Nrf2 regulates an adaptive response protecting against oxidative damage following diquat-mediated formation of superoxide anion

Mouse embryonic fibroblasts derived from Nrf2-/- mice (N0) and Nrf2+/+ mice (WT) have been used to characterize both basal and diquat (DQ)-induced oxidative stress levels and to examine Nrf2 activation during exposure to DQ-generated superoxide anion. Microarray analysis revealed that N0 cells have similar constitutive mRNA expression of genes responsible for the direct metabolism of reactive oxygen species but decreased expression of genes responsible for the production of reducing equivalents, repair of oxidized proteins and defense against lipid peroxidation, compared to WT cells. Nonetheless, the basal levels of ROS flux and oxidative damage biomarkers in WT and N0 cells were not different. Diquat dibromide (DQ), a non-electrophilic redox cycling bipyridylium herbicide, was used to generate intracellular superoxide anion. Isolated mitochondria from both cell lines exposed to DQ produced equivalent amounts of ROS, indicating a similar cellular capacity to generate ROS. However, N0 cells exposed to DQ for 24-h exhibited markedly decreased cell viability and aconitase activity as well as increased lipid peroxidation and glutathione oxidation, relative to WT cells. 2',7'-Dichlorofluorescein fluorescence was not increased in WT and N0 cells after 30-min of DQ exposure. However, increased levels of ROS were detected in N0 cells but not WT cells after 13-h of DQ treatment. Additionally, total glutathione concentrations increased in WT, but not N0 cells following a 24-h exposure to DQ. DQ exposure resulted in activation of an antioxidant response element-luciferase reporter gene, as well as induction of Nrf2-regulated genes in WT, but not N0 cells. Thus the enhanced sensitivity of N0 cells does not reflect basal differences in antioxidative capacity, but rather an impaired ability to mount an adaptive response to sustained oxidative stress.     

Lycium barbarum polysaccharide protects human keratinocytes against UVB-induced photo-damage

Ultraviolet B (UVB) irradiation plays a key role in skin damage, which induces oxidative and inflammatory damages, thereby causing photoaging or photocarcinogenesis. Lycium barbarum polysaccharide (LBP), the most biologically active fraction of wolfberry, possesses significant antioxidative and anti-inflammatory effects on multiple tissues. In the present study, the photoprotective effects and potential underlying molecular mechanisms of LBP against UVB-induced photo-damage were investigated in immortalized human keratinocytes (HaCaT cells). The data indicated that pretreatment with LBP significantly attenuated UVB-induced decrease in cell viability, increase in ROS production and DNA damage. LBP also significantly suppressed UVB-induced p38 MAPK activation, and subsequently reversed caspase-3 activation and MMP-9 expression. Notably, LBP was found to induce Nrf2 nuclear translocation and increase the expression of Nrf2-dependent ARE target genes. Furthermore, the protective effects of LBP were abolished by siRNA-mediated Nrf2 silencing. These results showed that the antioxidant LBP could partially protect against UVB irradiation-induced photo-damage through activation of Nrf2/ARE pathway, thereby scavenging ROS and reducing DNA damage, and subsequently suppressing UVB-induced p38 MAP pathway. Thus, LBP can be potentially used for skincare against oxidative damage from environmental insults.     

Cholesterol is more susceptible to oxidation than linoleates in cultured cells under oxidative stress induced by selenium deficiency and free radicals

Polyunsaturated fatty acids and their esters are known to be susceptible to free-radical mediated oxidation, while cholesterol is more resistant to oxidation. The present study focused on the relative susceptibilities of linoleates and cholesterol in Jurkat cells under oxidative stress induced by selenium deficiency and free radical insult, as assessed by total hydroxyoctadecadienoic acids (tHODE) and total 7-hydroxycholesterol (t7-OHCh) measured after reduction and saponification. It was observed that the levels of tHODE and t7-OHCh significantly increased by both oxidative insults. The increased amounts of t7-OHCh were higher than those of tHODE in both selenium-deficient and free radical-treated cells. These results suggest that, in contrast to plasma oxidation where cholesterol is much more resistant to oxidation than linoleates, cellular cholesterol is more susceptible to oxidation than cellular linoleates.     

