Nrf2通路在天麻素抑制油酸诱导的HL―7702-文档资料
Nrf2抗氧化的分子调控机制
Nrf2抗氧化的分子调控机制李慧;杨林【摘要】Nrf2是调控细胞氧化应激反应的重要转录因子,同时也是维持细胞内氧化还原稳态的中枢调节者.Nrf2通过诱导调控一系列抗氧化蛋白的组成型和诱导型表达,可以减轻活性氧和亲电体引起的细胞损伤,使细胞处于稳定状态,维持机体氧化还原动态平衡.本研究为了从分子层面深入探讨剖析Nrf2发挥抗氧化功能的作用机制,通过查找阅读大量相关文献并进行整理归纳,最终从Nrf2的结构与激活、Nrf2抗氧化功能以及Nrf2抗氧化的分子调控机制三个方面进行了概述分析.其中在对Nrf2抗氧化的分子调控机制的探讨部分,既探析了对Nrf2起激活作用的相关调节因子的作用机制,又分析了Nrf2被激活后对其下游多种抗氧化因子及谷胱甘肽氧化还原系统的诱导调控机制,以期较深入了解Nrf2抵抗机体氧化应激损伤作用及其抗氧化分子调控机制.【期刊名称】《生物信息学》【年(卷),期】2018(016)001【总页数】6页(P1-6)【关键词】Nrf2;氧化应激;抗氧化;分子调控,机制【作者】李慧;杨林【作者单位】哈尔滨工业大学化工与化学学院,哈尔滨150001;哈尔滨工业大学化工与化学学院,哈尔滨150001【正文语种】中文【中图分类】Q753氧化应激是由细胞过度产生的活性氧(Reactive oxygen species，ROS)和亲电体引起的，而过量的ROS又可以诱导自由基链反应，破坏细胞生物大分子如蛋白质、脂质和DNA等，并诱发一系列生活习惯性疾病，如心脑血管疾病、老化、II型糖尿病、癌症等[1-3]。
机体为控制ROS水平并防止其积累，形成了一套复杂的抗氧化防御体系，其中核因子NF-E2相关因子(Nuclear factor-erythroid 2-related factor 2，Nrf2)是一种重要的氧化还原敏感性转录因子，其通过诱导调控细胞内II 相解毒酶和抗氧化酶的组成型和诱导型表达，有利于改善机体氧化应激状态，促进细胞存活以及维持细胞的氧化还原稳态。
巨噬细胞中转录因子 Nrf2 同时调节一组氧化应激诱导的基因
YamamotoKatoh, Shiro Bannai and Masayuki Hideyo Sato, Toru Yanagawa, Yasutake Tetsuro Ishii, Ken Itoh, Satoru Takahashi,Stress-inducible Genes in Macrophages Regulates a Group of OxidativeTranscription Factor Nrf2 Coordinately REGULATION:GENES: STRUCTURE AND doi: 10.1074/jbc.275.21.160232000, 275:16023-16029.J. Biol. Chem./content/275/21/16023Access the most updated version of this article at.JBC Affinity Sites Find articles, minireviews, Reflections and Classics on similar topics on theAlerts:When a correction for this article is posted •When this article is cited • to choose from all of JBC's e-mail alertsClick here/content/275/21/16023.full.html#ref-list-1This article cites 40 references, 25 of which can be accessed free at by guest on January 25, 2014/Downloaded fromTranscription Factor Nrf2Coordinately Regulates a Group of Oxidative Stress-inducible Genes in Macrophages*Received for publication,September 10,1999,and in revised form,January 19,2000Tetsuro Ishii‡,Ken Itoh‡§,Satoru Takahashi,Hideyo Sato,Toru Yanagawa,Yasutake Katoh,Shiro Bannai,and Masayuki Yamamoto ¶From the Institute of Basic Medical Sciences and Center for Tsukuba Advanced Research Alliance,University of Tsukuba,1-1-1Tennoudai,Tsukuba 305-8577,JapanElectrophiles and reactive oxygen species have been implicated in the pathogenesis of many diseases.Tran-scription factor Nrf2was recently identified as a gen-eral regulator of one defense mechanism against such havoc.Nrf2regulates the inducible expression of a group of detoxication enzymes,such as glutathione S -transferase and NAD(P)H:quinone oxidoreductase,via antioxidant response ing peritoneal macro-phages from Nrf2-deficient mice,we show here that Nrf2also controls the expression of a group of electrophile-and oxidative stress-inducible proteins and activities,which includes heme oxygenase-1,A170,peroxiredoxin MSP23,and cystine membrane transport (system x c Ϫ)ac-tivity.The response to electrophilic and reactive oxygen species-producing agents was profoundly impaired in Nrf2-deficient cells.The lack of induction of system x c Ϫactivity resulted in the minimum level of intracellular glutathione,and Nrf2-deficient cells were more sensi-tive to toxic electrophiles.Several stress agents induced the DNA binding activity of Nrf2in the nucleus without increasing its mRNA level.Thus Nrf2regulates a wide-ranging metabolic response to oxidative stress.Oxidative stress conditions or enhanced production of reac-tive oxygen species (ROS)1result from a variety of stimuli including ionizing radiation,exposure to xenobiotics,inflam-mation,and phagocytosis (1).Treatment of mammalian cells with electrophilic agents usually provokes cellular responses,including transcriptional activation of genes encoding proteins that partake in the defense against oxidative stress.This proc-ess is referred to as the electrophile counterattack response (2).Through analyses of mouse and rat glutathione S -transferase (GST)Ya subunit genes and the rat NAD(P)H:quinone oxi-doreductase (NQO1)subunit gene,the cis -acting element re-sponsible for the induction by electrophiles was independently identified as an electrophile-responsive element (EpRE)(3)or antioxidant-responsive element (ARE)(4).The consensus ARE sequence has been extensively characterized (5).The consensus binding sequence of erythroid transcription factor NF-E2shows high similarity to the ARE/EpRE se-quence.Also,the expression profile of Nrf2,one of the NF-E2subunit factors,overlaps with those of drug-metabolizing en-zymes such as GST and NQO1.Based on these facts,we re-cently demonstrated that transcription factor Nrf2(6–8)is essential for the coordinated transcriptional activation of genes encoding the drug-metabolizing enzymes,such as GST and NQO1,via AREs/EpREs (9).Nrf2-deficient mice fed with bu-tylated hydroxyanisole,which normally leads to a pronounced up-regulation of Alpha,Pi,and Mu classes of GSTs and NQO1,failed to induce either of these detoxication enzymes in the liver or intestine (9).Since these detoxication enzymes decrease the level of oxidative stress by removing compounds capable of generating ROS or other highly reactive substances,they thereby constitute part of the defense mechanism against oxi-dative stress (10).Because ARE-type cis -acting sequences are frequently found in the regulatory regions of a number of other oxidative stress-inducible genes (5,11–13),we hypothesized that Nrf2might also serve as the key transcription factor activating these genes.A number of defense proteins and activities in murine peri-toneal macrophages are markedly induced upon exposure to electrophilic agents or other oxidative stresses.These proteins include heme oxygenase-1(HO-1)(14–16),peroxiredoxin MSP23(17),the cystine membrane transporter (system x c Ϫ)(18)and 60-kDa stress protein A170(19).HO-1is prominently induced under various oxidative stress conditions in many dif-ferent cell types (14).HO-1-deficient embryonic fibroblasts