The role of ANXA5 in DBP-induced oxidative stress through ERK/Nrf2 pathway

Di-N-butylphthalate (DBP) have given rise to more and more attention due to its unique endocrine toxicity to male reproductive system. Our previous studies have demonstrated antioxidative Nrf2 (nuclear factor erythroid related factor 2) pathway play a vital role in DBP induced oxidative stress injury. ANXA5 (annexin A5), which is highly expressed in testicular Leydig and Sertoli cells, was found upregulated after DBP stimulation. Mouse Leydig and Sertoli cells were exposed to different concentration of DBP for 24 h to examine the ROS (Reactive oxygen species), MDA (Malondialdehyde), SOD (superoxide dismutase) level and ANXA5, Nrf2, NQO1 (NAD(P)H-quinone oxidoreductase 1), HO-1 (heme oxygenase 1) and ERK/P-ERK protein expression by DHE (Dihydroethidium) staining, ELISA (enzyme-linked immunosorbent assay) and Western blot respectively. Firstly, the oxidative stress injury induced by DBP was re-validated. Then, we confirmed the change of Nrf2 pathway and ANXA5 level after DBP exposure to testicular cells. Additionally, overexpressed ANXA5 could activate Nrf2/HO-1/NQO1 antioxidant pathway and significantly attenuate DBP-induced oxidative stress. Ultimately, we demonstrated ANXA5 could increase ERK phosphorylated level and the activated role of ANXA5 on ERK/Nrf2 pathway could be reversed by ERK inhibitor. Overall, this study illuminated that ANXA5 could defend testicle Leydig and Sertoli cells against DBP-induced oxidative stress injury through ERK/Nrf2 pathway.     

Combination of chlorogenic acid and salvianolic acid B protects against polychlorinated biphenyls-induced oxidative stress through Nrf2

Caffeic acid derivatives (CADs) are well-known phytochemicals with multiple physiological and pharmacological activities. This study aimed to investigate the combined protective effects of CADs on PCB126-induced liver damages and oxidative stress in mice. Here, we used chemiluminescence and chose chlorogenic acid (CGA), salvianolic acid B (Sal B) as the best antioxidants. Then, mice were intragastrically administered with 60 mg/kg/d CGA, Sal B, and CGA plus Sal B (1:1) for 3 weeks before exposing to 0.05 mg/kg/d PCB126 for 2 weeks. We found that pretreatment with CGA, Sal B, and CGA plus Sal B effectively attenuated liver injury and cytotoxicity caused by PCB126, but improved the expressions of superoxide dismutase (SOD), glutathione reduced (GSH), heme oxygenase-1 (HO-1) and nuclear factor E2-related factor 2 (Nrf2), CGA plus Sal B especially, was found to have the best effects that indicated a synergetic protective effect. Taken together, as the Nrf2 regulates the cyto-protective response by up-regulating the expression of antioxidant genes, we suggested that CGA plus Sal B had a combined protection on PCB126-induced tissue damages and that the Nrf2 signaling might be involved.     

Sulforaphane protects kidneys against ischemia-reperfusion injury through induction of the Nrf2-dependent phase 2 enzyme

Reactive oxygen species are important mediators that exert a toxic effect during ischemia-reperfusion injury of various organs. Sulforaphane, which is a naturally occurring isothiocyanate that is present in cruciferous vegetables such as broccoli, is known to be an indirect antioxidant that acts by inducing Nrf2-dependent phase 2 enzymes. Phase 2 enzymes such as heme oxygenase-1, NAD(P)H: quinone oxidoreductase 1, glutathione reductase, and glutathione peroxidase participate in adaptive and protective responses to oxidative stress and various inflammatory stimuli. Therefore, we evaluated the preactivation of Nrf2 by sulforaphane to determine if it could inhibit ischemia-reperfusion-induced kidney damage. Treatment of HK2 renal tubular epithelial cells with sulforaphane effectively protected cells against cytotoxicity induced by hypoxia-reoxygenation, and sulforaphane dramatically induced phase 2 enzymes by decreasing the Keap1 protein levels and increasing Nrf2 nuclear translocation. Additionally, a second set of experiments using a renal ischemia-reperfusion model produced results that were essentially the same as those observed when HK2 cells were used; namely, that sulforaphane induced Nrf2-dependent phase 2 enzymes and thereby improved ischemia-reperfusion-induced changes in the lipid hydroperoxides, glutathione, creatinine clearance, kidney weight, and histologic abnormalities. Collectively, these results suggest that sulforaphane can be used as an effective adjunct for the prevention of renal oxidative insults during ischemia-reperfusion injury.     