7-Hydroxycholestrol as a possible biomarker of cellular lipid peroxidation: Difference between cellular and plasma lipid peroxidation

Polyunsaturated fatty acids and their esters are known to be susceptible to free radical-mediated oxidation, whereas cholesterol is thought to be more resistant to oxidation. In fact, it has been observed that in the case of plasma lipid peroxidation, the amount of oxidation products of polyunsaturated fatty acids such as linoleic acid was higher than that of cholesterol. In contrast, during oxidative stress-induced cellular lipid peroxidation, oxidation products of cholesterol such as 7-hydroxycholesterol (7-OHCh) were detected in greater amounts than those of linoleates such as hydroxyoctadecadienoic acid (HODE). There are several forms of oxidation products of cholesterol and linoleates in vivo, namely, hydroperoxides, as well as the hydroxides of both the free and ester forms of cholesterol and linoleates. To evaluate these oxidation products, a method used to determine the lipid oxidation products after reduction and saponification was developed. With this method, several forms of oxidation products of cholesterol and linoleates are measured as total 7-OHCh (t7-OHCh) and total HODE (tHODE), respectively. During free radical-mediated lipid peroxidation in plasma, the amount of tHODE was 6.3-fold higher than that of t7-OHCh. In contrast, when Jurkat cells were exposed to free radicals, the increased amount of cellular t7-OHCh was 5.7-fold higher than that of tHODE. Higher levels of t7-OHCh than those of tHODE have also been observed in selenium-deficient Jurkat cells and glutamate-treated neuronal cells. These results suggest that, in contrast to plasma oxidation, cellular cholesterol is more susceptible to oxidation than cellular linoleates. Collectively, cholesterol oxidation products at the 7-position may be a biomarker of cellular lipid peroxidation.     

Role of Galpha12 and Galpha13 as novel switches for the activity of Nrf2, a key antioxidative transcription factor

Galpha12 and Galpha13 function as molecular regulators responding to extracellular stimuli. NF-E2-related factor 2 (Nrf2) is involved in a protective adaptive response to oxidative stress. This study investigated the regulation of Nrf2 by Galpha12 and Galpha13. A deficiency of Galpha12, but not of Galpha13, enhanced Nrf2 activity and target gene transactivation in embryo fibroblasts. In mice, Galpha12 knockout activated Nrf2 and thereby facilitated heme catabolism to bilirubin and its glucuronosyl conjugations. An oligonucleotide microarray demonstrated the transactivation of Nrf2 target genes by Galpha12 gene knockout. Galpha12 deficiency reduced Jun N-terminal protein kinase (JNK)-dependent Nrf2 ubiquitination required for proteasomal degradation, and so did Galpha13 deficiency. The absence of Galpha12, but not of Galpha13, increased protein kinase C delta (PKC delta) activation and the PKC delta-mediated serine phosphorylation of Nrf2. Galpha13 gene knockout or knockdown abrogated the Nrf2 phosphorylation induced by Galpha12 deficiency, suggesting that relief from Galpha12 repression leads to the Galpha13-mediated activation of Nrf2. Constitutive activation of Galpha13 promoted Nrf2 activity and target gene induction via Rho-mediated PKC delta activation, corroborating positive regulation by Galpha13. In summary, Galpha12 and Galpha13 transmit a JNK-dependent signal for Nrf2 ubiquitination, whereas Galpha13 regulates Rho-PKC delta-mediated Nrf2 phosphorylation, which is negatively balanced by Galpha12.     

