专题报告-S4氧化应激与DNA损伤信号途径

Reactive oxygen species （ROS） and reactive nitrogen species （RNS） can cause cellular damage implicated in pathogenesis of a wide range of human disorders, including cancer, autoimmune disease, diabetes, cardiovascular disease and neurodegenerative disease.     

Eupatilin Protects Gastric Epithelial Cells from Oxidative Damage and Down-Regulates Genes Responsible for the Cellular Oxidative Stress

Purpose The formulated ethanol extract (DA-9601) of Artemisia asiatica has pronounced anti-inflammatory activities and exhibits cytoprotective effects against gastrointestinal damage. Here we investigated whether eupatilin, a major component of DA-9601, has a property of antioxidant activity and protects gastric epithelial cells from H₂O₂-induced damage. Methods The protective effect of eupatilin against H₂O₂-induced damages was studied in gastric epithelial AGS cells by measuring wound healing, cell proliferation, and cell viability. Global gene expression profiling was obtained by high-density microarray. Results Hydrogen peroxide significantly delayed epithelial migration in wounded area. In contrast, eupatilin prevented the reduction of epithelial migration induced by H₂O₂. Eupatilin also ameliorated H₂O₂-induced actin disruption in AGS cells. Interestingly, treatment with eupatilin dramatically inhibited FeSO₄-induced ROS production in a dose-dependent manner. In addition, eupatilin protected cells from FeSO₄-induced F-actin disruption. With high-density microarray, we identified dozens of genes whose expressions were up-regulated in H₂O₂-treated cells. We found that eupatilin reduces the expression of such oxidative-responsible genes as HO-1, PLAUR and TNFRSF10A in H₂O₂-treated cells. Conclusion These results suggest that eupatilin acts as a novel antioxidant and may play an important role in DA-9601-mediated effective repair of the gastric mucosa. Eun-Ju Choi and Hyun-Mee Oh contributed equally to this work.     

葛根素对青光眼视网膜氧化应激损伤的保护作用

【摘要】目的研究葛根素对青光眼视网膜氧化应激损伤的保护作用。方法24只健康雄性成年家兔随机分为对照组、模型组及治疗组,每组8只。将2.5%羟丙基甲基纤维素溶液0.2mL注入家兔前房内,制作模型组及治疗组兔青光眼模型,治疗组家兔腹腔注射葛根素10mL/kg,每日1次,共21d。模型建立后第4周,测定视网膜抗氧化酶超氧化物歧化酶（SOD）、谷胱甘肽过氧化物酶（GSH-Px）和过氧化氢酶（CAT）活性,检测抗氧化物质谷胱甘肽含量及氧化还原状态和丙二醛（MDA）、一氧化氮（NO）含量。结果模型组视网膜抗氧化酶SOD、GSH-Px和CAT活性、还原型谷胱甘肽（GSH）和GSH/氧化型谷胱甘肽（GSSG）均低于对照组,GSSG、MDA和NO含量高于对照组;治疗组视网膜上述抗氧化酶活性、GSH含量、GSH/GSSG均高于模型组,但低于对照组;GSSG、MDA和NO含量均低于模型组,GSSG含量与对照组差异无统计学意义。结论葛根素可以在一定程度上增强抗氧化酶活性及维持谷胱甘肽氧化还原状态,从而减轻青光眼视网膜的氧化应激损伤。      

黄杞苷保护DNA氧化损伤的活性及其可能机制

目的 研究黄杞苷保护DNA氧化损伤的活性与可能机制。方法 采用DNA保护分析法、超氧自由基（·O₂^-）清除法、Ferrozine显色法,探讨其保护DNA氧化损伤的活性与可能机制,并通过紫外可见光谱（UV-vis spectra）考察其与Fe²⁺络合的变化。结果 在DNA保护分析法、·O₂^-自由基清除法、Ferrozine显色法中,黄杞苷在一定浓度范围内,表现出浓度依赖性。其IC₅₀值分别（60.3±9.9）,（44.5±7.6）,（159.7±19.9）μg·mL^-1。UV-vis光谱分析表明,黄杞苷与Fe²⁺混合后,在波长475 nm处出现新的峰,摩尔消光系数为102.5 L·mol^-1·cm^-1。结论 黄杞苷能较好地保护DNA,免受羟基自由基（·OH）诱导的氧化损伤。其保护作用由直接清除ROS和间接清除ROS 2个途径实现。黄杞苷直接清除ROS,可能与氢转移和电子转移有关;间接清除ROS可能通过络合Fe²⁺的方式,阻断·OH自由基生成。不过,受3-位鼠李糖基的空间位阻影响,其Fe²⁺络合能力较弱。     