are hypersensitive to the cytotoxicity of both hemin and hydrogen peroxide (15).Induction of system x c Ϫactivity increases the intracellular cysteine pool,which consequently augments the synthesis of GSH (20),a potent antioxidant with a short half-life.MSP23is the murine peroxiredoxin I with antioxidative activity (21).It was recently shown that a mammalian perox-iredoxin isoform reduces the intracellular hydrogen peroxide level utilizing thioredoxin as an immediate electron donor (22)and protects cells from apoptosis by oxidative stress (23).A170has a structural domain that interacts with ubiquitin (24)and PKC-(25).Electrophilic agents,such as diethylmaleate (DEM),and other oxidative stress agents have been reported to induce the proteins HO-1,A170,MSP23,and system x c Ϫactiv-ity in peritoneal macrophages (20)and fibroblasts (26).To determine whether these antioxidant stress proteins are also under the regulation of Nrf2,we examined in this study the electrophilic induction of this group of genes in peritoneal macrophages from the nrf2-null mutant mouse.*This work was supported in part by grants-in-aid from the Ministry of Education,Science,Sports and Culture,the Japanese Society for Promotion of Sciences (JSPS),and CREST.The costs of publication of this article were defrayed in part by the payment of page charges.This article must therefore be hereby marked “advertisement ”in accordance with 18U.S.C.Section 1734solely to indicate this fact.‡These two authors contributed equally to this work.§Research Fellow of the JSPS.¶To whom correspondence should be addressed:Center for TARA,University of Tsukuba,1-1-1Tennoudai,Tsukuba 305-8577,Japan.Tel.:81-298-53-6158;Fax:81-298-53-7318;E-mail:masi@tara.tsukuba.ac.jp.1The abbreviations used are:ROS,reactive oxygen species;GST,glutathione S -transferase;NQO1,NAD(P)H:quinone oxidoreductase;EpRE,electrophile-responsive element;ARE,antioxidant-responsive element;HO-1,heme oxygenase-1;DEM,diethylmaleate;MTT,3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide;CDNB,1-chloro-2,4-dinitrobenzene;t -BHQ,t-butylhydroquinone;LPS,lipopo-lysaccharide;EMSA,electrophoretic mobility shift assay;MARE,Maf recognition element;StRE,stress-responsive element.T HE J OURNAL OF B IOLOGICAL C HEMISTRYVol.275,No.21,Issue of May 26,pp.16023–16029,2000©2000by The American Society for Biochemistry and Molecular Biology,Inc.Printed in U.S.A.This paper is available on line at 16023by guest on January 25, 2014/Downloaded fromEXPERIMENTAL PROCEDURESCulture of Macrophages—Female wild type ICR and nrf2mutant mice (9)weighing 20–25g received an intraperitoneal injection of 2ml of 4%thioglycollate broth.Four days later,macrophages were collected by peritoneal lavage (17).The cells were resuspended at 7.5ϫ105cells/ml and cultured in RPMI 1640medium containing 10%(v/v)fetal bovine serum as described previously (17).For the 3-[4,5-dimethylthia-zol-2-yl]-2,5-diphenyltetrazolium bromide (MTT)(27)assay,the cells were resuspended at 2ϫ105cells/ml and cultured in medium without fetal bovine serum.After 1h of culture,stress agents were added to the medium.Final concentrations of the agents in the medium were 100M for DEM,paraquat,and hydrogen peroxide (H 2O 2);80M for catechol;20milliunits/ml for glucose oxidase (GO);5M for CdCl 2and menadi-one (2-methyl-1,4-naphthoquinone);10M for 1-chloro-2,4-dinitroben-zene (CDNB);20M for iodoacetic acid;2.5M for t-butylhydroquinone (t-BHQ)and sodium arsenite (NaAsO 2);and 1ng/ml for lipopolysaccha-ride (LPS).The macrophages were harvested at the times indicated in the figure legends.Cell viability was measured by the MTT assay (27)and the trypan blue dye exclusion test.RNA Blot Hybridization Analysis—Total cellular RNA was extracted from macrophages by RNAzol TM B (TEL-TEST,Inc.,Friendswood,TX).The RNA samples (10g)were electrophoresed and transferred to Zeta-Probe GT membranes (Bio-Rad).The membranes were probed with 32P-labeled cDNA probes as indicated in the figure legends.-Ac-tin cDNA was used as a positive control.Immunoblotting—Macrophages were solubilized with SDS-sample buffer (without dye or 2-mercaptoethanol),and protein concentrations were estimated by the BCA protein assay (Pierce).The proteins were separated by SDS-polyacrylamide gel electrophoresis in the presence of 2-mercaptoethanol and electrotransferred onto Immobilon membrane (Millipore Corp.,Bedford,MA).To detect immunoreactive proteins,we used horseradish peroxidase-conjugated anti-rabbit IgG and ECL blot-ting reagents (Amersham Pharmacia Biotech).Polyclonal rabbit anti-sera raised against rat HO-1,rat MSP23,and recombinant murine A170were used as described previously (19,21).Specific antibody was raised against Nrf2by immunizing rabbits with recombinant Nrf2protein (amino acids 140–318fused with E.coli maltose-binding pro-tein).An anti-actin antiserum was purchased from Santa Cruz Biotech-nology,Inc.(Santa Cruz,CA).Densitometric analysis was performed using NIH Image software.Determination of Cystine Uptake and GSH Level—Cystine transport activity was measured using 14C-labeled cystine in phosphate-buffered saline containing 0.1%glucose as described previously (20).Total (i.e.reduced plus oxidized)GSH was extracted from macrophages with 5%trichloroacetic acid solution,and GSH content was measured as de-scribed previously (20).Transient Transfection Assay—The quail fibroblast cell line QT6(28)was maintained in Dulbecco’s modified Eagle’s medium supplemented with 10%fetal bovine serum and seeded in 24-well dishes 24h before transfection.The cells were transfected with reporter (pHO-1-Luc (see “Results”)and pRBGP2(7))plasmids and an effector plasmid (cNrf2;Ref.7)using calcium phosphate precipitation as described previously (29).The Luciferase assay was performed by utilizing the Luciferase Assay System (Promega,Madison,WI)following the supplier’s protocol and measured in a