内皮细胞和平滑肌细胞氧化应激时Nrf2/ARE信号通路对抗氧化基因表达的调控:与动脉粥样硬化和先兆子痫的关系

动脉粥样硬化、糖尿病、慢性肾功能衰竭和先兆子痫等血管疾病时活性氧(reactive oxygen species,ROS)生成增加,容易导致内皮依赖性血管舒张功能的损害和血管损伤,而细胞可以诱导多种编码Ⅱ相解毒酶和抗氧化蛋白的基因表达,从而减轻ROS和亲电子物质介导的细胞损伤。一个被称为抗氧化反应元件(antioxidant response element,ARE)或亲电子反应元件(electrophile response element,EpRE)的顺式转录调控元件,可以介导诸如亚铁血红素加氧酶1、γ-谷氨酰半胱氨酸合成酶、硫氧还蛋白还原酶、谷胱甘肽-S转移酶和NAD(P)H:苯醌氧化还原酶等基因的转录。其他抗氧化酶,如超氧化物歧化酶、过氧化氢酶和非酶清除剂(如谷胱甘肽)等也参与ROS的清除。转录因子NF-E2相关因子2(nuclear factor-erythroid 2-related factor 2, Nrf2)是属于Cap‘n’Collar家族的转录因子,具有碱性亮氨酸拉链(basic region-leucine zipper,bZIP),它在ARE介导的抗氧化基因表达中起重要的作用。在正常情况下,Kelch样环氧氯丙烷相关蛋白-1(Kelch-like ECH-associated protein-1,Keapl)与Nrf2耦联,并与肌动蛋白细胞骨架结合被锚定于胞浆,但是在半胱氨酸残基发生氧化的情况下,Nrf2和Keapl解耦联,进入细胞核并与ARE结合,从而激活多种抗氧化基因和Ⅱ相解毒酶基因的转录。蛋白激酶C、丝裂原活化蛋白激酶和磷脂酰肌醇-3激酶参与Nrf2／ARE信号转导的调控。本文综述了有关Nrf2／ARE信号转导通路在血管稳态和动脉硬化、先兆子痫等疾病情况下内皮及平滑肌细胞对抗持续性氧化应激中起的作用。     

Can nitroxides evoke the Keap1–Nrf2–ARE pathway in skin?

Nitroxides are stable cyclic radicals of diverse size, charge, and lipophilicity. They are cell-permeative, which effectively protects cells, tissues, isolated organs, and laboratory animals from radical-induced damage. The mechanisms of activity through which nitroxides operate are diverse, including superoxide dismutase-mimetic activity, oxidation of semiquinone radicals, oxidation of reduced metal ions, procatalase-mimetic activity, interruption of radical chain reactions, and indirect modulation of NO levels. Nitroxides possess both a nucleophilic (reducing properties) and an electrophilic (oxidizing properties) nature and, therefore, they may affect different cellular pathways. In the current study, a novel mechanism of action by which nitroxides provide skin protection based on their electrophilic nature is suggested. This study shows that nitroxides may act as electrophiles, directly or indirectly, capable of activating the Keap1–Nrf2–ARE pathway in human keratinocytes (HaCaT) and in human skin (human organ culture model). The high potency of oxoammonium cations versus hydroxylamines in activating the system is demonstrated. The mechanism of action by which nitroxides activate the Keap1–Nrf2–ARE pathway is discussed. Understanding the mechanism of activity may expand the usage of nitroxides as a skin protection strategy against oxidative stress-related conditions.     

Modulation of Nrf2 expression alters high glucose-induced oxidative stress and antioxidant gene expression in mouse mesangial cells

Reactive oxygen species (ROS) play an important role in the pathogenesis of diabetic nephropathy. Nuclear factor erythroid 2-related factor 2 (Nrf2) can up-regulate the expression of antioxidant genes and protect cells from oxidative damage. The current study is aimed at examining the effect of modulation of Nrf2 expression on high glucose-induced oxidative stress and Nrf2-targeting antioxidant expression in mouse mesangial cells. In this study, mouse mesangial cells were transiently transfected with Nrf2-plasmid or the Nrf2-specific siRNA. The high glucose-induced intracellular ROS, malondialdehyde, cell proliferation, and TGF-β1 secretion were measured. The levels of Nrf2, heme oxygenase-1 (HO-1), γ-glutamylcysteine synthethase (γ-GCS) expression, and nuclear expression of Nrf2 in mouse mesangial cells were determined. We found that high glucose induced ROS and malondialdehyde generation in mouse mesangial cells. Induction of Nrf2 over-expression reduced the high glucose-induced ROS and malondialdehyde production, inhibited cell proliferation and TGF-β1 secretion, accompanied by up-regulating the expressions of HO-1 and γ-GCS in mouse mesangial cells. However, knockdown of Nrf2 expression displayed reverse effects in mouse mesangial cells. All these results indicated that Nrf2 and its downstream antioxidants, HO-1 and γ-GCS, are negative regulators of high glucose-induced ROS-related mouse mesangial cell dysfunction.     