Protective role of sitagliptin against oxidative stress in a kainic acid-induced status epilepticus in rats models via Nrf2/HO-1 pathway

Aim: Sitagliptin (Sita) is a dipeptidyl peptidase-4 inhibitor which has been approved as a curing medicine for Type 2 diabetes (T2D) and has also reported its neuroprotective and antioxidant activity. This article describes the therapeutic effects of Sita on induced rat model of SE by kainic acid (KA) and investigated the antioxidative pathway of sita. Methods: Sprague–Dawley male rats were used randomly divided in four groups: vehicle control, KA and Sita + KA in a 5 and 10 mg/kg doses respectively in further groups. SE in rats was induced by the administration of KA in Phosphate buffered saline (PBS) intraperitoneally (IP) in a dose of 15 mg/kg. Seizure intensity, oxidative stress parameters, TUNEL assay, histopathology, and Nrf2/HO-1 expressions were evaluated. Results: Increment in the latency in SE results in delaying the initiation of disease in the pretreated rats by Sita compared to induce group (KA) as well the percentage of occurrence of SE was decreased. The content of MDA elevates whereas the SOD production decreases on administering the KA at various time intervals. Sita shows protective action against the KA-induced SE by reducing the oxidative stress thus inhibiting the change in SOD and MDA was observed after KA administration prior SE onset. Based on the above results, the study explains possible molecular mechanism of Sita. Sita Pretreatment showed significant elevation in expression of Nrf2 and HO-1 proteins in hippocampus region of the brain. Conclusion: Above parameters defines the potential effect of Sita on brain injury occurred due to SE by anti-oxidative pathway.     

Bisphenol A induces Nrf2-dependent drug-metabolizing enzymes through nitrosylation of Keap1

Bisphenol A (BPA) is an endocrine-disrupting chemical, and activates the aryl hydrocarbon receptor (AhR) and the estrogen receptor, leading to the induction of drug metabolizing enzymes. In this study, we found that BPA increased nitric oxide (NO) levels but not reactive oxygen species (ROS) levels in the human hepatoma cell line, Hep3B, and induced drug-metabolizing enzymes such as UDP-glucuronosyltransferase (UGT). Nuclear factor erythroid 2-related factor 2 (Nrf2) has been reported to be activated by ROS through inactivation of its regulating protein, Kelch-like ECH-associated protein (Keap1), and to be the key mediator of phase I and phase II drug metabolizing enzymes, and phase III drug transporters. Treatment of Hep3B with BPA increased the levels of nitrous oxide, a metabolite of nitric oxide and activated Nrf2 by nitrosylation of Keap1, leading to the induction of heme oxygenase-1 (HO-1) and UGT2B1 mRNAs. A nitric oxide donor, 1-Hydroxy-2-oxo-3-(N-methyl-3-aminopropyl)-3-methyl-1-triazene (NOC7), also activated Nrf2 and a NOS inhibitor, N^G-Monomethyl-l-arginine, monoacetate salt (L-NMMA), inhibited activation of Nrf2 by BPA. Furthermore, calcium efflux by BPA was observed. These results suggested the new idea that BPA increases NO levels and activates Nrf2 via Keap1 inactivation, leading to induction of Nrf2-dependent drug-metabolizing enzymes.     

姜黄素对D-半乳糖致衰老大鼠氧化应激及Nrf2/ARE通路的影响

目的 研究姜黄素对D-半乳糖致衰老模型大鼠氧化应激及Nrf2/ARE通路的影响.方法 将大鼠随机均分为空白组、模型组和姜黄素组于模型组和姜黄素组大鼠每日sc 125 mg· kg-1D-半乳糖造模,姜黄素组大鼠同时ip 10 mg·kg-1姜黄素,空白组大鼠给予生理盐水,连续7周.测定各组大鼠血清中丙二醛(MDA)、肝组织中蛋白质糖基、谷胱甘肽过氧化物(GSH)的含量和血清超氧化物歧化酶(SOD)、全血谷胱甘肽过氧化物酶(GSH-Px)的活性;用Western blot法分析肝组织中Nrf2蛋白和HO-1蛋白的表达水平.结果 与模型组比较,姜黄素组大鼠血清中SOD的活性和MDA、肝中GSH、蛋白质羰基的含量均降低,全血中GSH-Px的活性显著升高,Nfr2和HO-1蛋白的表达水平也明显升高.结论 姜黄素能够缓解D-半乳糖导致的大鼠氧化应激,Nrf2/ARE通路参与了姜黄素的抗氧化活性.     

