叔丁基对苯二酚对糖尿病小鼠肾脏氧化应激损伤及Nrf2蛋白表达的影响

目的研究叔丁基对苯二酚(tBHQ)对早期糖尿病小鼠肾脏Nrf2-ARE信号通路表达及氧化应激的影响,探讨tBHQ保护肾脏的部分机制。方法CD-1♂小鼠随机分为3组,对照组(C组)、糖尿病组(DM组)、tBHQ干预组(DM+tBHQ组)。腹腔注射STZ诱发糖尿病小鼠模型,DM+tBHQ组在成模3d后给予添加了1%tBHQ(W/W)的饲料喂养,4wk后检测3组动物的血糖、肾功能、24h尿白蛋白定量、血清及肾皮质丙二醛(MDA)含量。免疫组化和Westernblot检测Nrf2,HO-1蛋白在肾组织的定位和表达水平。结果①DM组小鼠血尿素氮、24h尿白蛋白定量、肾重/体重,血清及肾皮质MDA含量较C组升高。tBHQ干预组上述指标均降低。②与DM组相比,tBHQ干预组肾皮质总蛋白Nrf2、HO-1及核蛋白Nrf2表达水平明显增高;免疫组织化学检测显示Nrf2于肾小管及肾小球的胞质及胞核内均有表达;HO-1主要表达于肾小管上皮细胞胞质中。结论tBHQ减轻了早期糖尿病小鼠肾脏的氧化应激损伤,其作用机制可能是部分通过激活Nrf2-ARE信号通路进而增强抗氧化蛋白HO-1的表达而实现的。     

氧化苦参碱协同增加环磷酰胺诱导小鼠肝毒性的研究

环磷酰胺(cyclophosphamide,CPA)是治疗多种肿瘤的一线化疗药,但过量应用可引起肝损伤。本文旨在探讨氧化苦参碱(oxymatrine,OMT)与CPA的联合给药是否会加剧其肝毒性,并初步阐明其机制。小鼠单独给药OMT(100 mg·kg^-1)不同时间后,检测肝组织Cyp2b10 mRNA和CYP2B10蛋白表达。小鼠灌胃(intragastric adminis‐tration,ig)给药不同剂量OMT,同时隔天腹腔注射(intraperitoneal injection,ip)给予CPA(200 mg·kg^-1),10天后,检测血清谷丙/谷草转氨酶(alanine/aspartate aminotransferase,ALT/AST)活力,记录小鼠死亡率,检测肝组织Cyp2b10mRNA水平,并分析ALT/AST活力、死亡率和Cyp2b10 mRNA水平间的相关性。本文中动物福利和实验过程均遵循上海中医药大学实验动物伦理委员会的规定。结果发现,OMT单独给药可以显著提高小鼠肝组织中Cyp2b10mRNA和CYP2B10蛋白表...     

黄芩素激活核转录因子Nrf2拮抗肝毒性的研究

目的研究黄芩素(Baicalein,BAI)对核转录因子Nuclear factor erythroid 2-related factor 2(Nrf2)的转录激活,以及对四氯化碳(carbon tetrachloride,CCl4)、乙醇(ethanol)和对乙酰氨基酚(acetaminophen,APAP)诱导的人正常肝L-02细胞毒性的拮抗作用。方法在肝L-02细胞中,采用瞬时转染报告基因实验检测不同浓度黄芩素对Nrf2转录激活的影响。在L-02细胞上分别用APAP(10 mmol·L-1)、CCl4(10 mmol·L-1)、Ethanol(100 mmol·L-1)诱导肝细胞毒性。黄芩素1、10、25、50、100μmol·L-1分别与细胞预孵15 min后,加入上述肝毒性物质,48 h后采用MTT法检测细胞存活率。结果与对照组比较,黄芩素(25、50μmol·L-1)能明显提高Nrf2的转录激活(P<0.01,P<0.05)。与对照组比较,3种肝毒性物质均能明显降低细胞存活率(P<0.01),而黄芩素能剂量依赖性地提高给予APAP、CCl4和Ethanol后降低的L-02细胞存活率(P<0.01)。结论黄芩素可以诱导重要的抗氧化核转录因子Nrf2的转录激活,这可能是黄芩素拮抗外源性肝毒性物质APAP、CCl4和Ethanol诱导的L-02肝细胞毒性的机制之一。     

Nrf2-ARE信号通路功能与临床疾病及相关药物的研究新进展

Nrf2（NF-E2-related factor 2）核因子E2相关因子是一种机体抵抗内界和外界氧化或化学等刺激的中枢调节者。Nrf2-ARE则是近年来新发现的细胞氧化应激反应的关键传导通路,当其在体内被有毒有害物质激活后转位进入细胞核能与抗氧化反应元件（antioxidant response element,ARE）结合形成Nrf2-ARE信号通路,从而调控下游抗氧化蛋白、氧化酶和Ⅱ相解毒酶等。研究发现该通路在抗衰老、抗肿瘤、抗炎症、神经损伤、眼科等多方面均有重要作用。以Nrf2为靶点的药物有望用于肿瘤、糖尿病、神经退行性疾病等。本文综述了Nrf2-ARE信号通路功能及以其为靶点的药物研究的进展。     