Fucodiphlorethol G Purified from Ecklonia cava Suppresses Ultraviolet B Radiation-Induced Oxidative Stress and Cellular Damage

Fucodiphlorethol G (6’-[2,4-dihydroxy-6-(2,4,6-trihydroxyphenoxy)phenoxy]biphenyl-2,2’,4,4’,6-pentol) is a compound purified from Ecklonia cava, a brown alga that is widely distributed offshore of Jeju Island. This study investigated the protective effects of fucodiphlorethol G against oxidative damage-mediated apoptosis induced by ultraviolet B (UVB) irradiation. Fucodiphlorethol G attenuated the generation of 2, 2-diphenyl-1-picrylhydrazyl radicals and intracellular reactive oxygen species in response to UVB irradiation. Fucodiphlorethol G suppressed the inhibition of human keratinocyte growth by UVB irradiation. Additionally, the wavelength of light absorbed by fucodiphlorethol G was close to the UVB spectrum. Fucodiphlorethol G reduced UVB radiation-induced 8-isoprostane generation and DNA fragmentation in human keratinocytes. Moreover, fucodiphlorethol G reduced UVB radiation-induced loss of mitochondrial membrane potential, generation of apoptotic cells, and active caspase-9 expression. Taken together, fucodiphlorethol G protected human keratinocytes against UVB radiation-induced cell damage and apoptosis by absorbing UVB radiation and scavenging reactive oxygen species.     

内质网应激对Nrf2抗氧化系统调控与高脂诱导的胰岛素抵抗发病机理研究

目的：探讨内质网应激对氧化还原平衡作用及其诱导胰岛素抵抗机制信号机理。方法随机将30只C57BL／6J小鼠分为低脂饲料对照组（LFD组,n＝10）和高脂饲料模型组（HFD组,n＝20）,14周后,经葡萄糖耐量试验（IPGTT）确定胰岛素抵抗模型成功后,HFD 组随机选出10只开始进行4-PBA药物干预（HFD＋PBA 组,n ＝10）,持续1周后检测小鼠葡萄糖耐量（IPGTT）、肌肉和肝脏及相应组织线粒体中丙二醛（ MDA）含量;Western blot检测肌肉和肝脏组织中胰岛素信号、内质网应激信号和Nrf2内源性抗氧化系统相关蛋白的表达。结果4-PBA干预组胰岛素抵抗明显减轻,PTEN蛋白受到抑制从而上调胰岛素信号（ PKB Ser473）;4-PBA干预组MDA水平明显下降,Nrf2系统信号增强;高脂组Keap1蛋白明显上调,而4-PBA干预则减弱这一变化。结论内质网应激造成氧化应激并通过上调 PTEN蛋白和抑制 Nrf2抗氧化系统参与胰岛素抵抗。     

Oxidative Stress and Psychological Disorders

Oxidative stress is an imbalance between cellular production of reactive oxygen species and the counteracting antioxidant mechanisms. The brain with its high oxygen consumption and a lipid-rich environment is considered highly susceptible to oxidative stress or redox imbalances. Therefore, the fact that oxidative stress is implicated in several mental disorders including depression, anxiety disorders, schizophrenia and bipolar disorder, is not surprising. Although several elegant studies have established a link between oxidative stress and psychiatric disorders, the causal relationship between oxidative stress and psychiatric diseases is not fully determined. Another critical aspect that needs much attention and effort is our understanding of the association between cellular oxidative stress and emotional stress. This review examines some of the recent discoveries that link oxidative status with anxiety, depression, schizophrenia and bipolar disorder. A discussion of published results and questions that currently exist in the field regarding a causal relationship between oxidative and emotional stress is also provided.     

