【作者】 李晓楠；

【导师】 张运； 沈胡英；

【作者基本信息】 山东大学 ， 内科学， 2009， 博士

【摘要】 论文Ⅰ游离脂肪酸诱导血管内皮细胞活性氧产生的机制研究背景游离脂肪酸介导的氧化应激在代谢综合征心血管疾病的发生发展中起关键作用。过量活性氧（reactive oxygen species,ROS）的产生可通过直接氧化损伤DNA,蛋白质,脂质等大分子物质,或激活NF-κB,p38 MAPK,JNK等信号通路引起组织损伤和细胞功能异常。因此有效降低ROS产生,增加体内抗氧化能力对预防糖尿病心血管并发症的发生具有重要作用。AMPK可启动分解代谢途径从而增加ATP的产生,因此在能量代谢调控中发挥重要作用。目前研究证实AMPK信号通路的激活具有保护心血管效应。但机制不明。以往研究表明活性氧可激活AMPK通路。同时AMPK的激活可有效降低细胞内ROS水平。但是,AMPK通路的激活能否降低棕榈酸（palmitic acid,PA）介导的人血管内皮细胞内ROS产生的水平尚不清楚。硫氧还蛋白（thioredoxin,Trx）系统清除活性氧及还原二硫键的功能使该系统成为维持细胞氧化还原稳态的重要因素。许多研究表明,Trx在多种刺激（特别是氧化应激）下可诱导其表达的特性,提示Trx是细胞抵抗氧化应激的重要分子,能保护组织或细胞免受多种刺激的损伤。然而,Trx是否参与AMPK通路对细胞内ROS产生的调节以及AMPK通路如何调节Trx的表达,均需要进一步的验证。近年研究发现,FOXO转录因子在细胞周期、新陈代谢、DNA修复、细胞凋亡、抵抗氧化应激、长寿等方面发挥重要的调节作用。最近研究发现,FOXOs还可直接调节Gadd45和锰超氧化物歧化酶（manganese superoxide dismutase,MnSOD）基因表达,保护静息细胞免受氧化损伤,促使DNA修复。FOXO转录因子在细胞对氧化应激反应中的作用已部分阐明。现有研究表明ROS可激活FOXO,但同时FOXO也可抵抗生理性氧化应激,增加细胞存活。但是,FOXO是否为Trx的转录调节因子,以及AMPK通路对FOXO的转录活性的影响尚不明确。针对以上问题,我们提出如下假说:AMPK通路的激活能够通过增强抗氧化剂Trx的表达而降低细胞内ROS产生的水平。FOXO作为转录因子参与了该过程的调控。AMPK-FOXO信号通路可能是减少体内氧化应激损伤的重要防御机制,是治疗代谢综合征心血管疾病的潜在治疗靶点。方法1.细胞培养:原代人主动脉内皮细胞（human aortic endothelial cells,HAECs）,使用内皮细胞生长培养基-2（endothelial cell growth medium-2,EGM-2）,在37℃和5%CO₂孵育箱中进行培养。细胞被转染siRNAs,或者被刺激以不同浓度的AICAR或者PA以构建细胞模型。2.脂肪酸白蛋白复合物的配制:将可溶性棕榈酸（PA）溶解在200mM的酒精当中,并与10%的无游离脂肪酸,低内毒素的BSA混合,使其终浓度在1-5mM。所有溶液的PH值调整在7.5左右,使用滤器消毒,-20℃储存。将不含有PA的BSA溶液作为对照。在处理细胞之前,将储存的PA按照1:10的比例,使用培养基进行稀释。3.siRNA诱导的基因沉默:使用特异的siRNA进行基因沉默,包括AMPKsiRNA,Trx siRNA和FOXO3a siRNA。使用LipofectAMINE^TM 2000脂质体包裹,并转染HAECs。被转染的HAECs细胞,再使用PA和AICAR等进行处理。4.质粒DNA的转染:分别使用0.5μg野生型FOXO3表达质粒（HA-FOXO3aWT）,负显性型FOXO3a表达质粒（dominant-negative HA-FOXO3a TM delta DM,DN）和持续激活型FOXO3a表达质粒（constitutively active HA-FOXO3a TM,CA）处理内皮细胞。首先使用LipofectAMINE^TM 2000脂质体包裹,并转染HAECs。被转染的HAECs细胞,再使用PA和AICAR等进行处理。5.免疫荧光染色:将处理好的细胞使用含有一抗1%BSA进行孵育,并用得克萨斯红标记的二抗进行处理。使用DAPI染核,装备有Leica DC 100数字相机的Leic DMLS荧光显微镜采集图像。应用Image-Pro Plus V4.5分析软件进行分析。6.蛋白印迹分析:使用细胞裂解液提取细胞的总蛋白。将含有15μg微粒蛋白的每个标本和蛋白Marker一起分别加入不同的泳道。在10%SDS-聚丙烯酰胺凝胶上电泳移动,再用一湿电转染仪电转染到聚偏氟乙烯膜（PVDF）上。将膜封闭后,使用一抗孵育,清洗后,再使用HRP结合的二抗孵育。滴加显色液显色。强弱用表达的蛋白和β-actin条带积分光密度的比值表示。7.实时定量PCR:使用Trizol提取细胞的总RNA。应用iScript cDNA合成酶将mRNA逆转录成cDNA。使用iCycler iQ荧光探针系统进行实时PCR。采用Trx与β-actin的循环阈值（threshold cycle,Ct）的比值并通过公式2△^Ct（△Ct=β-actin Ct-gene of interest Ct）表示mRNA的相对水平。8.细胞内ROS的检测:应用CM-H₂DCFDA荧光探针检测细胞内ROS水平。使用装备有Leica DC 100数字相机的Leic DMLS荧光显微镜采集图像。应用Image-Pro Plus V4.5分析软件进行分析。9.Trx活性分析:采用胰岛素二硫还原法分析Trx的活性。应用412 nm的吸收光谱进行测量。Trx活性使用与对照组的相对比值进行表示。10.染色质免疫沉淀分析:我们使用Upstate公司生产的染色质免疫沉淀分析试剂盒。首先将处理过的HAECs用甲醛孵育,以形成DNA-蛋白质复合体。应用特异抗体-蛋白A-琼脂糖浆对DNA-蛋白质复合体进行免疫沉淀（IgG作为阴性对照）。将这些免疫复合物珠子进行清洗,沈脱和解链。使用苯酚/氯仿/易戊酯酒精混合物萃取DNA片段。然后,将免疫沉淀的DNA片段作PCR,使用1.5%的琼脂糖胶分离PCR产物。11.免疫沉淀分析:将处理过的细胞在冰上裂解60分钟。细胞裂解液与抗体和蛋白A/G-琼脂糖珠子共同孵育4℃过夜以进行免疫沉淀。使用提取液清洗珠子两遍,然后再使用含有0.5 M氯化锂的提取液清洗两遍。使用SDS样本试剂直接洗脱蛋白质,然后进行蛋白印迹分析。12.激酶活性分析:使用AMPK特异性抗体将AMPK从细胞裂解液中沉淀出来。并将含AMPK的免疫磁珠与重组FOXO3在含有100μM ATP的激酶活性分析液中孵育20分钟,温度控制在30℃。将样本跑胶转膜,使用抗苏氨酸和丝氨酸抗体进行标记。