Ginsenoside Rg1 prevents acetaminophen-induced oxidative stress and apoptosis via Nrf2/ARE signaling pathway

Inappropriate use of acetaminophen (APAP) can lead to morbidity and mortality secondary to hepatic necrosis. Ginsenoside Rg1 is a major active ingredient in processed Panax ginseng, which is proved to elicit biological effects. We hypothesized the beneficial effect of Rg1 on APAP-mediated hepatotoxicity was through Nrf2/ARE pathway. The study was conducted in cells and mice, comparing the actions of Rg1. Rg1 significantly improved cell survival rates and promoted the expression of antioxidant proteins. Meanwhile, Rg1 reduced the excessive ROS and the occurrence of cell apoptosis, which were related to Nrf2/ARE pathway. Expression of Nrf2 has a certain cell specificity.

     

Schisandrin A protects against lipopolysaccharide-induced mastitis through activating Nrf2 signaling pathway and inducing autophagy

Schisandrin A (Sch A), a dibenzocyclooctadiene lignan extracted from Schisandra chinensis (Turcz.) Baill., has anti-oxidant and anti-inflammatory effects, but the effect on masitits has not been studied. Therefore, we investigated the effect of Sch A in cell and mouse models of lipopolysaccharide (LPS)-induced mastitis. Studies in vivo showed that Sch A reduced LPS-induced mammary injury and the production of pro-inflammatory mediators. Sch A also decreased the levels of pro-inflammatory mediators and activated nuclear factor-E2 associated factor 2 (Nrf2) signaling pathway in mouse mammary epithelial cells (mMECs). The Nrf2 inhibitor partially abrogated the downregulation of Sch A on LPS-induced inflammatory response. In addition, LPS stimulation suppressed autophagy, while both Sch A and the autophagy inducer rapamycin activated autophagy in mMECs, which down-regulated inflammatory response. Sch A also restrained LPS-induced phosphorylation of mammalian target of rapamycin (mTOR) and activated AMP-activated protein kinase (AMPK) and unc-51 like kinase 1 (ULK1). In summary, these results suggest that Sch A exerts protective effects in LPS-induced mastitis models by activating Nrf2 signaling pathway and inducing autophagy and the autophagy is initiated by suppressing mTOR signaling pathway and activating AMPK-ULK1 signaling pathway.     

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。     

Protective effects of imperatorin against cerebral ischemia/reperfusion-induced oxidative stress through Nrf2 signaling pathway in rats

OBJECTIVE To investigates the effects of imperatorin on the oxidative stress in the cerebral cortex and hippocampus after focal cerebral ischemia/reperfusion injury.METHODS Transient focal cerebral ischemia/reperfusion model in male Sprague-Dawley rats was induced by 2 h middle cerebral artery occlusion followed by 24 h reperfusion.Imperatorin(1.25 and 2.5 mg·kg-1)or vehicle were administered intraperitoneally at 1,5 and 9 h after the onset of ischemia.At 24 h after reperfusion,the biomarkers of oxidative stress such as the levels of reactive oxygen species(ROS),lipid peroxidation products malondialdehyde(MDA),nitric oxide(NO)and total antioxidant capacity(T-AOC),the activities of inducible nitric oxide synthase(iN OS),superoxide dismutase(SOD)and catalase(CAT)in the cerebral cortex and hippocampus were observed.We also assessed the nuclear factor erythroid 2-related factor 2(Nrf2),heme oxygenase-1(HO-1),and the NAD(P)H-quinone oxidoreductase 1(NQO-1)protein expression by Western blot.RESULTS As compared to vehicle-treated animals,imperatorin treatment significantly reduced the ROS,MDA,NO levels and i NOS activity,increased T-AOC and the activities of SOD and CAT.Furthermore,imperatorin treatment also significantly induced the nuclear translocation of Nrf2,enhanced the protein expression of HO-1 and NQO-1 in the cerebral cortex and hippocampus.CONCLUSION Our findings indicate that imperatorin can protect the brain against the excessive oxidative stress induced by cerebral ischemia/reperfusion through activation of Nrf2 signaling pathway.     