Biolumat Luminometer (Berthold,Germany).Trans-fection efficiencies were routinely normalized to the activity of a co-transfected -galactosidase expression plasmid,pENL.Normally,three independent experiments,each carried out in duplicate,were per-formed,and the results were averaged.The cells were washed 12h after transfection,and then DEM (Wako,Tokyo)was immediately added to the culture medium.Electrophoretic Mobility Shift Assay (EMSA)—Nuclear extracts were prepared from macrophages as described previously (7).An oligonucleo-tide containing the stress-responsive element of the ho-1AB1enhancer (5Ј-TCTGTTTTCGCTGAGTCATGGTTCCCGTTG-3Ј)was labeled with [␥-32P]ATP by T4polynucleotide kinase.EMSA was performed as de-scribed previously (7).Where indicated,antibodies were included in the binding reaction at 1:10to 1:100dilutions.The anti-chicken MafK antibody was described previously (9),and that against human Nrf2was purchased from Santa Cruz Biotechnology.In competition experi-ments,a 100-fold excess of unlabeled double-stranded oligonucleotides was included in the reaction (7).RESULTSImpaired Induction of Antioxidative Stress Proteins in Nrf2-deficient Macrophages—A number of proteins or activities are induced by electrophilic agents in murine peritoneal macro-phages.To test whether the electrophilic induction of this group of genes shares a common regulatory mechanism with that of the drug-metabolizing enzymes,we examined their expression in Nrf2-deficient macrophages.Peritoneal macro-phages were harvested from nrf2-homozygous and -heterozy-gous mutant (or wild type)female ICR siblings.The macro-phages were then independently challenged with DEM (anelectrophilic agent),paraquat (an O 2.generator),GO (an H 2O 2generator),or CdCl 2(heavy metal).After challenging macro-phages with these agents,we examined expression levels of three oxidative stress-inducible proteins (below)by immuno-blotting and RNA blotting analyses.Since wild type and het-erozygous mutant mice did not show large differences in induc-tion of the antioxidative proteins,we used both types of macrophages as positive controls.We first measured the levels of HO-1,MSP23,and A170proteins 1h after the start of in vitro culture and found that the basal levels of these proteins in the heterozygous macrophages were similar to those of nrf2-homozygous mutant macrophages (Fig.1A ,lanes 1and 7).After transfer of the nrf2-heterozygous cells to in vitro culture,these stress-inducible proteins were gradually induced by unknown mechanisms (compare lanes 1and 2).The gradual induction of HO-1and MSP23expression was not seen in Nrf2-deficient cells (lanes 2and 8).The impor-tant finding here was that whereas all of the stressagentsF IG .1.Stress agent-dependent loss of HO-1,MSP23,and A170gene induction in Nrf2-deficient macrophages.A ,immunoblot analysis of the effects of oxidative stress agents on the expression levels of HO-1(34kDa),MSP23(23kDa),and A170(60kDa).Macrophages from nrf2-heterozygous (ϩ/Ϫ)(lanes 1–6)and homozygous (Ϫ/Ϫ)(lanes 7–12)mutant mice were cultured for 1h (lanes 1and 7)or 9h (lanes 2and 8).Stress agents were added to the culture medium after a 1-h culture,and the macrophages were incubated with the agents for an-other 8h (lanes 3–6and 9–12).The stress agents added were DEM (lanes 3and 9),paraquat (lanes 4and 10),GO (lanes 5and 11),and CdCl 2(lanes 6and 12).B ,RNA blot analysis of the effects of oxidative stress agents on the levels of mRNAs for HO-1,MSP23,and A170.Total RNAs were prepared from macrophages cultured for 1h (lanes 1and 7)or 5h (other lanes ).Stress agents were added at 1h after the start of the ne arrangements are as in A .Nrf2Regulates a Group of Oxidative Stress-inducible Genes16024 by guest on January 25, 2014/Downloaded fromtested induced HO-1,MSP23,and A170in nrf2-heterozygous cells,induction was largely canceled in Nrf2-deficient cells.Closer examination revealed that in nrf2-null mutant cells (lanes 9–12)induction of HO-1and A170by DEM and GO was severely affected,but induction by paraquat and CdCl 2was less impaired.In contrast,while MSP23was markedly induced by these agents in nrf2-heterozygous cells,induction was largely absent in nrf2-null mutant cells.Quantitative Analysis of Antioxidative Stress Protein Induc-tion by Various Stress Agents—We also examined induction of these proteins by other stress agents:catechol,CDNB,H 2O 2,iodoacetic acid,sodium arsenite,menadione,and t-BHQ.Quan-titative analysis by densitometry of the stained bands is shown in Table I.Menadione and catechol induced HO-1in the Nrf2-deficient cells at levels comparable with those in nrf2-heterozy-gous mutant cells,suggesting the involvement of signal-trans-ducing pathway(s)other than the Nrf2system.Apparently,induction of HO-1by sodium arsenite,t -BHQ,CDNB,and iodoacetic acid is largely,if not exclusively,dependent on the presence of Nrf2.A significant increase of MSP23by all stress agents except sodium arsenite,H 2O 2,and iodoacetic acid was also observed in the heterozygous mutant cells but not in homozygous mutant cells (Table I).These results thus indicate that inducible expression of MSP23is regulated mainly through the ARE/Nrf2system.To assess the induction process at the level of transcription,RNA was extracted from macrophages that had been treated with various stress agents and analyzed by the RNA blot anal-ysis.Treatment with stress agents significantly increased the levels of HO-1and MSP23mRNA in nrf2-heterozygous cells (Fig.1B ,lanes 1–6),but the induction was markedly impaired in nrf2-null mutant cells (lanes 7–12).In contrast,whereas the lack of the induction of A170mRNA in nrf2-null mutant cells was evident when DEM was used as the stress agent,the induction was only partially affected in Nrf2-deficient cells when paraquat,GO,or CdCl 2was used as an inducer.Marked induction of the genes was also observed in the