多种砷化物对Chang肝细胞抗氧化酶的诱导作用

目的探讨不同无机砷和有机砷化物对Chang肝细胞的毒性,以及对Ⅱ相解毒酶和抗氧化酶蛋白的诱导作用。方法采用Alamar Blue还原法测定细胞活力;Western blot免疫印迹法检测细胞内HO-1、NQO1、GST和GR的蛋白表达。结果通过Alamar Blue还原法测定细胞增殖活力,几种砷化物的毒性大小依次为As2O3〉NaAsO2〉Na2HAsO4〉DMA;不同浓度的无机砷染毒Chang肝细胞6 h后,发现随着染毒浓度的升高,HO-1和NQO1的蛋白表达水平逐渐增加;GST和GR的蛋白表达与染毒浓度无关,3个组都表达且相同;而有机砷DMA正相反,随着染毒浓度的升高,GST和GR的蛋白表达水平逐渐增加,HO-1、NQO1的蛋白表达水平与染毒浓度无关,染毒组与对照组相比无差异。结论不同砷化物对肝细胞的毒性不同;对于抗氧化酶和Ⅱ相解毒酶的蛋白表达,4种砷化物的针对性和诱导强度明显不同。     

无机砷对角质细胞红系相关因子及其下游抗氧化酶蛋白表达的影响

目的探讨无机砷暴露对皮肤角质细胞红系相关因子2(Nrf2)及其调控的下游抗氧化酶NAD(P)H醌氧化还原酶1(NQO1)和血红素单加氧酶-1(HO-1)蛋白表达的影响。方法 25μmol/L亚砷酸钠(NaAsO2)作用于人类皮肤角质细胞系HaCaT细胞株0.5、3、6、12、24 h,采用western blot法检测细胞内Nrf2、NQO1和HO-1的蛋白表达情况。结果25μmol/L NaAsO2暴露0.5、3、6、12、24 h均能够显著诱导HaCaT细胞的Nrf2蛋白表达(P<0.01),Nrf2蛋白在NaAsO2暴露3、6、12 h表达量呈现高峰,24 h则呈下降趋势,但仍显著高于对照组;NQO1的蛋白表达仅在暴露后3 h显著高于对照组(P<0.01),暴露时间延长后则显著下降,6、12、24 h的NQO1蛋白表达水平明显低于对照组(P<0.05或P<0.01);HO-1蛋白则从暴露3 h开始呈现明显的诱导表达,且随染毒时间的延长(6、12、24 h)表达持续增强,具有明显的时间-效应关系(P<0.01)。结论无机砷暴露能够诱导人类皮肤角质细胞系HaCaT细胞的Nrf2及其调控的下游抗氧化酶NQO1和HO-1的蛋白表达。     

姜黄素干预对慢性饮水砷暴露小鼠肝脏Nrf2信号通路的影响

目的观察姜黄素干预对慢性饮水砷暴露小鼠肝脏核转录因子Nrf2信号通路的影响。方法实验小鼠自由饮用不同浓度亚砷酸钠(NaAsO2,10、50、100 mg/L)6周,再分别给予姜黄素灌胃干预(200 mg/kg和600 mg/kg,每周2次),采用Western blotting和免疫组化技术分别检测肝脏Nrf2、NQO1和HO-1的蛋白表达水平及Nrf2的细胞定位。结果与单纯砷染毒组相比,姜黄素干预组的肝脏Nrf2、NQO1和HO-1蛋白表达均显著增高,同时肝脏细胞的胞浆和胞核中棕褐色阳性颗粒均显著增多,且Nrf2明显入核增多。结论姜黄素干预能诱导饮水砷暴露小鼠肝脏Nrf2蛋白活化,并进一步激活Nrf2下游信号通路。     