尿酸激活Nrf2-ARE信号通路对帕金森病小鼠的保护作用

目的 探讨尿酸对帕金森病(PD)小鼠的神经保护作用以及对核因子E2相关因子2(Nrf2)的调控作用。方法 雄性C57BL/6J小鼠(6~8周、20~25 g)30只,采用随机数字表法分为三组:对照组(生理盐水)、1-甲基-4-苯基-1,2,3,6-四氢吡啶(MPTP)组、尿酸组(尿酸+MPTP),每组各10只。MPTP组小鼠经腹腔注射MPTP(20 mg/kg),每天1次,连续7 d。尿酸组于MPTP给药之前2 h腹腔注射尿酸(250 mg/kg),共13 d(MPTP注射前3 d,MPTP注射同时的7 d,MPTP结束后3 d)。对照组接受0.9%生理盐水代替MPTP和尿酸。在第14天测量各组小鼠的行为和认知功能,处死小鼠获取脑组织,免疫荧光染色测定黑质酪氨酸羟化酶(TH)的表达。实时荧光定量PCR检测Nrf2及下游基因mRNA的表达。免疫组化检测海马白细胞介素-1β(IL-1β)表达。酶联免疫吸附试验(ELISA)检测血清和海马IL-1β、白细胞介素-6(IL-6)和肿瘤坏死因子-α(TNF-α)水平。结果 尿酸能改善PD小鼠的行为学和认知功能,增加黑质TH阳性多巴胺能神经元数目和纹状体多巴胺含量。尿酸增加Nrf2和Nrf2下游基因mRNA的表达,包括γ-GCLC、HO-1和NQO1。尿酸显著提高MPTP处理小鼠黑质区SOD、CAT、GSH含量,降低MDA含量。尿酸抑制海马组织IL-1β蛋白的表达,并降低血清和海马中IL-1β、IL-6和TNF-α水平。结论 尿酸对PD小鼠多巴胺能神经元显示神经保护特性,其机制可能与尿酸通过激活Nrf2-ARE通路及调节神经炎症和氧化应激有关。     

黄连素激活Nrf2通路缓解新生大鼠哮喘模型氧化应激反应和对心肺组织的保护作用

目的：探讨黄连素（berberine,BB）对新生大鼠哮喘模型氧化应激反应的影响和对心肺组织的保护作用以及潜在的分子机制。方法：将新生SD雄性大鼠随机分成健康对照组、健康加药组、哮喘模型组和模型加药组;卵清蛋白诱导新生大鼠哮喘模型;收集肺泡灌洗液（broncho alveokar lavage fluid,BALF）并分析细胞类型和细胞数目;苏木伊红（hematoxylin eosin,HE）染色观察心肌组织和肺组织病理变化;末端标记法（terminal deoxynucleoitidyl transferase mediated nick end labeling,TUNEL）染色检测心肌组织和肺组织细胞凋亡情况;试剂盒检测血清中超氧化物歧化酶（superoxide dismutase,SOD）的活性和丙二醛（malondialdehyde,MDA）、一氧化氮（nitric oxide,NO）的含量;蛋白质印记检测肺组织中Kelch样ECH相关蛋白1（Kelch-like ECH-associated protein 1,Keap1）、NFE2相关因子2（NF-E2 related factor 2,Nrf2）和血红素氧合酶1（heme oxygenase 1,HMOX-1）的表达水平。结果：哮喘模型组与对照组相比,肺泡灌洗液中炎性细胞的数目显著增加;心肌组织细胞排列不规则,部分细胞结构不清晰;肺组织中有大量的炎性细胞浸润,黏膜下水肿,气道上皮断裂脱落,支气管壁明显增厚;心肌组织和肺组织中凋亡细胞比例显著增加;血清中SOD的活性显著降低,MDA和NO的含量显著增加;肺组织中Keap1、Nrf2和HMOX-1的表达水平显著升高。模型加药组与模型组相比,肺泡灌洗液中炎性细胞的数目明显减少;心肌组织较规则,细胞排列较整齐;肺组织中仍有部分炎性细胞浸润,部分肺泡壁增厚;心肌组织和肺组织中凋亡细胞比例显著降低;SOD的活性显著升高,MDA和NO的含量显著降低;Keap1、Nrf2和HMOX-1的表达水平显著升高。结论：黄连素可激活Nrf2通路,缓解新生大鼠哮喘模型的氧化应激反应,对心肺组织具有保护作用。     

Induction of the Nrf2-driven antioxidant response by tert-butylhydroquinone prevents ethanol-induced apoptosis in cranial neural crest cells

Previous studies have shown that ethanol exposure causes apoptosis in cranial neural crest cells (NCCs), an ethanol-sensitive cell population implicated in Fetal Alcohol Spectrum Disorders (FASD). Additionally, induction of endogenous antioxidants through activation of nuclear factor-erythroid 2-related factor 2 (Nrf2) has been shown to prevent oxidative stress and apoptosis in ethanol-exposed mouse embryos. The objective of this study was to test whether tert-butylhydroquinone (tBHQ), an Nrf2 inducer, can protect NCCs against ethanol-induced apoptosis. Ethanol exposure was shown to cause a moderate increase in the protein expression of Nrf2 and its downstream antioxidants in the NCCs. Treatment of NCCs with tBHQ alone significantly increased the protein expression of Nrf2 and its downstream antioxidants and also significantly increased the activities of the antioxidant enzymes. In NCCs exposed to ethanol, the tBHQ-mediated antioxidant response prevented oxidative stress and apoptosis. These results clearly demonstrate that the activation of Nrf2 signaling confers protection against ethanol-induced apoptosis in NCCs.