nrf2-heterozy-gous cells treated with menadione,t -BHQ,catechol,or CDNB (data not shown).The induction of HO-1mRNA by menadione and catechol was diminished in Nrf2-null mutant cells,while that by t -BHQ and CDNB was largely abolished.Induction of MSP23mRNA by all of these agents was markedly impaired in nrf2-null mutant cells.The three oxidative stress-inducible genes we addressed here showed roughly comparable variation in the mRNA and protein levels in response to various stressagents (Fig.1,compare A and B ,and data not shown).These results thus indicate that Nrf2regulates the stress agent-mediated induction of HO-1,MSP23,and A170gene expres-sion.The results also clearly show that the contribution of Nrf2to the transcriptional activation of these genes differed based upon the stress-inducing agent.Induction of the Cystine Transporter x c ϪSystem Is Defective in Nrf2-deficient Cells—Because oxidative stress agents tran-scriptionally induce system x c Ϫactivity in macrophages (20,26),the stress induction of system x c Ϫactivity in nrf2-null mutant cells was examined next (Fig.2A ).Whereas nrf2-heterozygous cells show system x c Ϫactivity comparable with that of wild type cells under both basal and induced conditions (data not shown),the oxidative stress agents DEM,paraquat,GO,and CdCl 2barely induced system x c Ϫactivity in nrf2-null mutant cells (Fig.2A ).In contrast,LPS,a well known inducer of system x c Ϫactivity (30),significantly induced the system x cϪactivity even in nrf2-null mutant cells,indicating that LPS induction is mediated through an alternative regulatory pathway rather than the Nrf2pathway.These results argue that the transcrip-tion of the cystine transporter gene may be under the regula-tory influence of Nrf2.It should be noted that system x c Ϫactivity is necessary to maintain a high GSH level in cultured macrophages,since cysteine is easily oxidized to cystine upon exposure to air (18).While the addition of 500M to 1m M of DEM to the culture medium depletes the intracellular stores of GSH significantly,the addition of 100M DEM only diminishes the GSH level minimally,and then its level increases as a result of induced system x c Ϫactivity (26).As expected,the defect in inducibleT ABLE IQuantitative analysis of the induction of HO-1and MSP23proteinsby various stress agentsThe protein levels were calculated from densitometrical analysis of the immunoblot bands.Results are mean ϮS.D.of 3–5separate exper-iments.The standard levels (1.00)are those of cells cultured for 9h without agents.The culture conditions are the same as in Fig.1A .AgentsHO-1MSP23Nrf2(ϩ/Ϫ)Nrf2(Ϫ/Ϫ)Nrf2(ϩ/Ϫ)Nrf2(Ϫ/Ϫ)relative absorbanceNone (1h)0.58Ϯ0.020.50Ϯ0.190.60Ϯ0.170.50Ϯ0.25None (9h) 1.000.57Ϯ0.12 1.000.50Ϯ0.16DEM 1.67Ϯ0.170.69Ϯ0.04 1.83Ϯ0.040.54Ϯ0.30CDNB 2.94Ϯ0.210.77Ϯ0.02 1.51Ϯ0.690.34Ϯ0.08GO 1.22Ϯ0.020.57Ϯ0.07 1.51Ϯ0.020.34Ϯ0.21H 2O 2 1.26Ϯ0.100.55Ϯ0.060.97Ϯ0.270.38Ϯ0.14t -BHQ1.91Ϯ0.770.86Ϯ0.47 1.58Ϯ0.070.44Ϯ0.27Menadione 6.38Ϯ3.93 4.54Ϯ2.76 1.94Ϯ0.100.58Ϯ0.21Catechol3.70Ϯ0.02 1.77Ϯ0.06 1.75Ϯ0.060.52Ϯ0.17CdCl 21.57Ϯ0.14 1.21Ϯ0.08 1.38Ϯ0.220.35Ϯ0.15Paraquat 1.34Ϯ0.030.88Ϯ0.03 1.50Ϯ0.280.55Ϯ0.25NaAsO 23.70Ϯ1.30 1.08Ϯ0.51 1.01Ϯ0.110.43Ϯ0.16Iodoacetic acid 2.05Ϯ0.100.98Ϯ0.27 1.15Ϯ0.170.60Ϯ0.27F IG .2.Oxidative stress agents do not induce system x c ؊or GSHin Nrf2-deficient macrophages.A ,inability to induce the system x c Ϫcystine transport activity in the nrf2-deficient macrophages.Macro-phages were incubated for 12h with DEM,paraquat,GO,CdCl 2,and LPS as described in the legend to Fig.1A .B ,decreased cellular GSH level in nrf2-deficient macrophages treated with 100M DEM.The values in A and B represent mean ϮS.E.of three independent exper-iments,each carried out in duplicate.Nrf2Regulates a Group of Oxidative Stress-inducible Genes16025by guest on January 25, 2014/Downloaded fromexpression of system x c Ϫactivity in nrf2-null mutant cells re-sulted in a decrease in cellular GSH content;after a 24-h incubation with DEM,the GSH level dropped to less than half its original level (Fig.2B ).Overexpression of Nrf2Up-regulates the Activities of the Dis-tal Enhancer of the ho-1Gene—The AB1and SX2enhancers of the ho-1gene (31)contain three and two copies,respectively,of the Maf recognition element (MARE),which largely overlaps with ARE (32).Due to their responsiveness to a wide variety of stress agents including oxidative stress,the MAREs were also named stress-responsive elements (StREs)(31–34).To ask whether Nrf2can regulate the expression of this antioxidative stress gene via the ho-1AB1enhancer,we co-transfected a Nrf2expression plasmid and a ho-1AB1enhancer-luciferase (pHO-1-Luc)reporter or -globin enhancer-luciferase (pRBGP2)reporter into QT6fibroblasts (Fig.3A ).DEM (Fig.3B )and Nrf2overexpression (Fig.3C )both activated HO-1-Luc reporter gene expression,with the highest concentrations of these agents generating more than 10-fold activation.This increase was strictly dependent on the presence of the AB1enhancer (data not shown).We also tested a pRBGP2reporter that contains three tandem copies of the NF-E2binding se-quence of the chicken -globin enhancer (7),a sequence that is very similar to the AB1enhancer sequence.Interestingly,the pRBGP2reporter responded more efficiently than the pHO-1-Luc reporter did to both DEM and the overexpression of Nrf2(Fig.3,B and C ).These results indicate that StREs in the AB1enhancer are actually responsible for the activation of ho-1gene expression and that Nrf2can activate the ho-1gene expression through StREs.Stress Agents Post-transcriptionally Induce DNA Binding Activity of Nrf2—Given the clear lack of inductive response in a number of electrophile-inducible genes,it was of interest to determine how oxidative stress agents activate Nrf2and thereby induce a group of genes to counteract oxidative stress.To this end,we first examined the level of Nrf2mRNA in