依达拉奉通过Nrf2信号分子调节氧化应激减轻脑缺血再灌注诱导的神经损伤

本研究旨在探讨依达拉奉(edaravone,ED)在脑缺血再灌注损伤中发挥神经元保护作用与Nrf2信号分子间的关系。体内实验利用脑内脑中动脉闭塞(middle cerebral artery occlusion model,MCAO)建立SD大鼠脑缺血再灌注损伤模型,体外实验采用过氧化氢(H2O2)损伤PC12细胞建立氧化应激模型。通过TTC染色、HE染色、Nissl染色来检测大脑的病理状态。测定活性氧(reactive oxygen species,ROS)、丙二醛(malondialdehyde,MDA)含量、超氧化物歧化酶(superoxide dismutase,SOD)活性,来反映氧化应激水平。此外,通过Hoechst 33342染色和线粒体膜电位(mitochondrial membrane potential,MTP)测定,探究细胞水平的损伤。采用免疫组织化学和蛋白质印记测定Nrf2的表达。构建Nrf2敲除的PC12细胞系,证实Nrf2信号分子抑制氧化应激损伤的作用。结果提示,经依达拉奉给药后,在动物体内水平,TTC染色证实,脑缺血再灌注损伤(cerebral ischemia reperfusion injury,CIRI)大鼠的脑组织梗死体积减小(P<0.001),ROS和MDA水平下降(P<0.01),SOD活性上升(P<0.01);在细胞水平,凋亡细胞减少(P<0.05),MTP上升(P<0.01),ROS和MDA水平下降,SOD活性上升(P<0.01);在分子水平,免疫组化和Western印迹结果均提示,Nrf2蛋白质含量较正常组增加。H2O2诱导Nrf2基因敲除的PC12细胞损伤加重,且依达拉奉的治疗效果明显削弱。综上所述,Nrf2在依达拉奉减轻脑缺血再灌注诱导的氧化应激损伤中发挥关键作用。     

低氧运动对Nrf2基因敲除大鼠运动能力和氧化应激的影响

Nrf2可调节多种抗氧化酶的表达,Nrf2的缺失可能影响机体的运动能力,而低氧可提高机体的抗氧化能力并改善运动能力。为了考察低氧运动对Nrf2基因敲除大鼠运动能力和氧化应激的影响,本研究分别在常氧和低氧环境(12%氧浓度)中对野生型大鼠和Nrf2敲除大鼠进行4周的跑台运动。研究显示,低氧运动可提高野生型大鼠的跑台运动力竭时间,Nrf2敲除可缩短大鼠的力竭时间;低氧运动可上调大鼠的Nrf2 m RNA表达量;Nrf2敲除明显抑制HIF-1α蛋白表达,而低氧运动可上调野生型和Nrf2敲除大鼠的HIF-1α蛋白表达;Nrf2敲除大鼠的骨骼肌ROS水平明显升高,并且低氧均可降低野生型和Nrf2敲除大鼠骨骼肌ROS水平。低氧运动可上调Nrf2敲除大鼠的CAT和GSH-PX蛋白表达。苏木精和伊红(HE)染色显示,Nrf2敲除大鼠在力竭跑台运动完成后出现更严重的骨骼肌病理改变,而低氧运动可减轻骨骼肌损伤。本研究认为,Nrf2敲除导致了大鼠骨骼肌中抗氧化酶的抑制及ROS的过量累积,从而造成了骨骼肌损伤并降低了运动能力。此外,低氧可通过上调Nrf2的表达,进而激活HIF-1α及抗氧化酶活性,从而提高运动能力,并防止骨骼肌损伤。