肺炎支原体LAMPs经PI3K/Nrf2诱导人单核细胞表达HO-1

目的 探讨肺炎支原体(Mycoplasma pneumoniae,Mp)脂质相关膜蛋白(lipid-associated membrane protein,LAMPs)对人单核细胞表达血红素氧合酶-1(heme oxygenase-1,HO-1)的影响,并探讨可能的调控机制。方法 体外培养THP-1细胞,用不同浓度的LAMPs作用后,分别采用realtime-PCR和Western blot检测HO-1 mRNA和蛋白的表达。同时采用不同浓度的放线菌素D(ActD)和放线菌酮(CHX)预处理细胞,观察其对HO-1表达的影响,以证实HO-1的表达是否通过转录和翻译水平;提取LAMPs作用前后的核蛋白,凝胶迁移率实验检测Nrf2的核转位、Western blot检测Akt的磷酸化情况;同时采用PI3K抑制剂LY294002处理细胞,观察其对LAMPs诱导Nrf2核转位及HO-1表达的影响;最后采用 siRNA干扰Nrf2表达,以证实Nrf2是否参与调控HO-1表达。结果 (1)0~5 μg/mL LAMPs能以剂量依赖性方式诱导THP-1表达HO-1 mRNA和蛋白;(2)5 μg/mL LAMPs能诱导THP-1细胞Akt磷酸化,并能增强其DNA结合活性;PI3K抑制剂LY294002处理后,可明显抑制LAMPs诱导的HO-1表达和Nrf2核转位;(3)干扰Nrf2表达后,可明显下调LAMPs诱导THP-1细胞表达HO-1。结论 LAMPs可诱导THP-1细胞表达HO-1,其机制可能与PI3K/Nrf2通路有关。     

无机砷对红系相关因子-2及其抑制因子mRNA表达的影响

目的观察亚砷酸钠（NaAsO2,sodium arsenite）对Chang肝细胞株核转录因子红系相关因子（nuclear factor ery-throid 2-related factor 2,Nrf2）及其胞浆抑制因子Keap1（Kelch-like ECH-associated protein 1）的mRNA表达水平的影响。方法分别以不同浓度NaAsO2（0、50、200、400μmol/L）暴露人类Chang肝细胞株12 h,采用AlamarBlue法测定细胞增殖活性,采用RT-PCR法测定Nrf2和Keap1的mRNA表达水平。结果 50μmol/L NaAsO2暴露组的细胞增殖活性与对照组相比差异无统计学意义（P〉0.05）,而200μmol/L和400μmol/L NaAsO2暴露组的细胞增殖活性均显著低于对照组（P〈0.05）;50、200、400μmol/L的NaAsO2暴露12 h,Nrf2和Keap1的mRNA表达水平与对照组比较均显著下降（P〈0.05）,且呈剂量-反应关系。结论高浓度无机砷暴露能抑制Chang肝细胞株Nrf2和Keap1的mRNA表达水平,并可能与无机砷造成机体的高氧化应激水平有关。     

Nrf2/ARE/HO-1通路:阿尔茨海默病神经保护的潜在靶点

<正>近年研究表明,调节Nrf2/ARE/HO-1通路在各种神经系统疾病中具有神经保护作用。通过遗传学或药理学手段上调Nrf2或HO-1的表达,在动物模型和细胞体系中具有神经保护作用。因此有必要就Nrf2/ARE/HO-1在阿尔茨海默病(AD)中作用的研究进行梳理。本文对Nrf2/ARE/HO-1在AD中作用的研究历史进行了梳理。调节Nrf2/ARE/HO-1通路的激活     

香豆素通过调节Keap1/Nrf2/ARE信号通路减轻脑缺血-再灌注大鼠神经损伤

目的 探讨香豆素通过调节Kelch样环氧氯丙烷相关蛋白(Keap)1/核因子E2相关因子(Nrf)2/抗氧化反应元件(ARE)信号通路减轻脑缺血-再灌注大鼠神经损伤。方法 大鼠随机分为假手术(Sham)组、脑缺血-再灌注损伤模型(CIRI)组,低(2.5 mg/kg)、中(5 mg/kg)、高剂量(10 mg/kg)香豆素组,Keap1/Nrf2/ARE信号通路抑制剂组(ML385)组及ML385+高剂量香豆素组,脑缺血2 h、再灌注24 h后采用Longa5分制法评估大鼠神经行为评分;2,3,5氯化三苯基四氮唑(TTC)染色测量大鼠脑梗死体积;苏木素-伊红(HE)染色观察大鼠脑皮质组织病理学变化;Western印迹检测大鼠脑皮质组织中Keap1/Nrf2/ARE信号通路相关因子[Keap1、Nrf2、血红素氧合酶(HO)-1、醌氧化还原酶(NQO)1]蛋白水平。结果 与Sham组比较,CIRI组神经行为评分、脑梗死体积,脑皮质组织中胞核Nrf2/Nrf2及HO-1、NQO1蛋白增加,Keap1蛋白减少,差异有统计学意义(P<0.05),且神经细胞可见明显病理学改变。与CIRI组...     