macrophages under various oxidative stresses and found thatthe mRNA level was not changed significantly by any of the oxidative stress agents tested (Fig.4A ).In contrast,we found that these same stress agents significantly enhanced the DNA binding activity of Nrf2to the StRE of HO-1AB1enhancer.StRE binding activity in nuclear extracts was strongly in-duced by DEM treatment of macrophages,as revealed by the retarded band (arrow in Fig.4B ,lanes 1and 2).The induction of binding activity was not observed in nuclear extracts pre-pared from nrf2-deficient cells (Fig.4B ,lanes 3and 4).The complex was effectively competed by the addition of an excess of unlabeled StRE,mouse GST Ya gene ARE,or chicken -glo-bin enhancer NF-E2binding site but not by a yeast Gal4binding consensus sequence (Fig.4D ).This DNA-protein com-plex was markedly diminished by treatment with antibodies against mouse Nrf2(Fig.4C ,lane 3)and chicken MafK (lane 4),which are partner molecules together comprising the het-erodimeric transcription factor complex (34),but the decrease was not obvious using a normal rabbit IgG (lane 5).These results indicate that the DNA-protein complex contains both Nrf2and small Maf proteins.The same binding activity was also induced in nuclear extracts of macrophages treated with GO,paraquat,or CdCl 2(Fig.4E ).The Nrf2expression level was also examined by immunoblotting analyses of nuclear ex-tracts prepared from the macrophages treated with these stress agents.We found that DEM,GO,paraquat,CdCl 2,and CDNB all increased the Nrf2level 1.5–3-fold (Fig.4F ).We therefore concluded that post-transcriptional regulation might be involved in the activation of Nrf2by electrophilic agents and ROS.Lack of Nrf2Renders Macrophages Sensitive to Oxidative Stress—The analysis thus far clearly indicates that macro-phages invoke an electrophile-inducible response upon expo-sure to oxidative stress agents and that the response is medi-ated by Nrf2.To ask whether this response has a major impact on cell viability,we incubated both nrf2-null and heterozygous control macrophages with 5–20M CDNB for 12h and meas-ured cell viability by the colorimetric MTT assay.WhilecellF IG .3.Overexpression of Nrf2is sufficient to activate reporter gene expression driven by the AB1en-hancer of the ho-1gene.A ,schematic representation of the luciferase reporter constructs.In the HO-1-Luc reporter con-struct,a 161-base pair fragment of the AB1enhancer of the ho-1gene is placed upstream of the rabbit -globin TATA box and luciferase reporter gene.The pRBGP2construct is similar,but three copies of the NF-E2binding sequence from the chicken -globin enhancer pre-cede the TATA box and luciferase gene (7).B ,pRBGP2or pHO-1-Luc reporter construct was transfected into QT6fibro-blasts in the presence or absence of the electrophilic agent DEM.Luciferase ac-tivity in the absence of DEM was set at 100%,and results of three independent experiments each carried out in duplicate are shown with S.E.C ,an increasing amount of chicken Nrf2was transfected with pRBGP2or pHO-1-Luc.Experi-ments were performed as in B .Nrf2Regulates a Group of Oxidative Stress-inducible Genes16026 by guest on January 25, 2014/Downloaded fromviability was decreased in both nrf2-null mutant and control macrophages treated with 20M CDNB,with 10M CDNB,nrf2-null mutant cells were more sensitive to the CDNB treat-ment than the heterozygous control cells (Table II).Notably,10M CDNB treatment resulted in an approximately 2-fold dif-ference in the MTT assay,and this difference is statistically significant (p Ͻ0.05).The difference in the sensitivity to CDNB between the nrf2-null and -heterozygous cells was much clear when we measured the cell viability by the trypan blue dye exclusion test after 12h of the CDNB treatment.The viability of the cells was 77and 16%(mean of two independent experi-ments)for nrf2-heterozygous and nrf2-null mutant cells,re-spectively.Nrf2thus appears to contribute significantly to cellular defense mechanisms against toxic electrophiles.To highlight the Nrf2contribution to cellular defense mech-anisms,we pretreated macrophages for 36h with 100M DEM,a concentration that potently induces the antioxidative stress response but with low cytotoxicity.The macrophages were sub-sequently treated with 5or 10M CDNB for an additional 12h.After incubation,we measured cell viability by trypan blue dye exclusion.The Nrf2-deficient macrophages were more sensitive to treatment with CDNB than the heterozygous control cells.After CDNB treatment,less than 20%of the Nrf2-deficient macrophages were viable (Fig.5B ),whereas more than 95%of the heterozygous cells were viable (Fig.5A ).These results unequivocally demonstrate that there are electrophile-induci-ble responses mediated by ARE/EpRE and Nrf2and that the response machinery protects cells against toxic electrophiles and ROS stresses.DISCUSSIONWe demonstrate in this study that,in addition to the drug metabolizing enzymes that have already been shown to be regulated by the Nrf2pathway (9),a group of oxidative stress-inducible genes is also under the immediate transcriptional influence of Nrf2-small Maf heterodimer regulatory proteins.The fact that Nrf2regulates a group of stress-inducible protein genes via ARE/EpRE,as schematically illustrated in Fig.6,is intriguing in the context of the physiological origin of these defense mechanisms.In an evolutionary sense,the acquisition of the ARE regulatory mechanism by genes that protect against oxidative stress seems to confer a significant advantage on the survival of living creatures.It should also be noted that,reaction.The complex containing Nrf2is indicated by an arrow .E ,increase in the DNA binding activity of Nrf2-small Maf in stress agent-treated macrophages.Nuclear extracts were prepared from the perito-neal macrophages treated with DEM (lane 2),GO (lane 3),paraquat (lane 