结果1.AMPK降低PA所致的血管内皮细胞ROS升高:AICAR本身对基础ROS水平影响很小。PA能显著增加细胞内ROS水平。AICAR可降低PA所致的细胞内ROS水平的升高,呈剂量依赖性,最高浓度500umAICAR可降低ROS水平高达60%。同时用特异小分子干扰核糖核酸（siRNA）来抑制AMPK表达不仅增加基础ROS水平,同时增加PA所致的ROS水平增高。AICAR诱导的ROS减低可由AMPK SiRNA阻断。2.AMPK增加抗氧化剂Trx的表达:AICAR引起的AMPK激活无论是在PA存在还是不存在的情况下均可显著增加Trx的表达。PA本身可暂时性引起Trx表达的增加,但长期PA处理可降低Trx表达。用特异siRNA敲除AMPK可抑制基础状态Trx的表达及AICAR引起的Trx表达增加。3.Trx沉默可阻止AICAR诱导的ROS降低:Trx siRNA不仅增加基础ROS水平,同时增强PA所致的ROS水平的增加。同时Trx siRNA阻止AICAR诱导的ROS降低。4.AMPK在转录水平调节Trx表达:通过实时定量PCR,发现AICAR可显著增加Trx mRNA并呈剂量依赖性。特异性siRNA敲除AMPK可降低基础Trx表达。同时降低AICAR诱导的Trx mRNA水平。5.FOXO3a参与AMPK诱导的Trx上调:Trx基因5‘端含有多个高度保守的转录因子结合区域。我们验证转录因子FOXO3是否参与AMPK诱导的Trx表达。特异性siRNA敲除AMPK可阻止AICAR诱导的Trx表达,无论是在蛋白还是转录水平。6.FOXO3a直接与Trx启动子结合:为验证FOXO3a是否直接调节Trx转录,我们在体外验证FOXO3a是否直接与Trx启动子结合。FOXO3a的结合位点是5’-（C/G）（A/T）AAA（C/T）A,-3’。Trx基因启动子区域包含6个FOXO3a结合位点,（tgAAAGAgtga at-1346/-1342,tgAAAGAagga at-1339/-1335,gaAAACAcagaat-1236/-1232,caAAATAccgc at-859/-855,ggAAACActga at-807/-803,和tgAAAGAacag at-613/-609）。CHIP检测结果显示FOXO 3a与位点4和位点5结合能力弱,但与位点6结合能力强。更重要的是AICAR处理可显著增加FOXO 3a与位点6的结合,表明FOXO3a参与了Trx的转录调节。7.AMPK增加FOXO3a的核转位:免疫荧光显示AICAR显著增加FOXO3a的核转位同时AMPK SiRNA可阻止这一过程。同时我们检测AMPK是否能直接磷酸化FOXO3a在体外激酶检测中,以纯化的AMPK作为激酶,重组FOXO3a作为底物,结果显示AMPK可直接在丝氨酸和缩氨酸位点磷酸化FOXO3a,AICAR可增加FOXO3a的磷酸化。以上研究表明FOXO3a可直接被AMPK磷酸化,进而发生核转位,与Trx启动子结合,增加Trx转录。8.FOXO3a结合p300在Trx启动子区形成转录激活复合物:ChIP检测发现AICAR处理可显著增加p300与Trx启动子在保守位点6的结合。我们进一步验证体内FOXO3是否与p300相关。采用免疫共沉淀方法证实了FOXO3与p300相关,AICAR处理可显著增加这种相关性,表明p300与Trx启动子区域的结合至少部分是由FOXO3介导的。同时采用双CHIP检测FOXO3与p300是否在Trx启动子区域同一转录复合体内。首先用FOXO3抗体进行免疫沉淀,后用p300进行免疫沉淀。在免疫复合物内相关的DNA由PCR扩增。结果表明FOXO3与p300在Trx启动子同一区域内形成转录激活复合物。结论1.PA能显著的增加人主动脉内皮细胞内ROS水平。2.AMPK激活可通过上调Trx表达显著降低PA所致的细胞内ROS的升高。3.转录因子FOXO3a介导了AMPK对Trx的调节。4.AMPK通过增加FOXO3a核转位,促进其DNA与Trx启动子区域结合而增加Trx的转录。5.AMPK-FOXO3a信号通路对代谢应激下细胞内超氧化物水平有保护作用,是治疗糖尿病心血管并发症的潜在治疗靶点。更多还原

【Abstract】 BaekgroudOxidative stress induced by free fatty acids（FFA） plays a key role in the development of cardiovascular diseases in metabolic syndrome.Excessive generation of reactive oxygen species（ROS） can cause tissue injury and cellular dysfunction by directly oxidizing and damaging DNA,proteins,and lipids as well as by activating several cellular stress-sensitive pathways such as nuclear factor-kappa beta,p38 MAPK,and JNK.Reducing ROS production and increasing antioxidant availability are important in preventing diabetic cardiovascular complications.The AMPK pathway responds to energy depletion by stimulating metabolism for ATP generation and plays an important role in controlling metabolism.It has been increasingly recognized that activation of this pathway has vascular protective effects; however,the mechanisms involved are not completely understood.Reactive oxygen species can activate the AMPK pathway.Previous studies have shown that activation of the AMPK pathway reduces intracellular ROS levels.However,it is still unknown whether the activation of the AMPK pathway may affect PA（palmitic