富马酸二甲酯对抗β淀粉样蛋白诱导的大鼠星形胶质细胞的氧化应激反应

目的研究富马酸二甲酯(dimethylfumarate,DMF)通过调节核转录因子-E2相关因子2(Nrf2)对β淀粉样蛋白(Aβ)诱导的氧化应激的防御作用和作用机制。方法将大鼠星形胶质细胞分为Aβ组、DMF组、Nrf2组和Nrf2+DMF组。采用RT-PCR检测各组细胞Nrf2、醌氧化还原酶1(Nqo1)、血红素氧化酶1(HO-1)和Kelch样环氧氯丙烷相关蛋白1(Keap1)mRNA表达水平,采用Western blot检测组蛋白去乙酰酶(HDAC)表达水平。结果 Nrf2组较Aβ组、Nrf2+DMF组较DMF组Nrf2、Nqo1和HO-1 mRNA表达显著降低(P0.05)。DMF组较Aβ组Nrf2、Nqo1和HO-1mRNA表达显著升高,Keap1 mRNA表达显著降低(P0.05)。Nrf2+DMF组较Nrf2组Keap1mRNA表达显著降低(P0.05)。DMF组较Aβ组HDAC表达显著降低(6.41±0.43 vs 9.01±1.54,P0.05),Nrf2+DMF组较Nrf2组HDAC表达显著降低(6.72±0.30 vs 8.76±0.74,P0.05)。结论 DMF可能通过抑制HDAC表达,提高Nrf2水平,从而减弱Aβ诱导的大鼠星形胶质细胞的氧化应激反应。     

蓝莓对急性肝损伤大鼠抗氧化能力的影响

目的探讨蓝莓对四氯化碳(CCl4)所致急性肝损伤大鼠肝组织核转录相关因子2(Nrf2)和Ⅰ型血红素氧化酶(HO-1)mRNA表达的影响。方法 SD大鼠48只随机分为正常对照组10只、蓝莓汁低、高剂量各组10只(1g/100g、2g/100g)、齐墩果酸(7.5mg/100g)组及模型组各9只,用CCl4致大鼠急性肝损伤模型。采用TRIzol提取肝组织总RNA,实时荧光定量PCR技术测定大鼠肝组织Nrf2及HO-1 mRNA的表达。结果与正常对照组比较,模型组肝组织Nrf2、HO-1 mRNA表达有所增加,差异无统计学意义。与模型组比较,蓝莓汁低、高剂量组及齐墩果酸组肝组织Nrf2、HO-1 mRNA表达明显增高,P<0.05。结论蓝莓可激活大鼠肝脏Nrf2增加HO-1的表达,提高机体的抗氧化应激能力,这可能是蓝莓预防CCl所致大鼠急性肝损伤的机制之一。     

核因子E2相关因子2/血红素加氧酶-1(Nrf2/HO-1)信号通路在酒精性肝病中的作用

酒精性肝病(ALD)在我国的发病率逐年上升，国民的疾病负担日益增加。肝细胞的氧化应激反应是ALD的重要致病机制。核因子E2相关因子2/血红素加氧酶-1(Nrf2/HO-1)信号通路是人体重要的内源性抗氧化应激通路，在氧化应激作用下,Nrf2被激活并发挥其转录活性诱导HO-1高表达。HO-1是体内重要的氧化应激反应蛋白，与其血红素酶解产物(胆红素、CO、铁)共同发挥着抗炎、抗氧化及调控细胞凋亡的作用。本文将对近年来Nrf2/HO-1信号通路在ALD中的研究进展进行综述，力求为ALD的发生发展寻找理论依据及治疗切入点。     

Induction of HO-1 and redox signaling in endothelial cells by advanced glycation end products: A role for Nrf2 in vascular protection in diabetes

Background and aims

Hyperglycemia and diabetes are associated with increased formation of advanced glycation end products and enhanced oxidative stress, leading to the progression of diabetic vascular disease. We have investigated the mechanisms by which AGE-modified bovine albumin (AGE-BSA) induces reactive oxygen species (ROS) generation, leading to nuclear factor-erythroid 2-related factor (Nrf2) dependent induction of the antioxidant genes heme oxygenase-1 (HO-1) and NADPH:quinone oxidoreductase 1 (NQO1) in bovine aortic endothelial cells.