Nrf2活化正向调控紫草素诱导的HL-60细胞分化

目的探讨核因子E2相关因子2(Nrf2)在紫草素诱导HL-60细胞分化中的作用。方法取对数生长期HL-60细胞,分别采用不同浓度紫草素、Nrf2激动剂叔丁基对苯二酚(t BHQ)、Nrf2抑制剂鸦胆子苦醇作用。通过Hoechst 33258染色、MTT法观察HL-60细胞形态和增殖情况,NBT试验检测HL-60细胞还原能力,流式细胞术检测HL-60细胞分化率。ELISA法测定HL-60细胞核内Nrf2含量,RT-PCR或real-time PCR法检测HL-60细胞Nrf2、CD11b、CD14 m RNA表达水平及Nrf2下游基因的表达水平。结果经紫草素(50~200μg·L-1)作用,HL-60细胞形态、还原能力均趋向成熟,与空白对照相比细胞抑制率、分化率、Nrf2含量及其下游基因GCLC、NQO1、GCLM m RNA的表达量均升高(P<0.05或P<0.01),且随紫草素浓度增高有上升的趋势。紫草素与t BHQ联合作用,HL-60细胞CD11b、CD14、NQO1、GCLM m RNA表达量高于紫草素组(P<0.05或P<0.01);紫草素与鸦胆子苦醇联合作用,HL-60细胞CD11b、CD14、GCLM m RNA表达量低于紫草素组(P<0.05或P<0.01)。结论 Nrf2活化正向调控紫草素诱导的HL-60细胞分化。     

飞燕草素葡萄糖苷通过上调SOX2OT减轻高糖诱导的心肌细胞损伤

目的:探讨飞燕草素葡萄糖苷(DPg)对高糖(HG)诱导的心肌细胞H9C2损伤的影响及可能机制.方法:体外培养H9C2细胞,不同剂量(1,10,100μmol·L-1)的DPg作用于33.3 mmol·L-1葡萄糖诱导的H9C2细胞24 h,CCK-8法检测细胞增殖,流式细胞术检测细胞凋亡,Western Blot检测细...     

丹参酮Ⅰ基于Akt-Nrf2抗氧化通路减轻多柔比星诱导的心脏毒性

多柔比星(doxorubicin, DOX)是一种蒽环类抗生素,广泛用于治疗肿瘤,但其长期使用会产生严重不良反应,尤其是急性和慢性心脏毒性。本研究探索了丹参酮Ⅰ(tanshinone Ⅰ, Tan Ⅰ)对DOX诱导的急性心脏毒性的保护作用及其潜在的分子机制。动物福利和实验过程均遵循北京中医药大学实验动物伦理委员会的规定。采用小鼠尾静脉注射DOX (6 mg·kg-1,每周2次)和DOX刺激H9C2心肌细胞方法制备在体和离体急性心脏毒性模型。在体实验于尾静脉注射前5天,灌胃给药Tan Ⅰ (10 mg·kg-1),直至实验结束,检测Tan Ⅰ对小鼠心功能、心肌组织形态学、血清学指标的影响。离体实验进一步研究Tan Ⅰ抗氧化应激的具体机制。应用免疫荧光技术检测核因子E2相关因子2 (nuclear erythroid factor 2-related factor 2, Nrf2)的表达量和入核情况,并用Western blot方法检测氧化应激相关蛋白蛋白激酶B (protein kinase B, Akt)、Nrf2、血红素加氧酶1 (heme oxygenase-1, HO-1)、NA...     