acid） induced intracellular ROS levels in human aortic endothelial cells.The regulation of cellular redox balance is critically determined by the activity of Trx（thioredoxin） system.Some studies have shown that the expression of Trx was dramatically increased by several stimuli,especially the oxidative stress.It seems that Trx is an important antioxidant protein and protect the tissues and cells from several oxidative stress injuries.It is unclear that whether Trx is involved in the AMPK pathway reduced intracellular ROS production and how the AMPK pathway regulates the expression of Trx.FOXO transcription factors are important regulators of metabolism,cell-cycle progression,DNA repair,apoptosis,oxidative stress resistance,and longevity.The role of FOXO in regulating cellular functions under oxidative stress has been partially clarified.Recent findings found that FOXOs could regulate the expression of Gadd45 and MnSOD（manganese superoxide dismutase） directly and protect cell from oxidative injure,thus promote the DNA damage repair.This suggests that ROS can activate FOXO.Although FOXOs mediate ROS-induced apoptosis under lethal conditions,they can increase cell survival in response to physiologic oxidative stress, a function that is required for long-term regenerative potential and cell longevity. However,whether FOXO is the transcriptional factor of Trx and whether the AMPK pathway affects the activity of FOXO are still unclear.In the present study,we hypothesized that the activation of AMPK pathway may reduced the intracellular ROS levels by increasing the expression of antioxidant Trx. FOXO may be the transcriptional factor of Trx.Thus,the AMPK-FOXO pathway may be an important defense mechanism against excessive ROS levels induced by metabolic stress and could be a therapeutic target in treating cardiovascular diseases in metabolic syndrome.Methods1.Cell culture:Primary human aortic endothelial cells（HAECs） were cultured at 37℃in 5%CO₂ in endothelial cell growth medium-2（EGM-2）.The cells were transfected with siRNAs,treated with the AICAR or PA at various concentrations for the time periods indicated in the text.2.Preparation of fatty acid-albumin complexes:Saturated palmitic acid（PA） was dissolved in ethanol at 200 mM and then combined with 10%FFA-free, low-endotoxin BSA to final concentrations of 1-5 mM.The pH of all solutions was adjusted to approximately 7.5,and the stock solutions were filter-sterilized and stored at-20℃until used.Control solutions containing ethanol and BSA were prepared similarly.Working solutions were prepared fresh by diluting the stock solution（1:10） in 2%fetal calf serum-EBM.There was 1%BSA in all PA media;however,the PA/BSA ratio varied with the PA concentrations.3.siRNA-indueed gene silencing:Silencing gene expression was achieved using specific siRNA including,AMPK siRNA,Trx siRNA