung tissue, as well as the level of ROS in the lung tissue and BALF. The degree of eosinophilia and the levels of IL-5, ROS, and NFκB were significantly increased, whereas the endogenous levels of vitamin E and Nrf2 were decreased in the lung and BALF in the older mice compared to younger mice. The administration of vitamin E attenuated AHR, airway inflammation, and the level of IL-13 and ROS and enhanced the Nrf2 level in the older mice compared to the younger mice. Taken together, vitamin E treatment may have the therapeutic potential through restoration of the Nrf2 level, especially in elderly asthma.     

Polymyxin B exerts nephrotoxicity effects via inhibition of the Nrf2/NQO1 pathway-mediated antioxidant response

Polymyxin B (PMB) is a polypeptide antibiotic widely used in treating multidrug-resistant Gram-negative bacteria. However, nephrotoxicity is a serious adverse effect that limits its clinical use. Therefore, clarification of the molecular mechanism of PMB-induced renal injury is essential. Our study aimed to explore possible mechanisms of PMB-induced nephrotoxicity in vivo and in vitro. Mice were treated with PMB to construct the kidney injury model. The antioxidant capacity was assessed by measuring the superoxide dismutase (SOD) and catalase (CAT) activities and the glutathione (GSH) and malondialdehyde (MDA) contents. The pathway of the nuclear factor erythroid 2-related factor 2/NADH quinone oxidoreductase 1 (Nrf2/NQO1) was examined after PMB treatment in NRK-52E cells and mice. Finally, the expressions of genes and proteins (Bax, Bcl-2, Caspase-3, Caspase-9) related to apoptosis were evaluated through quantitative polymerase chain reaction and western blot assay. The study verified PMB-induced nephrotoxicity in mice and NRK-52E cells in a dose- and time-dependent manner. PMB treatment significantly decreased the expression of Nrf2 and its downstream target gene NQO1 and increased the apoptosis-related proteins expression. In summary, our results suggested that PMB-induced oxidative stress damage by inhibiting the Nrf2/NQO1 pathway and promoting apoptosis in kidney tissues.