4),or CdCl 2(lane 5)and examined by EMSA.An arrow indicates the complex containing Nrf2.F ,increase of Nrf2protein in the nucleus of the stress agent-treated macrophages.Immunoblotting analysis was performed with macrophage nuclearextracts.F IG .4.Increase in DNA binding activity of Nrf2-small Maf heterodimers upon exposure to stress agents.A ,RNA blot analy-sis of Nrf2mRNA in macrophages treated with various stress agents.The nrf2-heterozygous mutant cells were incubated for 4h with the stress agents as in Fig.1B .Total RNAs extracted from the macrophages were analyzed with Nrf2and -actin cDNA probes.B ,EMSA analysis of Nrf2-small Maf DNA binding activity in macrophage nuclear ex-tracts.Nuclear extracts were prepared from wild type (lanes 1and 2)or nrf2-deficient peritoneal macrophages (lanes 3and 4).These macro-phages were either treated with DEM for 4h (lanes 2and 4)or not treated (lanes 1and 3).A double-stranded oligonucleotide of the StRE sequence in the ho-1gene AB1enhancer (ho-1probe)was used as a probe.The complex containing Nrf2is indicated by an arrow .C ,Nrf2-small Maf heterodimer binds to StRE.The ho-1probe was incubated with the nuclear extract from control macrophages (lane 1)or the nuclear extract of macrophages treated with DEM (lane 2).Anti-Nrf2(lane 3),anti-MafK (lane 4),or normal rabbit IgG (lane 5)was included in the reaction.The complex containing Nrf2is indicated by an arrow .D ,the binding specificity of the Nrf2-small Maf heterodimer was con-firmed by an EMSA competition experiment.The ho-1probe was incu-bated with the nuclear extract of macrophages treated with DEM (lane 1).An excess of unlabeled probe oligonucleotide (lane 2)or double-stranded oligonucleotides from mouse GST Ya gene ARE (lane 3),chicken -globin enhancer NF-E2binding site (lane 4),or yeast tran-scription factor Gal4cognate site (lane 5)was included in the bindingT ABLE IICDNB sensitivity of Nrf2-null mutant macrophagesBoth nrf2-heterozygous (ϩ/Ϫ)and -homozygous (Ϫ/Ϫ)mutant macro-phages were incubated with 0,5,10,and 20M CDNB for 12h.The cell viability was examined with an MTT assay.Values are mean ϮS.E.of relative absorbance of three independent experiments each carried out in duplicate.Both heterozygous and homozygous mutant cells showed similar absorbance in the MTT assay without CDNB.*p Ͻ0.05.CDNBSensitivityNrf2(ϩ/Ϫ)Nrf2(Ϫ/Ϫ)Mrelative absorbance0 1.00 1.0050.85Ϯ0.090.86Ϯ0.15100.74Ϯ0.06*0.46Ϯ0.13*200.19Ϯ0.020.22Ϯ0.05Nrf2Regulates a Group of Oxidative Stress-inducible Genes 16027by guest on January 25, 2014/Downloaded from。
天麻素保护过氧化氢诱导的神经元氧化损伤的研究
doi:10.3969/j.issn.1000⁃484X.2020.15.009天麻素保护过氧化氢诱导的神经元氧化损伤的研究①尹明姬 池永学 李今子 (延边大学附属医院,延吉133002) 中图分类号 R742.1 文献标志码 A 文章编号 1000⁃484X (2020)15⁃1836⁃04①本文受中央财政支持地方高校发展专项(吉财教指[2016]566号)和吉林省卫生科技创新项目(2017J097)资助㊂作者简介:尹明姬,女,硕士,住院医师,主要从事小儿神经系统方面的研究,E⁃mail:mingji36@㊂通讯作者及指导教师:李今子,女,博士,主任医师,主要从事小儿神经系统方面的研究,E⁃mail:yjzli329@㊂[摘 要] 目的:探究天麻素对过氧化氢引起的神经细胞氧化损伤的保护作用及其作用机制㊂方法:培养SH⁃SY5Y 细胞,并分为3组:对照组㊁氧化损伤组和天麻素处理组㊂用流式细胞术检测细胞凋亡率及细胞活性氧(ROS)水平,用ELISA 检测细胞内超氧化物歧化酶(SOD)表达水平,用蛋白质免疫印迹实验检测细胞内PI3K㊁AKT 磷酸化水平㊂结果:过氧化氢处理引起细胞凋亡率升高,胞内ROS 平上升,SOD 表达下调以及PI3K㊁AKT 磷酸化水平下降;天麻素处理后可以显著抑制过氧化氢引起的细胞凋亡㊁ROS 水平上升及SOD 表达下调,并缓解氧化损伤对PI3K /AKT 磷酸化的抑制作用㊂结论:天麻素通过激活PI3K /AKT 信号通路保护过氧化氢诱导的神经细胞氧化损伤㊂[关键词] 天麻素;神经细胞;氧化损伤;癫痫Study on gastrodin protects neuron from hydrogen peroxide⁃induced oxidative damageYIN Ming⁃Ji ,CHI Yong⁃Xue ,LI Jin⁃Zi .The Affiliated Hospital of Yanbian University ,Yanji 133002,China[Abstract ] Objective :To investigate the effect of gastrodin on hydrogen peroxide⁃induced neuron oxidative damage,and toreveal the mechanism.Methods :Human neuroblastoma cell line SH⁃SY5Y was cultured and divided into three groups:control group,oxidative damage group and gastrodin treated group.The apoptotic cells and the reactive oxygen species were detected by flow cytometry;the SOD expression level was measured by ELISA assay;the phosphorylations of PI3K and AKT were measured by Western blot.Results :Hydrogen peroxide⁃induced oxidative damage caused cell apoptosis and ROS burst,while SOD expression level and PI3K /AKT phosphorylation were decreased.Gastrodin treatment decreased cell apoptosis rate and ROS levels,and reduced the inhibitoryeffects on PI3K /AKT phosphorylation by oxidative damage.Conclusion :Gastrodin protects neuron from hydrogen peroxide⁃inducedoxidative damage via PI3K /AKT signaling pathway.[Key words ] Gastrodin;Neuron;Oxidative damage;Epiepsy 癫痫是一类常见的中枢神经系统疾病,其主要发病表现为大脑海马区神经元异常电位变化引起短暂的中枢神经功能异常[1]㊂癫痫的发病受到多种遗传因素和环境因素的影响,但其深入的致病机制还未阐明[2]㊂既往研究已经表明,癫痫的发作常常伴随细胞氧化应激水平的升高,而且在癫痫的病理组织中,活性氧(reactive oxygen species,ROS)水平显著升高,因此氧化损伤与癫痫的发病密切相关[3,4]㊂天麻素(gastrodin)是兰科植物天麻块根中提取的生物活性物质,很多研究表明天麻素具有抗氧化㊁抗细胞凋亡㊁抗炎等功效,被用于癫痫的辅助治疗[5,6]㊂有研究表明天麻素可以通过调控p38/Nrf2信号通路抑制神经细胞SH⁃SY5Y 的凋亡[7,8],但其在癫痫治疗中的具体机制还需要深入研究㊂本研究通过建立过氧化氢诱导的SH⁃SY5Y 细胞氧化损伤模型,阐释了天麻素是否通过调控PI3K /AKT 信号通路缓解氧化损伤引起的细胞凋亡㊂1 材料与方法1.1 材料 细胞培养所需的DMEM 培养基㊁胎牛血清㊁青霉素/链霉素㊁Annexin⁃V /PI 凋亡检测试剂盒㊁细胞活性氧探针H2FCAD 均购自美国ThermoFisher 公司;人超氧化物歧化酶(superoxide dismutase,SOD)ELISA 试剂盒购自美国Abcam 公司,丙二醛(malondialdehyde,MDA)㊁乳酸脱氢酶(lactate dehydrogenase,LDH)及谷胱甘肽过氧化物酶(glutathione peroxidase,GSH⁃Px)ELISA 试剂盒均购自上海樊克生物技术公司;兔源抗Phospho⁃PI3Kinase p85(Tyr458)抗体购自美国CST 公司(#17366),鼠源抗p⁃Akt1/2/3(Thr308)抗体(#㊃6381㊃中国免疫学杂志2020年第36卷sc271964)㊁辣根过氧化物酶标记的山羊抗兔IgG及山羊抗鼠IgG均购自美国SantaCruz公司,天麻素购自美国Sigma公司,过氧化氢溶液购自上海生工公司㊂人视网膜神经母细胞瘤细胞系SH⁃SY5Y购自中国医学科学院细胞库SH⁃SY5Y细胞㊂1.2 方法1.2.1 细胞培养及实验分组 SH⁃SY5Y细胞培养在含有10%胎牛血清和青霉素/链霉素的DMEM培养基中,置于37℃㊁5%CO2培养箱中培养㊂设置对照组㊁氧化损伤组(H2O2)和天麻素处理组(H2O2+天麻素),其中对照组细胞在正常条件下培养,氧化损伤组细胞在培养基中加入200μmol/L过氧化氢(H2O2),天麻素处理组同时加入200μmol/L H2O2和10μmol/L天麻素㊂1.2.2 流式细胞术检测细胞凋亡 SH⁃SY5Y细胞经过胰酶消化㊁离心弃上清,用Annexin⁃V结合缓冲液重悬细胞,并调整细胞密度至1×106个/ml,取100μl细胞悬液加入5μl FITC⁃Annexin⁃V和1μl PI,室温孵育15min,加400μl磷酸盐缓冲液,随后使用流式细胞仪检测㊂1.2.3 流式细胞术检测细胞ROS水平 SH⁃SY5Y 细胞经胰酶消化㊁离心后用磷酸盐缓冲液重悬,清洗1次㊂随后加入终浓度1μmol/L H2DCFDA活细胞ROS指示剂,室温孵育30min,随后离心去除染料,用DMEM培养基重悬细胞,使用流式细胞仪检测488nm波长处激发荧光信号㊂1.2.4 ELISA 