and FOXO3a siRNA. Transfection of HAECs with siRNAs was carried out using LipofectAMINE^TM 2000, according to the manufacturer’s instruction.Transfected cells were then treated with PA and AICAR at the designated concentrations for the time periods indicated in the text.4.In Vitro Transfection of Plasmid DNA:HAECs were transfected with 0.5ug of FOXO3a plamid DNA including wide type（HA-FOXO3A WT）,constitutively active（HA-FOXO3a TM） and dominant-negative（HA-FOXO3a TM deltaDM） The transfection was carried out using LipofectAMINETM 2000 according to the manufacturer’s instructions.Transfected cells were then treated with PA and AICAR at the designated concentrations for the indicated time periods.5.Immunofluorescent staining and microscopy:Cells were grown on glass coverslips and treated with PA and AICAR.The coverslips were blocked with 1% BSA,incubated with the primary antibody,washed with PBS,and then incubated with Texas Red-labeled secondary antibody.The cells were incubated for 15 minutes with the DNA stain,4’,6-diamidino-2-phenylindole dihydrochloride（DAPI;0.1 ug/mL）.The slides were examined with a Leica DMLS epifluorescence microscope equipped with a Leica DC 100 digital camera,and the data were analyzed with Image-Pro Plus V4.5 software.6.Western blot analysis:Cell extracts were prepared with lysis buffer.Protein samples（15μg per lane） were subjected to SDS-polyacrylamide gel electrophoresis and transferred to PVDF membranes.The membranes were blocked,treated with primary antibody,washed,and then incubated with the secondary horseradish peroxidase-labeled antibody.Bands were visualized with Enhanced Chemiluminescence.The expression of cytokine protein was demonstrated by the ratio of integral optical density（IOD） between cytokine andβ-actin.7.Real-time quantitative PCR:Total RNA from treated cells was extracted with Trizol,according to the manufacturer’s protocol.The mRNAs were reverse-transcripted into cDNAs using iScript cDNA synthesis kit.Real-time PCR was performed using iCycler iQ real-time PCR detection system.The mRNA levels were estimated from the value of the threshold cycle（Ct） of the real-time PCR adjusted by that ofβ-actin through the formula 2^ΔCt（ΔCt=β-actin Ct-gene of interest Ct）.8.Intracellular ROS level detection:Intracellular ROS level was determined using the oxidant-sensitive fluorogenic probe CM-H₂DCFDA（5-（and-6）-chloromethyl-2’,7’-dichlorodihydrofluorescein diacetate,acetyl ester）.The slides were examined with a Leica DMLS epifluorescence microscope equipped with a Leica DC 100 digital camera and the data were analyzed with Image-Pro Plus V4.5 software.9.Trx Activity Assay:Trx activity was measured using the insulin disulfide reduction assay.The absorption at 412 nm was measured spectroscopically.T10.Chromatin immunoprecipitation assay:We used the ChIP assay kit, according to the Upstate Chromatin Immunoprecipitation Protocol.In brief,treated HAECs were first incubated with formaldehyde to cross-link DNA-protein complexes. Protein-DNA complex was immunoprecipitated with antibody-protein A-agarose slurry.