Oxidative Stress in the Hypothalamus: the Importance of Calcium Signaling and Mitochondrial ROS in Body Weight Regulation

A considerable amount of evidence shows that reactive oxygen species (ROS) in the mammalian brain are directly responsible for cell and tissue function and dysfunction. Excessive reactive oxygen species contribute to various conditions including inflammation, diabetes mellitus, neurodegenerative diseases, tumor formation, and mental disorders such as depression. Increased intracellular calcium levels have toxic roles leading to cell death. However, the exact connection between reactive oxygen production and high calcium stress is not yet fully understood. In this review, we focus on the role of reactive oxygen species and calcium stress in hypothalamic arcuate neurons controlling feeding. We revisit the role of NPY and POMC neurons in the regulation of appetite and energy homeostasis, and consider how ROS and intracellular calcium levels affect these neurons. These novel insights give a new direction to research on hypothalamic mechanisms regulating energy homeostasis and may offer novel treatment strategies for obesity and type-2 diabetes.     

五味子木脂素缓解AngⅡ诱导人脐静脉内皮细胞氧化应激损伤的研究

目的 考察五味子木脂素缓解血管紧张素Ⅱ（angiotensin Ⅱ,AngⅡ）诱导的人脐静脉内皮细胞（human umbilical veinendothelial cells,HUVEC）氧化应激损伤,并探究其内在机制。方法 HUVEC分别于一定浓度梯度的五味子乙素（schisandrin B,Sch B）、五味子丙素（schisandrin C,Sch C）共孵育24 h,利用CCK8确定药物作用浓度; Annexin V-FITC/PI双染法检测细胞凋亡变化情况; DCFH-DA和DHE探针检测胞内活性氧表达情况;采用RT-qPCR检测Bax、Bcl-2 mRNA水平,抗氧化蛋白核因子E2相关因子（Nrf-2）、血红素氧合酶-1（HO-1）和醌氧化还原酶（NQO-1）的mRNA表达情况;采用Western blotting检测Bax、Bcl-2、Nrf-2和Kelch样环氧氯丙烷相关蛋白-1（Keap-1）的表达情况;利用特异性的siRNA构建低表达Nrf-2的HUVEC,探究五味子木脂素是否靶向Nrf-2缓解Ang II诱导氧化应激损伤。结果 Sch B（50 μmol·L^-1）和Sch C（26 μmol·L^-1）作用于AngⅡ诱导的HUVEC损伤模型中,发现Sch B、Sch C可通过调控抗氧化轴Nrf-2/Keap-1,抑制细胞凋亡（P < 0.01或P < 0.001）和ROS水平（P < 0.05）,促进HO-1（P < 0.05）、NQO-1（P < 0.05）等抗氧化酶的表达,并有效缓解H₂O₂诱导的氧化应激损伤。在特异性沉默Nrf-2的HUVEC中,发现Sch B、Sch C可靶向Nrf-2激活下游抗氧化酶（NQO-1、HO-1）的表达,缓解AngⅡ诱导的氧化应激损伤。结论 Sch B、Sch C可通过调控Nrf-2/Keap-1抗氧化通路缓解AngⅡ诱导的HUVEC氧化应激损伤。     

Oxidative Stress in Smokers and Non-smokers

Oxidative stress plays an important role in the pathogenesis of some diseases such as lung cancer, chronic obstructive pulmonary disease, and atheroscleorosis. Smoking may enhance oxidative stress not only through the production of reactive oxygen radicals in smoke but also through weakening of the antioxidant defense systems. In this study, we investigated the effects of smoking on lipid peroxidation and paraoxonase activity in a healthy population. The study consisted of (n = 30) smokers and (n = 30) nonsmokers. The age of the population which is studied was 44.74 ± 10.59 yr. The levels of serum malondialdehyde (MDA) and paraoxonase (PON1) activities were measured by the modified Buege method and the Eckerson method, respectively. Student's t-test was used to analyze the data. The serum MDA levels were significantly higher (p < .05) and serum PON1 activities were significantly lower (p < .001) in smokers than in nonsmokers. Thus, increased levels of serum MDA and decreased PON1 activities may be important in determining the oxidant/antioxidant imbalance in smokers.     