大承气汤调控Nrf2/TGF-β1/ERK通路来减轻脂多糖诱导的急性肺损伤

目的：研究大承气汤（Dachengqi Decoction,DCQD）减轻脂多糖诱导的急性肺损伤模型的作用及机制。方法：选择无特定病原体（specific pathogen free,SPF）级8周龄SD雄性大鼠,采用脂多糖腹腔注射诱导急性肺损伤模型。将48只大鼠随机分为空白组（blank group）、模型组（model group）、大承气汤低浓度组（L-DCQD group,2.5 g·kg^-1）、中浓度组（M-DCQD group,5 g·kg^-1）、高浓度组（H-DCQD group,10 g·kg^-1）和大承气汤高浓度合Nrf2抑制剂组（H-DCQD＋ML385 group）,每组8只。采用脂多糖10 ml·kg^-1腹腔注射造模,每天灌胃给药1次,连用5 d,并于造模后2 h给予不同浓度的大承气汤灌胃,6 h后收集标本。ELISA检测肺泡灌洗液中肿瘤坏死因子-α（TNF-α）、白介素-1β（IL-1β）、髓过氧化物酶（MPO）、丙二醛（MDA）、转化生长因子-β（TGF-β1）表达量,HE染色观察各组大鼠肺脏的病理变化,Western blot检测肺组织核因子E2相关因子2（Nrf2）、p-Nrf2、血红素氧合酶1（HO-1）、细胞外调节蛋白激酶（ERK）、p-ERK的表达,免疫荧光检测Nrf2核转位情况。结果：与blank组相比,model组出现严重的炎症反应,肺泡损伤,肺水肿形成,大量炎症细胞浸润;相关促炎因子TNF-α、IL-1β及氧化指标MPO、MDA明显升高（P<0.05）;大鼠肺组织p-Nrf2及HO-1、p-ERK蛋白表达均上调（P<0.05）。与model组相比,DCQD组炎症反应减轻,肺组织损伤改善,TNF-α、IL-1β及氧化指标MPO、MDA下降,p-Nrf2及HO-1蛋白表达均上调（P<0.05）,p-ERK蛋白表达下调（P<0.05）,且DCQD高浓度组变化趋势更明显（P<0.05）。结论：大承气汤可减轻脂多糖引起的急性肺损伤,其机制与激活Nrf2/TGF-β1/ERK信号通路及抑制炎症反应、氧化应激、上皮间质转化有关。     

圣草酚调控MAPK和Nrf2/HO-1信号通路缓解非酒精性脂肪肝的作用及机制

目的 研究圣草酚缓解非酒精性脂肪肝（NAFLD）的作用及可能机制。方法 将16只C57BL/6J小鼠按体重随机分为对照组、NAFLD模型组和圣草酚低、高剂量组（50、100 mg/kg）,每组4只。除对照组外,其余各组均使用高脂饲料诱导NAFLD模型。在预处理4周后,采取腹腔注射方式（0.01 mL/g）进行给药,每日1次,连续6周。测定小鼠体重、肝脏质量,观察小鼠肝组织病理损伤情况,测定小鼠血清中天冬氨酸转氨酶（AST）、丙氨酸转氨酶（ALT）和甘油三酯（TG）水平及肝组织中核转录因子红系2相关因子2(Nrf2)、血红素加氧酶1(HO-1)蛋白表达。使用0.5 mmol/L油酸诱导HepG2细胞建立体外NAFLD模型,实验设置正常对照组、NAFLD模型组和圣草酚低、中、高浓度组（50、100、150μmol/L）;给药组在诱导模型的同时加入圣草酚,培养24 h。观察细胞中脂质沉积情况,检测细胞中TG、丙二醛（MDA）、活性氧（ROS）水平以及丝裂原活化蛋白激酶（MAPK）信号通路相关蛋白[细胞外信号调节激酶（ERK）、c-Jun氨基末端激酶（JNK）]磷酸化水平和Nrf2、HO-1蛋...     

Protective effect of lipoic acid on micronuclei induction by cyclophosphamide

The present study investigated the protective efficacy of DL-α-lipoic acid on the cyclophosphamide (CP)-induced clastogenicity using the in vivo micronucleus assay. Male Wistar rats of 140 ± 20 g were categorized into eight groups. Five groups were administered CP (40 mg/kg body weight, intraperitonealy) to induce genotoxicity; four of these groups received a single intraperitoneal injection of lipoic acid at a dose of either 100 or 200 mg/kg body weight, and either 30 or 60 min prior to CP administration. A vehicle-treated control group and lipoic acid control groups were also included. The number of micronucleated polychromatic erythrocytes (MNPCEs) was determined at 24 h after CP administration. In rats injected with CP, the frequency of MNPCEs in bone marrow and peripheral blood was increased significantly in comparison with the controls, and in rats treated with lipoic acid and CP, the number of MNPCEs was decreased significantly in comparison to those given CP alone. The chemoprotective effect was found to be stronger after the administration of lipoic acid at a dose of 200 mg/kg body weight than 100 mg/kg body weight dosage, indicating the dose-dependent protective effect of lipoic acid. However, the protection by lipoic acid was not dependent on the time intervals between lipoic acid and CP administration. Our results illustrate the protective effect of lipoic acid on the in vivo clastogenicity induced by CP.     