（IgG served as the negative control.） The immunocomplex beads were washed, eluted and reversed the cross-link.The DNA was recovered by extraction with the phenol/chloroform/isoamyl alcohol mixture.The immunoprecipitated DNA was used as a template for PCR.The PCR products were separated by 1.5%agarose gel.11.Immunoprecipitation:Treated cells were lysed for 60 min in ice-cold extraction buffer.For immunoprecipitation,cleared cell lysates were incubated with the appropriate antibody precoupled to protein A/G-agarose beads at 4℃overnight. The beads were washed twice with extraction buffer and twice with extraction buffer containing 0.5 M LiCl.Proteins were eluted directly in SDS sample buffer for Western blot analysis.12.Kinase assays:AMPK was precipitated from cell lysate using an anti-AMPK antibody.AMPK-containing beads were incubated with recombinant FOXO 3 in kinase assay buffer supplemented with 100μM ATP for 20 minutes at 30℃.Samples were separated on a 10%SDS-PAGE and transferred to PVDF membranes. Anti-serine and anti-threonine antibodies were used to detect phosphorylated serines and threonines incorporated into the FOXO 3.Results1.AMPK reduces ROS levels induced by PA in endothelial cells:AICAR by itself had minimal effects on basal ROS levels.Palmitic acid significantly increased intracellular levels of ROS,an observation consistent with previous reports.The PA-induced increase of intracellular ROS levels was reduced by AICAR in a dose-dependent manner with up to a 60%reduction at the highest dose 500μM.This result indicates that activation of AMPK can reduce intracellular ROS levels. Additionally,suppression of AMPK by specific siRNA not only increased basal ROS levels,but also augmented PA-induced increase of ROS levels.Furthermore,AICAR induced reduction of ROS was abolished by AMPK siRNA.2.AMPK increases the expression of the antioxidant Trx:Activation of the AMPK pathway by AICAR,significantly up-regulated the expression of Trx in a dose-dependent manner in the absence and the presence of PA.Prolonged PA exposure decreased Trx expression.Importantly,knockdown of AMPK by its specific siRNA inhibited the basal Trx expression and the upregulation of Trx by AICAR, implicating the involvement of the AMPK pathway in Trx upregulation.3.Trx silencing prevents AICAR-induced ROS reduction:Trx siRNA not only increased basal ROS levels,but also amplified PA-induced increase of ROS levels.Furthermore,AMPK siRNA prevented AICAR induced reduction of ROS.4.AMPK regulates Trx expression at the mRNA level:Using quantitative RT-PCR,we found that AICAR significantly increased Trx mRNA in a dose-dependent manner.Knockdown