Methods and results

AGE-BSA (100 μg ml⁻¹, 0-24 h), but not native BSA, elicited time-dependent increases in ROS generation, Nrf2 nuclear translocation and enhanced mRNA and protein expression of HO-1 and NQO1, but not glutathione peroxidase-1. Inhibition of ROS production with the superoxide scavenger Tiron or inhibitors of flavoproteins (diphenylene iodonium) and NADPH oxidase (apocynin), but not eNOS (l-NAME) or mitochondria complex I (rotenone) abrogated HO-1 induction by AGE-BSA. Although AGE-BSA induced rapid phosphorylation of JNK and Akt, only inhibition of JNK abrogated HO-1 expression, implicating the involvement of the JNK signaling pathway in AGEs activation of Nrf2/ARE-linked antioxidant gene expression.

Conclusion

Our findings establish that AGEs activate redox sensitive Nrf2-dependent antioxidant gene expression in bovine aortic endothelial cells, providing an adaptive endogenous defense against oxidative stress in diabetes.     

Liver expression of Nrf2-related genes in different liver diseases

BACKGROUND: The KEAP1-Nrf2 antioxidant signaling pathway is important in protecting liver from various insults. However,little is known about the expression of Nrf2-related genes in human liver in different diseases.METHODS: This study utilized normal donor liver tissues(n=35), samples from patients with hepatocellular carcinoma(HCC, n=24), HBV-related cirrhosis(n=27), alcoholic cirrhosis(n=5) and end-stage liver disease(n=13). All of the liver tissues were from the Oriental Liver Transplant Center, Beijing,China. The expressions of Nrf2 and Nrf2-related genes, including its negative regulator Kelch-like ECH-associated protein 1(KEAP1), its targeted gene NAD(P)H-quinone oxidoreductase 1(NQO1), glutamate-cysteine ligase catalytic subunit(GCLC) and modified subunit(GCLM), heme oxygenase 1(HO-1) and peroxiredoxin-1(PRDX1) were evaluated. RESULTS: The expression of Nrf2 was decreased in HCC, increased in alcoholic cirrhosis and end-stage liver disease. The expression of KEAP1 was increased in all of the liver samples.The most notable finding was the increased expression of NQO1 in HCC(18-fold), alcoholic cirrhosis(6-fold), endstage liver disease(5-fold) and HBV-related cirrhosis(3-fold).Peri-HCC also had 4-fold higher NQO1 m RNA as compared to the normal livers. GCLC m RNA levels were lower only in HCC, as compared to the normal livers and peri-HCC tissues.GCLM m RNA levels were higher in HBV-related cirrhosis and end-stage liver disease. HO-1 m RNA levels were increased in all liver tissues except for HCC. Peri-HCC had higher PRDX1 m RNA levels compared with HCC and normal livers.CONCLUSION: Nrf2 and Nrf2-related genes are aberrantly expressed in the liver in different diseases and the increase of NQO1 was the most notable finding, especially in HCC.     

Age-related changes of Nrf2 and phosphorylated GSK-3β in a mouse model of accelerated aging (SAMP8)

SAMP8 mice show spontaneously accelerated aging and a short life span with systemic accumulation of oxidative stress. Nrf2 translocates into the nucleus upon oxidative stress and induces the expression of detoxifying and antioxidant enzymes. Recently, several studies reported that Nrf2 is associated with aging and various diseases. In the present study, we investigated the levels of Nrf2 nuclear translocation and phosphorylation of Akt and GSK-3β in livers of SAMP8 and normal aging SAMR1 mice. The protein level of Nrf2 in the nucleus of the liver was significantly decreased in SAMP8 at 10 months old compared with that in age-matched SAMR1. The protein level of Keap1, which anchors Nrf2 in the cytoplasm, did not differ between SAMP8 and SAMR1. In addition, the mRNA expression of Nrf2 in the liver of SAMP8 was significantly lower than that of SAMR1. Moreover, mRNA levels of detoxification and antioxidant enzymes, GSTa1 and NQO1, were significantly decreased in SAMP8 compared with SAMR1. These results indicate that a higher level of oxidative stress in SAMP8 might be caused by a lower level of Nrf2. Furthermore, the phosphorylation of Akt and GSK-3β was significantly decreased in the liver of SAMP8 at 10 months old. Recent studies have suggested that the Akt/GSK-3β signaling pathway is involved in the nuclear translocation of Nrf2. Therefore, it is suggested that the reduction of the translocation of Nrf2 into the nucleus might be induced by a decrease of GSK-3β phosphorylation, resulting in an increase of oxidative stress in SAMP8 mice.