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.     

氧化和化学应激的防御性转导通路——Nrf2/ARE

Nrf2/ARE是近年新发现的机体抵抗内外界氧化和化学等刺激的防御性转导通路．生理条件下,NF-E2相关因子2(Nrf2,NF-E2-related factor 2)在细胞质中与Keap1结合处于非活性、易降解的状态.在内外界自由基和化学物质刺激时,Keap1的构象改变或者Nrf2直接被磷酸化,导致Nrf2与Keap1解离而活化．活化的Nrf2进入细胞核,与抗氧化反应元件(ARE)结合,启动ARE下游的Ⅱ相解毒酶、抗氧化蛋白、蛋白酶体/分子伴侣等基因转录和表达以抵抗内外界的有害刺激．MAPK、PI3K/AKT、PKC等信号通路分子广泛参与了Nrf2的活化和核转位过程,但是具体何种通路被激动、何种通路发挥主导作用,取决于刺激物种类、刺激方式和细胞类型．本文就Nrf2分子结构、Nrf2活化机制、Nrf2/ARE调控的下游基因、与Nrf2相关的信号通路分子以及其在肿瘤、炎症、衰老等应用领域的最新进展进行综述．     

Quercetin protects human granulosa cells against oxidative stress via thioredoxin system

Granulosa Cells (GCs) are sensitive to excessive production of reactive oxygen species (ROS). Quercetin (QUR) is a free radical scavenger which can alleviate oxidative stress through nuclear factor (erythroid-derived 2)-like 2 (Nrf2)/antioxidant response element (ARE) pathway and thioredoxin (Trx) system. We aimed to explore the probable protective role of QUR on cultured human GCs treated with hydrogen peroxide (H₂O₂) as an inducer of oxidative stress. MTT assay was applied for evaluating the cell cytotoxicity of QUR and H₂O₂. The rate of apoptotic cells and intracellular ROS generation were determined by Annexin V-FITC/PI staining and 2′-7′-dichlorodihydro?uorescein diacetate ?uorescent probes (DCFH-DA), respectively. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis and western blot analysis were used to evaluate the gene and protein expression of Nrf2 and kelch-like ech-associated protein 1 (Keap1)1. The Nrf2 and Trx activities were measured by Enzyme-linked Immunosorbent Assay (ELISA). The results indicated that QUR pretreatment can decrease ROS production and apoptosis induced by H₂O₂. In addition, QUR increased Nrf2 gene and protein expression, as well as its nuclear translocation. Moreover, in QUR-treated group, a lower level of Keap1 protein was observed, which was not reported as significant. The results also indicated a significant correlation between the expression of Nrf2 and Keap1 in QUR-treated group. Further, QUR protected GCs from oxidative stress by increasing Trx gene expression and activity. This study suggests that QUR as a supplementary factor may protect GCs from oxidative stress in diseases related to this condition.     

Nrf2及天然产物导向的Nrf2激活剂研究进展

氧化应激能够破坏细胞内氧化还原平衡,造成系统和组织损伤,最终引起一系列疾病的产生.转录因子E2相关因子2(Nrf2),受Kelch样环氧氯丙烷相关蛋白1(Keap1)蛋白的调控,是细胞氧化应激反应中的关键因子,在氧化应激条件下,Nrf2从Keap1中分离,然后进入细胞核与抗氧化反应元件(ARE)结合,增加了Ⅱ相解毒酶的...     

Keap1/Nrf2/ARE通路相关基因在热诱导的氧化应激小鼠肝脏中的表达

为了研究热应激对小鼠肝脏抗氧化功能及Keap1 (kelch-like ECH-associated protein- 1)/Nrf2（NF-E2-related factor 2）/ARE (antioxidant response element)通路相关基因表达的影响,选用30只8周龄雄性小鼠随机分成6组,每 d连续42 ℃热处理2 h,分别在热处理0 d(对照组)、1 d、2 d、4 d、8 d和12 d时观察肝脏组织形态学和免疫组织化学分析,另取一部分肝脏组织保存于-80 ℃用于后续荧光定量PCR实验,检测肝脏抗氧化指标及Keap1/Nrf2/ARE通路相关基因的表达．结果显示：小鼠的体表温度和直肠温度在热处理后都极显著高于热处理前．组织形态学观察发现,热处理导致小鼠肝脏组织充血和肝细胞水肿．小鼠肝脏氧化应激指标 MDA (malondialdehyde)含量在热处理第2 d较对照组显著升高,GSH (glutathione)含量、GSH-PX (glutathione peroxidase)活力和总SOD (superoxide dismutase)活力在第4 d和12 d都有升高．免疫组织化学发现,与对照组和第12 d组相比,Nrf2蛋白在第1 d,2 d,4 d,8 d表达明显,其中Nrf2蛋白在第4 d表达最为显著. 荧光定量RT PCR结果表明,与对照组比较Keap1基因的表达量从热处理第1 d开始显著降低,Nrf2基因的表达量在第4 d和12 d显著升高,HO-1 (Heme oxygenase-1)基因的表达量在第1 d显著升高,NQO1 (Quinone oxidoreductase)和GCLC (Glutamate cysteine ligase catalytic)基因的表达量在第1 d和4 d显著升高．上述结果表明,热应激引起了小鼠肝脏氧化损伤, Keap1/Nrf2/ARE通路可能参与了肝脏自身缓解热应激的过程.     

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.     

Oxidative and electrophilic stresses activate Nrf2 through inhibition of ubiquitination activity of Keap1

The Keap1-Nrf2 system is the major regulatory pathway of cytoprotective gene expression against oxidative and/or electrophilic stresses. Keap1 acts as a stress sensor protein in this system. While Keap1 constitutively suppresses Nrf2 activity under unstressed conditions, oxidants or electrophiles provoke the repression of Keap1 activity, inducing the Nrf2 activation. However, the precise molecular mechanisms behind the liberation of Nrf2 from Keap1 repression in the presence of stress remain to be elucidated. We hypothesized that oxidative and electrophilic stresses induce the nuclear accumulation of Nrf2 by affecting the Keap1-mediated rapid turnover of Nrf2, since such accumulation was diminished by the protein synthesis inhibitor cycloheximide. While both the Cys273 and Cys288 residues of Keap1 are required for suppressing Nrf2 nuclear accumulation, treatment of cells with electrophiles or mutation of these cysteine residues to alanine did not affect the association of Keap1 with Nrf2 either in vivo or in vitro. Rather, these treatments impaired the Keap1-mediated proteasomal degradation of Nrf2. These results support the contention that Nrf2 protein synthesized de novo after exposure to stress accumulates in the nucleus by bypassing the Keap1 gate and that the sensory mechanism of oxidative and electrophilic stresses is closely linked to the degradation mechanism of Nrf2.