胰酶消化㊁离心并用磷酸盐缓冲液重悬SH⁃SY5Y细胞,置于冰上,使用超声破碎细胞,随后12000r/min离心10min,去除细胞碎片,保留上清细胞裂解液㊂每孔加入100μl细胞裂解液或标准品加入96孔包被板中,室温孵育2h后洗涤3次,后加入酶标试剂100μl,室温孵育1h后洗涤3次,分别加入显色液避光放置15min后加入终止液终止显色㊂用酶标仪读取每孔在450nm的A值,并通过标准曲线计算SOD浓度㊂1.2.5 蛋白质免疫印迹实验 细胞裂解液加入5×SDS上样缓冲液后,100℃沸水浴10min,得到的蛋白样品经过SDS⁃PAGE凝胶电泳分离,并使用湿转法将蛋白样品转移到PVDF膜上,随后用5%脱脂奶粉封闭PVDF膜,并分别孵育一抗和辣根过氧化物酶标记的二抗,洗涤3次后加入化学发光底物,在凝胶成像仪下显影㊂1.3 统计学分析 所以数据使用SPSS17.0软件进行统计分析,数据以x±s表示,并用t检验分析组间差异,以P<0.05为差异具有统计学意义㊂2 结果2.1 Annexin⁃V/PI双染法检测细胞凋亡水平 与对照组相比,H2O2处理细胞8h㊁24h后,SH⁃SY5Y 细胞凋亡水平显著上升(8h组:t=5.713,P= 0.0046;24h组:t=8.77,P=0.0009)㊂而H2O2联合天麻素处理组细胞相较于H2O2处理组细胞凋亡水平显著下降(8h组:t=2.883,P=0.0449;24h 组:t=4.145,P=0.0143),见表1㊂2.2 H2DCFDA染色检测细胞ROS水平 与对照组相比,H2O2处理显著提升了细胞内ROS水平(t=15.9, P<0.0001),而H2O2联合天麻素处理后细胞内ROS水平显著下降(t=7.294,P=0.0019),见图1㊁表2㊂2.3 ELISA检测细胞SOD㊁MDA㊁GSH⁃Px和LDH 含量 与对照组相比,H2O2处理引起的氧化损伤导致细胞内SOD表达水平下降(t=5.969,P=0.004),表1 流式细胞术检测各组细胞凋亡水平(x±s)Tab.1 Apoptotic rates in each group were measured by flow cytometry(x±s)Groups Apoptotic cells(%)8h24hControl4.31±0.734.91±0.64H2O28.72±1.141)17.67±2.441) H2O2+Gastrodin6.39±0.842)9.63±2.312) Note:Compared with control group,1)P<0.05;compared with H2O2 group,2)P<0.05.图1 流式细胞术检测SH⁃SY5Y细胞内ROS水平Fig.1 ROS levels in SH⁃SY5Y cells were measured by flow cytometry表2 流式细胞术检测各组细胞ROS水平(x±s)Tab.2 ROS levels in each group were measured by flow cytometry(x±s)Groups Mean fluorescence intensity(MFI)Control8.41±0.35H2O262.86±5.921)H2O2+Gastrodin34.48±3.222) Note:Compared with control group,1)P<0.05;compared with H2O2 group,2)P<0.05.㊃7381㊃尹明姬等 天麻素保护过氧化氢诱导的神经元氧化损伤的研究 第15期表3 ELISA 检测各组细胞SOD ㊁MDA ㊁LDH 和GSH⁃Px表达(x ±s )Tab.3 Expressions of SOS ,MDA ,LDH and GSH⁃Px ineach group were measured by ELISA (x ±s )Groups SOD(ng /ml)MDA(nmol /ml)LDH(ng /ml)GSH⁃Px(ng /ml)Control 2.48±0.311.56±0.2422.85±4.219.27±0.68H 2O 21.17±0.221)3.47±0.421)49.69±4.771)6.75±0.331)H 2O 2+Gastrodin 1.92±0.302)2.41±0.212)36.13±3.162)7.98±0.352)Note:Compared with control group,1)P <0.05;compared with H 2O 2group,2)P <0.05.图2 Western blot 检测各组细胞AKT ㊁PI3K 磷酸化修饰水平Fig.2 Expression of AKT and phosphorylation of PI3Kin each group were measured by Western blotNote:Compared with control group,*.P <0.05;compared with H 2O 2group,#.P <0.05.MDA 水平上升(t =6.839,P =0.0024),LDH 水平上升(t =7.307,P =0.0019),GSH⁃Px 水平下降(t =5.775,P =0.0045);而H 2O 2联合天麻素处理后细胞内SOD 表达水平有一定程度的回升(t =3.492,P =0.0251),MDA 水平下降(t =3.91,P =0.0174),LDH 水平下降(t =4.105,P =0.0148),GSH⁃Px 水平上升(t =4.429,P =0.014),见表3㊂2.4 蛋白质免疫印迹实验检测PI3K /AKT 信号通路 与对照组相比,H 2O 2处理引起的氧化损伤的细胞中,磷酸化AKT(p⁃AKT,分子量约60kD)蛋白表达水平下降(t =25.1,P <0.0001),磷酸化PI3K (p⁃PI3K,分子量约80kD)表达水平下降(t =39.35,P <0.0001);相较于H 2O 2单独处理组,H 2O 2联合天麻素处理组细胞p⁃AKT 表达水平回升(t =3.905,P =0.0175),p⁃PI3K 水平回升(t =4.11,P =0.0147)㊂3 讨论天麻素在癫痫治疗中的作用早有报道,并已经作为辅助药物应用于癫痫的临床治疗之中,然而天麻素作用于神经细胞的分子机制还缺少深入研究㊂本研究发现了天麻素在过氧化氢诱导的SH⁃SY5Y细胞的氧化损伤中能够发挥保护作用,抑制过氧化氢诱导的细胞凋亡㊂过氧化氢处理的细胞会产生大量自由基,进而引起细胞的氧化应激损伤和细胞凋亡㊂在癫痫的发病过程中通常会伴随着ROS 升高导致的神经细胞的氧化损伤[3]㊂细胞内高水平的ROS 可以引发细胞的氧化应激,使细胞内蛋白氧化失活㊁DNA 损伤以及线粒体等细胞器的功能紊乱[9]㊂过去的一些研究已经利用动物模型实验验证了天麻素的在治疗神经系统相关疾病中的作用,如在戊四唑诱导的小鼠惊厥模型中,天麻素可以抑制模型动物大脑中相关炎症因子如IL⁃1β㊁TNF⁃α等的表达,并缓解小鼠症状[10]㊂此外,天麻素还被发现能够抑制α⁃突触核蛋白以及β淀粉样蛋白在神经元中的积累,对神经退行性疾病如阿兹海默氏症㊁帕金森症中具有一定的治疗作用[11,12]㊂本研究发现,天麻素处理的SH⁃SY5Y 细胞可以通过上调SOD 和GSH⁃Px 的表达帮助细胞清除自由基,降低细胞内的ROS 水平,但天麻素是如何调控SOD 和GSH⁃Px 的表达还需要深入的研究㊂本研究还发现天麻素能够激活氧化损伤细胞的PI3K /AKT 信号通路㊂PI3K /AKT 是调控细胞活性的重要信号通路,PI3K 的磷酸化引发下游AKT 蛋白的激活,进而影响细胞有丝分裂,促进细胞的增殖活性[13]㊂FoxO3是细胞响应氧化应激的一个重要转录因子,在活性氧刺激的细胞中,FoxO3发生磷酸化从胞浆转运到细胞核中,并调控包括SOD 在内的多种靶基因的转录,进而影响细胞的氧化应激[14,15]㊂在神经干细胞中,激活的AKT 能够磷酸化FoxO3,并促进其向细胞核转运[16]㊂因此可以推测,在本研究所使用的过氧化氢诱导的SH⁃SY5Y 的氧化损伤模型中,天麻素很可能通过PI3K /AKT 信号通路激活FoxO3并上调SOD 表达,清除细胞内活性氧,抑制细胞凋亡㊂最近有研究表明,炎症反应在癫痫发病过程中也发挥了重要作用,而神经元细胞的氧化损伤也是激活神经系统免疫反应的主要因素之一[17]㊂天麻素是否能够通过对免疫反应的调控影响癫痫的疾病进展还需深入探讨㊂参考文献:[1] Xiang J,Wen F,Zhang 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天麻和天麻素改善糖脂代谢紊乱的药理学研究进展
[] 天麻和天麻素具有镇痛、镇静、改善神经紊乱等药理作用，目前临床上主要用于神经系统疾病的治疗。近年来的研究发现，天麻和天麻素还能有效调控糖脂代谢，具有显著的降血脂、改善非酒精性脂肪性肝病NAFLD、减肥、降血糖、增加胰岛素敏感性以及抑制氧化应激和炎性反应的作用，具有广阔的应用前景。因此，本文对天麻和天麻素改善糖脂代谢紊乱的药理学进行了综述。
天麻素改善胰岛素抵抗、增加胰岛素敏感性的机制尚不明确。有研究发现同型半胱氨酸水平的增高与糖尿病、胰岛素抵抗的发生密切相关[2728] 胱硫醚合成酶CBS是同型半胱氨酸的代谢酶，其表达下降会导致同型半胱氨酸水平的增高[2728]。有报道在高糖高脂饲料诱导的SD大鼠胰岛素抵抗模型中，剂量为60 mgkg的天麻素腹腔注射给药1个月能显著上调肝脏组织中CBS基因的mRNA和蛋白表达水平，并使血浆中同型半胱氨酸的浓度显著下降，提示天麻素可能通过减少同型半胱氨酸来发挥改善胰岛素抵抗的作用[29]。
天麻和天麻素不仅在动物实验中具有降血脂的作用，其临床上的降血脂疗效也有报道。见表1。如口服天麻细粉片6.75 gd用于治疗单纯性高脂血症，45 d后患者血清中TC和TG水平出现有统计学意义的下降[15]。另有临床研究报道，天麻素口服或静脉滴注用于治疗单纯性高脂血症或合并脑梗死、眩晕症的高脂血症患者，均能有效降低TC、TG和LDLC[13，1617]。其中有一篇报道发现天麻素能显著增加患者血清HDLC的水平[16]，而其他临床报道和动物实验均不支持，提示天麻和天麻素对HLDC代谢的影响需要深入研究。天麻和天麻素用于临床治疗高脂血症安全性良好，未发现明显不良反应。见表1。
天麻素用于治疗DM也有良好的抗氧化作用。在STZ诱导的昆明种小鼠DM模型中，天麻素灌胃给药能显著增加动物血清中的SOD活力并减少MDA含量[14]。在INS1细胞中，天麻素作用后可抑制STZ诱导的细胞氧化应激，使TAOC显著升高、MDA显著减少，从而发挥细胞保护作用[26]。
天麻素通过激活AMPK通路减少油_省略_诱导的HL_7702细胞脂肪蓄积_耿雅娜
网络出版时间:2014－12－413:45网络出版地址:http ://www．cnki．net /kcms/doi /10．3969/j．issn．1001－1978．2015．01．010．html天麻素通过激活AMPK 通路减少油酸诱导的HL-7702细胞脂肪蓄积耿雅娜，于滨，孔维佳(中国医学科学院医药生物技术研究所，北京100050)doi ：10．3969/j．issn．1001－1978．2015．01．010文献标志码：A 文章编号：1001－1978（2015）01－0039－06中国图书分类号：Ｒ284．1；Ｒ322．47；Ｒ329．24；Ｒ344．3；Ｒ575.5；Ｒ589.2摘要：目的研究天麻素（GSTD ）对油酸（OA ）诱导的HL-7702细胞脂肪蓄积的抑制作用，并探讨其可能的细胞信号通路。
方法以噻唑蓝（MTT ）法测定GSTD 对HL-7702细胞存活率的影响；以1mmol ·L －1的OA 处理24h 诱导细胞脂肪变性，同时加入不同浓度的GSTD ，油红O （OＲO ）染色观察细胞脂肪蓄积情况并测定细胞内三酰甘油（TG ）含量；以Western blot 检测GSTD 处理后细胞中AMPK α和ACC 的磷酸化水平；以compound C 处理细胞，研究其对GSTD 药效的影响。
结果GSTD 浓度≤3386.5μmol ·L－1时对HL-7702细胞没有明显毒性。
OA 处理24h 后细胞中出现大量脂滴蓄积，TG 含量明显增加，但同时加入浓度为169.3或338.7μmol ·L －1的GSTD 可明显抑制HL-7702细胞的脂肪蓄积，并使TG 含量分别平均下降35%和43.6%（与单加OA 的细胞比较P ＜0.01）。
GSTD 处理后能时间和浓度依赖性地增加细胞中的p-AMPK α和p-ACC 水平，compound C 能完全阻断GSTD 对AMPK 通路的激活作用以及减少肝细胞TG 蓄积的作用。
Nrf2通路在天麻素抑制油酸诱导的HL—7702
Nrf2通路在天麻素抑制油酸诱导的HL—7702作者：曲丽丽于滨孔维佳来源：《中国医药导报》2016年第19期[摘要] 目的研究核因子E2相关因子2（Nrf2）在天麻素（GSTD）抑制油酸（OA）诱导的肝细胞氧化应激中的作用。
方法体外培养HL-7702细胞，以Nrf2特异性的小干扰RNA （siRNA）转染细胞以沉默其表达，以浓度为100 μg/mL的GSTD处理细胞24 h后提取细胞总蛋白以及核、质蛋白，以免疫印迹法检测Nrf2和血红素加氧酶-1（HO-1）的表达水平。
在抗氧化实验中，Nrf2 siRNA转染后以浓度为0.6 mmol/L的OA与GSTD共同处理细胞24 h，以试剂盒检测细胞内超氧化物歧化酶（SOD）的活性以及活性氧簇（ROS）和丙二醛（MDA）的含量。
结果与对照组细胞比较，Nrf2 siRNA转染细胞后Nrf2总蛋白、细胞核内Nrf2蛋白和HO-1蛋白的基础表达水平均显著下降（P < 0.01）。
与对照组细胞比较，GSTD使细胞核内Nrf2蛋白和HO-1蛋白的表达水平分别平均增加约90.2%和80.3%（P < 0.01）。