Aluminium Phosphide Poisoning and Oxidative Stress

According to previous animal studies, aluminium phosphides (AlPs) may induce oxidative stress leading to generation of free radicals and alteration in antioxidant defense system. This study was conducted to evaluate the existence and degree of oxidative stress in patients with acute AlP ingestion. A total of 44 acute AlP ingested patients as well as 44 age- and sex-matched controls were included. All patients had acute poisoning symptoms with AlP at the time of presentation and had blood samples analyzed for lipid peroxidation, total antioxidant capacity and total thiol. Our findings showed that there is a significant increase in lipid peroxidation in AlP ingested group along with a reduction in total antioxidant capacity and total thiols groups. These clinical data confirm previous experimental models that showed AlP exposure might significantly augment lipoperoxidative damage with simultaneous alterations in the antioxidant defense system. Hence, our findings might justify use of antioxidants in treatment of acute AlP poisoning which needs to be clarified by additional clinical trials.     

萝卜硫素对肾小管上皮细胞氧化应激及Nrf2/HO 1信号通路的影响

摘 要 目的：探讨萝卜硫素（SFN）对肾小管上皮细胞（HK-2）的氧化应激及核转录因子E2相关因子2（Nrf2）/血红素加氧酶（HO）-1信号通路的影响。方法: 体外培养HK-2细胞,将细胞分为空白对照组、H2O2处理组、H2O2+10 μmol·K^-1 SFN组、H2O2+20 μmol·K^-1 SFN组和H2O2+40 μmol·K^-1 SFN组;测定HK-2细胞中丙二醛（MDA）、一氧化氮（NO）、谷胱甘肽（GSH）的含量及超氧化物歧化酶（SOD）的活性;RT-PCR和Western Blot法分别检测Nrf2和HO-1 mRNA及蛋白表达水平。结果: 与空白对照组相比,H2O2处理组中MDA、NO含量明显升高,GSH水平和SOD酶活性显著降低（P<0.05）;经高、中、低3种浓度的萝卜硫素处理后MDA、NO含量显著降低,GSH水平显著升高,SOD酶活性也显著升高,Nrf2和HO 1mRNA及蛋白表达明显上调（P<0.05）。结论:萝卜硫素有抗HK 2细胞氧化应激损伤的作用,其作用机制可能与激活Nrf2/HO-1信号通路有关。     

Oxidative Stress and the HIV-Infected Brain Proteome

Human immunodeficiency virus (HIV) is capable of infiltrating the brain and infecting brain cells. In the years following HIV infection, patients show signs of various levels of neurocognitive problems termed HIV-associated neurocognitive disorders (HAND). Although the introduction of highly active antiretroviral therapy (HAART) has reduced the incidence of HIV-dementia, which is the most severe form of HAND, the milder forms have become more prevalent today due to the increased life expectancy of infected individuals. Pre-HAART era markers such as HIV RNA level, CD4+ count, TNF-α, MCP-1 and M-CSF are not able to clearly distinguish mild from advanced HAND. One promising approach for new biomarker discovery is the identification and quantitation of proteins that are post-translationally modified by oxidative and nitrosative species. The occurrence of oxidative and nitrosative stress in HIV-infected brain, both through the early direct and indirect effects of viral proteins and through the later effect on mitochondrial integrity during apoptosis, is well-established. This review will focus on how the reactive species are produced in the brain after HIV infection, the specific oxidative and nitrosative species that are involved in the post-translational modification of the brain proteome, and the methods that are currently used for the detection of such modified proteins. This review also provides an overview of related research pertaining to oxidative stress-related HAND using cerebrospinal fluid and human brain tissue.     