of AMPK by its specific siRNA reduced basal Trx expression.Moreover,the AICAR-induced Trx mRNA was reduced in the presence of AMPK siRNA5.FOXO 3a is required for AMPK-induced upregulation of Trx:We identified FOXO 3a as one of these transcriptional factors that may mediate AMPKinduced Trx transcription.Silencing FOXO 3 with siRNA significantly prevented AICAR induced expression of Trx at both protein and mRNA levels.6.FOXO 3a binds directly to the Trx promoter:The promoter region in the Trx gene contains 6 putative FOXO 3a binding sites（tgAAAGAgtga at-1346/-1342, tgAAAGAagga at-1339/-1335,gaAAACAcaga at-1236/-1232,caAAATAccgc at -859/-855,ggAAACActga at-807/-803,and tgAAAGAacag at-613/-609）.Results of the ChIP assay performed with a FOXO 3a antibody showed that FOXO 3a weakly binds sites 4 and 5,but strongly binds site 6.Importantly,the binding of FOXO 3a to site 6 was significantly increased by AICAR treatment,indicating that FOXO 3a may mediate AMPK-induced Trx transcription.7.AMPK increases FOXO 3a nuclear translocation:The immunostaining assay shows that AICAR significantly increased the nuclear translocation of FOXO 3a, which was prevented by AMPK siRNA.The in vitro kinase assay with purified AMPK as the kinase and recombinant FOXO 3a as the substrate showed that AMPK directly phosphorylated FOXO 3a at serine and threonine sites and that AICAR increased phosphorylation of FOXO 3a.8.FOXO 3a recruits p300 and forms a transcription activator complex on the Trx promoter:Using the ChIP assay,we found that AICAR treatment significantly increased p300 binding to the Trx promoter at consensus site 6.Using coimmunoprecipitation assay,we observed that FOXO 3a was associated with p300 and that the association was increased by AICAR treatment,indicating that p300 recruitment to the Trx promoter may be mediated at least in part by FOXO 3a.The double-ChIP assay showed that the Trx promoter sequence could be recovered from the immunocomplexes precipitated by FOXO 3a and p300 antibodies,indicating the simultaneous association of FOXO 3a and p300 within that region of the Trx promoter.Conclusions1.PA significantly increased intracellular ROS levels in human aortic endothelial cells（HAECs）.2.The ativation of the AMPK pathway significantly reduced PA-induced intracellular ROS levels by increasing the expression of the antioxidant Trx.3.Transcriptional factor FOXO 3a mediated AMPK’s effect on Trx expression.4.AMPK upregulated Trx transcription by increasing FOXO3a nuclear translocation and promoting its DNA binding to the Trx promoter.The activated FOXO3a may recruit p300 and form a transcription activation complex in the Trx promoter.5.AMPK-FOXO3a pathway has protective effects against cellular superoxide levels induced by metabolic stress and could be a therapeutic target in treating cardiovascular complications in diabetes.更多还原