与单用GSTD 处理的细胞比较，GSTD促进Nrf2蛋白细胞核转位和上调HO-1表达的作用能被Nrf2 siRNA 完全阻断（P < 0.001）。
与对照组细胞比较，OA处理后可诱导细胞产生氧化应激（P <0.01）；与单加OA的细胞比较，GSTD与OA共同处理细胞后能使SOD的活力显著增加，ROS和MDA的水平显著下降（P < 0.01）；与OA+GSTD组细胞比较，GSTD的抗氧化作用能被Nrf2 siRNA完全阻断（P < 0.001）。
结论 Nrf2通路在GSTD抑制OA诱导的HL-7702细胞氧化应激的活性中发挥关键作用。
[关键词] 天麻素；核因子E2相关因子2；基因沉默；油酸；氧化应激[中图分类号] R965 [文献标识码] A [文章编号] 1673-7210（2016）07（a）-0010-04[Abstract] Objective To investigate the role of nuclear factor erythroid-2-related factor-2（Nrf2） in the activity of gastrodin （GSTD） in suppressing oleic acid- （OA-） induced oxidative stress in hepatocytes. Methods HL-7702 cells were cultured in vitro， small interfering RNA （siRNA） specific for Nrf2 was used to transfect the cells in order to silence its expression. GSTD at a concentration of 100 μg/mL was used to treat the cells for 24 h； cell total proteins，nuclear and cytoplasmic proteins were extracted for Western blot analysis of the expression levels of Nrf2 and heme oxygenase-1 （HO-1）. In the antioxidant experiments， cells were treated with 0.6 mmol/L of OA together with GSTD for 24 h after Nrf2 siRNA transfection. Intracellular superoxide dismutase （SOD） activity， reactive oxygen species （ROS） and malondialdehyde （MDA）levels were determined by kits. Results Compared with the control cells， after the transfection of Nrf2 siRNA， the cellular baseline expression levels of total Nrf2， nuclear Nrf2 and HO-1 proteins declined greatly （P < 0.01）. Compared with the control cells， GSTD increased the nuclear Nrf2 protein and HO-1 protein levels averagely by 90.2% and 80.3%， respectively （P < 0.01）.Compared with cells treated with GSTD alone， the stimulating activities of GSTD on the nuclear translocation of Nrf2 protein and the expression of HO-1 were totally blocked by Nrf2 siRNA （P < 0.001）. Compared with the control cells， the cells developed oxidative stress after the administration of OA （P < 0.01）. Compared with cells treated with OA alone， when GSTD was co-administered with OA， the SOD activity increased but the levels of ROS and MDA decreased greatly in HL-7702 cells （P < 0.01）. Compared with cells treated with OA+GSTD， the antioxidant activity of GSTD could be blocked by Nrf2 siRNA completely （P < 0.001）. Conclusion The Nrf2 pathway plays a critical role for the activity of GSTD in suppressing OA-induced oxidative stress in HL-7702 cells.[Key words] Gastrodin； Nuclear factor erythroid-2-related factor-2； Gene silencing； Oleic acid； Oxidative stress天麻素（gastrodin，GSTD）是中药天麻（Gastrodia elata Bi.）的主要有效成分之一，目前在临床上主要用于神经系统疾病如癫痫、头痛等的治疗[1]。
Nrf2在液压冲击脑损伤抗神经细胞凋亡中的作用以及姜黄素对其的影响的开题报告
Nrf2在液压冲击脑损伤抗神经细胞凋亡中的作用以及姜黄素对其的影响的开题报告1. 研究背景液压冲击脑损伤是一种常见的枪击、爆炸等暴力事件引起的脑部损伤，在临床上难以治愈，损伤后往往引起神经细胞凋亡、脑部炎症等不良反应，严重影响患者的生命质量和健康。
因此，寻找液压冲击脑损伤的治疗方法是当前研究的热点之一。
2. 研究问题Nrf2是一种重要的转录因子，可通过调节抗氧化反应、抗炎反应等方式参与细胞的应激反应。
然而，目前对Nrf2在液压冲击脑损伤中的作用还不清楚。
本研究旨在探究Nrf2在液压冲击脑损伤抗神经细胞凋亡中的作用及其调节机制。
另外，姜黄素是一种常用的中药成分，具有抗氧化、抗炎、抗肿瘤等多种生物活性。
近年来的研究表明，姜黄素可以影响Nrf2通路的调节，但其对Nrf2在液压冲击脑损伤中的作用尚未明确。
因此，本研究还将探究姜黄素对Nrf2通路的调节及其对液压冲击脑损伤的治疗作用。
3. 研究方法实验中将采用液压冲击脑损伤模型，以大鼠为实验对象，分别对正常组、液压冲击脑损伤组、液压冲击脑损伤+姜黄素组、液压冲击脑损伤+Nrf2干扰组进行分组探究。
实验中将采用细胞学和分子生物学等方法对各组进行生化指标、病理学变化、细胞凋亡等方面的分析，以探究Nrf2在液压冲击脑损伤中的作用及姜黄素对其的影响。
4. 研究意义本研究可为液压冲击脑损伤的治疗提供新的思路和方法。
探究Nrf2在液压冲击脑损伤中的作用及姜黄素对其的影响，有助于深入理解Nrf2调节机制，并为中药药理学的研究提供新的依据。
同时，本研究也将延伸Nrf2在神经退行性疾病中的作用、抗氧化和抗炎反应机制等方面的研究，进一步拓展Nrf2的应用领域。
基于Nrf2抗氧化通路的表观遗传调控机制的研究进展
基于Nrf2抗氧化通路的表观遗传调控机制的研究进展张志城苏庆盛指导老师：邹志辉摘要：当今环境问题日益突出，对人体健康的影响越来越大，机体自身有一定的防御能力，但一旦超过其所能承受范围，机体就会患病，甚至死亡。
Nrf2是一种氧化应激基本表达的关键转录因子，存在于全身多个器官，它的缺失或激活障碍直接引起细胞对应激源的敏感性变化。
表观遗传（epigenetics）是指DNA 序列不发生变化，但基因表达却发生了可遗传的改变。
这种改变是细胞内除了遗传信息以外的其他可遗传物质发生的改变，且这种改变在发育和细胞增殖过程中能稳定传递。
在表观遗传调控机制中，有众多的基因能间接或直接的调控Nrf2抗氧化通路。
现就基于Nrf2抗氧化通路的表观遗传调控机制的研究进展作一综述。
关键词：Nrf2；表观遗传；抗氧化通路；DNA甲基化；组蛋白修饰；miRNA机体与外界的持续接触时，包括呼吸（氧化反应）、外界污染、放射线照射等因素不断的在人体体内产生自由基。
科学研究表明，癌症、衰老或其它疾病大都与过量自由基的产生有关联。
自由基具有强氧化性，可直接或间接地损害细胞内蛋白质、脂质、核酸等大分子物质的生理功能，是众多疾病发生的病理生理基础。
而近年来的研究发现，核因子NF-E2相关因子（nuclear factor erythroid2-related factor，Nrf2）是外源性有毒物质和氧化应激的感受器，是细胞众多抗氧化反应的中枢调节者，在参与细胞抗氧化应激和外源性有毒物质诱导的主要防御机制中发挥重要的作用。
表观遗传（epigenetics）的两个主要方面DNA甲基化、组蛋白修饰对调控基因表达有相当重要的作用,近年来发现非编码MicroRNA（miRNA）在基因调控中也有十分重要地位，miRNA广泛存在于生物体内、长度在19~25nt之间、高度保守的一类非编码小RNA，通过与靶基因信使RNA特定区域不同程度的互补结合而引起后者的翻译抑制或降解,从而在转录后水平调控基因表达,是包括细胞增殖、分化、发育、免疫调节、凋亡等在内的众多生物学进程的重要调节因子[1-2]。
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Nrf2通路在天麻素抑制油酸诱导的HL―7702
天麻素（gastrodin，GSTD）是中药天麻（Gastrodia elata Bi.）的主要有效成分之一，目前在临床上主要用于神经系统疾病如癫痫、头痛等的治疗[1]。
笔者实验室前期的研究工作发现天麻和GSTD具有降血脂、降血糖和改善肝脏脂肪变性的药理学作用[2-9]。
肝细胞的脂肪变性是非酒精性脂肪性肝病（NAFLD）的最主要病理学特点。
在NAFLD的发生和发展过程中，氧化应激起着非常关键的作用[10]。
笔者实验室近期研究发现，GSTD在NAFLD的体内外模型中均可显著抑制氧化应激[11]。
GSTD可通过腺苷酸活化蛋白激酶（AMPK）依赖的方式来激活肝细胞中的核因子E2相关因子2（Nrf2）通路，促进Nrf2的细胞核转位[11]。
Nrf2是一种抗氧化转录因子，被激活后能进入细胞核，促进其靶基因如血红素加氧酶-1（HO-1）的表达，从而发挥抗氧化作用[12-13]。
笔者实验室上述的研究工作阐明了AMPK在GSTD的抗氧化活性中所发挥的关键作用，但Nrf2的作用仍需进一步确证。
本研究以Nrf2特异性的小干扰RNA（siRNA）来沉默其表达，并观察对GSTD抗氧化药效的影响。
1 材料与方法
1.1 材料和仪器
GSTD购自浙江诚意药业XX公司，批号20120509，纯度≥98%。
油酸（OA）和牛血清白蛋白（BSA）购自美国Sigma公司。
胎牛
血清（FBS）、DMEM培养基、非必需氨基酸、1%双抗（青霉素与链霉素）购自美国GIBCO公司。
人源的Nrf2 siRNA购自美国Santa Cruz公司；X-treme Gene siRNA Transfection Reagent购自美国R&D公司；Opti-MEMR培养基购自美国Invetrogen公司。
M-PERR Mammalian 蛋白裂解液、HaltR蛋白酶&磷酸酶双重抑制剂、细胞核质蛋白分离提取试剂盒、BCA蛋白浓度测定试剂盒购自美国Thermo Scientific公司。
Nrf2、HO-1、β-肌动蛋白（ACTB）、组蛋白H3（histone H3）的单克隆抗体购自美国Cell Signaling 公司。
PVDF膜购自美国Millipore 公司。
总超氧化物歧化酶（SOD）活性检测试剂盒、丙二醛（MDA）检测试剂盒和活性氧（ROS）检测试剂盒均购自上海碧云天生物技术XX公司。
1.2 实验方法
1.2.1 细胞培养 HL-7702细胞常规培养于含10%FBS、1%非必需氨基酸和双抗（100 U/mL青霉素+100 U/mL链霉素）的DMEM 培养基中，在37°C含5% CO2的培养箱中培养。
每次实验前将细胞按2×105个/孔接种至6孔板，待长至70%～80%满时，换成无血清培养基，饥饿24 h后进行实验。
1.2.2 siRNA沉默实验用X-treme Gene siRNA Transfection Reagent按照试剂盒说明，将Nrf2 siRNA转染细胞。
转染6 h后，弃去原培养基，换为含10%FBS的完全培养基，继续培养24 h，细胞饥饿后进行相应处理。
1.2.3 药物处理 GSTD以灭菌生理盐水溶解，浓度为10
mg/mL，以DMEM培养基稀释100倍后（100 μg/mL）处理细胞，时间为24 h。
OA以灭菌磷酸缓冲盐溶液（PBS）+5%BSA溶解，制备成浓度为6 mmol/L的储备液，分装后-20℃保存。
在GSTD 的抗氧化实验中，OA以DMEM培养基稀释10倍（0.6 mmol/L）后与GSTD共同处理细胞24 h。
实验中每个处理孔重复3～4次。
1.2.4 免疫印迹实验使用相应试剂盒按照说明书要求提取细胞总蛋白或分离细胞核质蛋白；蛋白定量后，使用含30 μg 蛋白的样品以10%分离胶进行聚丙烯酰胺凝胶电泳。