DL-Selenomethionine Alleviates Oxidative Stress Induced by Zearalenone via Nrf2/Keap1 Signaling Pathway in IPEC-J2 Cells

Zearalenone (ZEN) is a kind of nonsteroidal mycotoxin that is considered a risk affecting the safety of human food and livestock feed that causes oxidative damages in mammalian cells. Selenomethionine (SeMet) was indicated to have antioxidant activity and received great interest in investigating the role of SeMet as a therapeutic agent in oxidation. Therefore, the aim of this study was to investigate the hormetic role of DL-SeMet in porcine intestinal epithelial J2 (IPEC-J2) cells against ZEN-induced oxidative stress injury. As a result of this experiment, 30 μg/mL of ZEN was observed with significantly statistical effects in cell viability. Following the dose-dependent manner, 20 μg/mL was chosen for the subsequent experiments. Then, further results in the current study showed that the ZENinduced oxidative stress with subsequent suppression of the expression of antioxidant stress pathway-related genes species. Moreover, SeMet reversed the oxidative damage and cell death of ZEN toxins to some extent, by a Nrf2/Keap1-ARE pathway. The finding of this experiment provided a foundation for further research on the ZEN-caused cell oxidative damage and the cure technology.     

罗汉果甜苷干预棕榈酸致胰岛B细胞氧化应激相关损伤的机制研究

目的:研究罗汉果甜苷干预棕榈酸致胰岛B细胞氧化应激相关损伤的机制。方法:以小鼠胰岛B细胞系NIT-1细胞作为细胞模型。实验分为空白对照、罗汉果甜苷对照、模型、罗汉果甜苷干预4组,使用流式细胞术测定细胞内活性氧自由基(ROS)含量和细胞凋亡率,半定量逆转录PCR测定葡萄糖转运受体(GLUT)-2和丙酮酸激酶编码基因表达水平。结果:与模型组比较,罗汉果甜苷组NIT-1细胞内ROS含量显著降低,GLUT-2和丙酮酸激酶编码基因的表达水平显著增强(P<0.05),细胞凋亡无显著改变(P>0.05)。结论:罗汉果甜苷可显著降低NIT-1细胞内ROS水平,显著上调受抑制的GLUT-2和丙酮酸激酶编码基因表达,其机制可能是通过降低细胞内ROS含量,逆转由氧化应激激活的转录因子叉头框蛋白-1(FOXO1)导致的葡萄糖代谢障碍和其他效应,产生对胰岛B细胞的保护作用。     

Oxidative Damage of Sulfur Dioxide on Various Organs of Mice: Sulfur Dioxide Is a Systemic Oxidative Damage Agent

Cu,Zn-superoxide dismutase (SOD), Se-dependent glutathione peroxidase (GSH-Px), catalase (CAT), and glutathione (GSH) play an important role in attenuating free radical-induced oxidative damage. The purpose of this research was to determine (1) whether sulfur dioxide (SO 2) increases levels of lipid peroxidation and alters intracellular redox status in multiple organs of mice, and (2) whether SO 2 is a systemic toxic agent. The effect of SO 2 on levels of thiobarbituric acid-reactive substances (TBARS) and GSH and activities of SOD, GSH-Px, and CAT were investigated in nine organs (brain, lung, heart, liver, stomach, intestine, spleen, kidney, and testis) of Kunming albino mice of both sexes. SO 2 at 20 ppm (56 mg/m 3) was administrated to the animals of SO 2 groups in an exposure chamber for 6 h/day for 7 days while control groups were exposed to filtered air in the same condition. Results show that SO 2 inhalation decreased significantly activities of SOD and GSH-Px in all organs tested in all SO 2 groups, with respect to their corresponding control groups; CAT activities in all organs tested of both sexual mice were significantly unaltered, except CAT activities in livers were significantly lowered by SO 2; SO 2 exposure decreased significantly GSH contents and significantly increased TBARS levels of all organs tested, in comparison with their respective control groups. These results lead to two conclusions: (1) SO 2 is a systemic oxidative damage agent. It results in a significant increase in the lipid peroxidation process in all organs tested of mice of both sexes, which is accompanied by changes of antioxidant status in these organs. (2) SO 2 may cause toxicological damage to multiple organs of animals, and it is suggested that the oxidative damage produced by SO 2 inhalation may influence or promote the progression or occurrence of some disease states of various organs, not only to respiratory system. Further work is required to understand the toxicological role of SO 2 on multiple or even all organs in mammals.