Nrf2/ARE signaling protects against oxaliplatin-induced hepatotoxicity in mice

Nuclear factor erythroid 2-related factor 2 (Nrf2) controls the expression of a wide array of antioxidant response element (ARE)-driven genes, which are involved in stress response and metabolism regulation. The role of Nrf2/ARE signaling in resistances of cancer cells to radiotherapy and chemotherapy has been widely accepted. However, much less is known about the relevance of Nrf2 to chemotherapy-associated toxicities, such as hepatotoxicity. In the present study, nine chemotherapeutic agents were firstly tested in embryonic fibroblasts (MEFs) and hepatocytes isolated from Nrf2 deficient or wild-type mice. The results indicate that the cytotoxicity of oxaliplatin in hepatocytes was significantly higher than that in MEFs and enhanced by Nrf2 deficiency. Furthermore, oxaliplatin treatment caused more pronounced steatosis and severer liver injury in Nrf2^–/–mice compared with wild-type counterparts, as evidenced by dramatically elevated serum transaminase and bilirubin, increased accumulation of fat, inflammatory infiltration and blood congestion. The increased hepatotoxicity in Nrf2 deficient mice was possibly caused by decreased expression of antioxidant genes and glutathione depletion. Our results demonstrated that oxaliplatin-inducedhepatotoxicity was significantly impacted by Nrf2 status, therefore Nrf2 could potentially serve as a biomarker to predict or a target to prevent hepatotoxicity of oxaliplatin.     

Hydrogen gas alleviates blood-brain barrier impairment and cognitive dysfunction of septic mice in an Nrf2-dependent pathway

Sepsis-associated encephalopathy (SAE) is a cognitive impairment caused by sepsis and is related to increased morbidity and mortality. Damage to the blood–brain barrier (BBB) has been proved to be one of the important causes of SAE. Molecular hydrogen (H₂) is a promising method for the treatment of SAE, yet the underlying mechanism is not clear. This study was designed to demonstrate whether H₂ can alleviate SAE by protecting the BBB, and whether it is protected by Nuclear factor erythroid-2-related factor 2 (Nrf2) and its downstream signaling pathways. Either a sham or a cecal ligation and puncture (CLP) procedure was applied to female wild-type (WT) and Nrf2-knock-out (Nrf2^-/-) C57BL/6J mice. H₂ (2%) was given for 60 min starting at 1 h and 6 h after the sham or CLP procedure. In addition, bEnd.3 cells cultured with medium which contained LPS, Saline, DMSO or ML385 (a Nrf2 inhibitor) were also used in the research. The 7-day survival rates were recorded. The Morris water maze was used to determine cognitive function. Pro-inflammatory and anti-inflammatory cytokines [tumor necrosis factor-alpha (TNF-α), interleukin 6 (IL-6), HMGB1, and IL-10), antioxidant enzymes, and oxidation products [superoxide dismutase (SOD), chloramphenicol acetyltransferase (CAT), malondialdehyde (MDA), and (8-iso-PGF2α)] were determined by enzyme-linked immunosorbent assay (ELISA). Brain water content, Dextran tracer, and Evans blue extravasation were used to detect the damage of the BBB. Western blot analysis was used to detect β-catenin, phosphorylated β-catenin, adhesion-linked protein VE-cadherin, and associated tight junction protein ZO-1. We found that H₂ can improve survival in septic mice, decrease escape latency and platform crossing times, decrease pro-inflammatory cytokines and oxidative product levels in the mouse cortex, and increase the expression of anti-inflammatory factors in WT, but not Nrf2^-/-, mice. Moreover, H₂ can also decrease brain water content, extravascular dextran, extravascular Evans blue dye, and β-catenin level, and increase ZO-1 and VE-cadherin expressions in WT mice, but not in Nrf2^-/- mice. Our result shows that H₂ can protect the BBB by decreasing its permeability, thereby reducing SAE and improving cognitive function, which is mediated through Nrf2 and its downstream signaling pathways.     

Amelioration of cyclophosphamide-induced hepatotoxicity by the root extract of Decalepis hamiltonii in mice

Hepatoprotective potential of the aqueous extract of the roots of Decalepis hamiltonii (DHA) against cyclophosphamide (CP)-induced oxidative stress has been investigated in mice. Administration of CP (25 mg/kg b.w., i.p) for 10 days induced hepatic damage as indicated by the serum marker enzymes aspartate and alanine transaminases (AST, ALT), alkaline phosphatase (ALP) and lactate dehydrogenase (LDH). Parallel to these changes CP induced oxidative stress in the liver as evident from the increased lipid peroxidation (LPO), reactive oxygen species (ROS), depletion of glutathione (GSH), and reduced activities of the antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), and glutathione-S-transferase (GST). Treatment with DHA (50 and 100 mg/kg b.w., po) mitigated the CP-induced oxidative stress. Moreover, expression of genes for the antioxidant enzymes, were down-regulated by CP treatment which was reversed by DHA. Our study shows the DHA protected the liver from toxicity induced by CP and therefore, it could be serve as a safe medicinal supplement during cyclophosphamide chemotherapy.