【作者】 黄丹；

【导师】 廖玉华；

【作者基本信息】 华中科技大学 ， 内科学， 2009， 博士

【摘要】 心血管系统相关细胞(如心肌细胞、心脏成纤维细胞、血管平滑肌细胞、内皮细胞等)在各种致病因素(如心脏缺血再灌注损伤、心脏负荷增加、神经内分泌系统激活、炎症系统激活、感染、老龄化等)的作用下均会产生过量的活性氧或活性氮分子，出现氧化应激。这些活性氧／氮分子影响了细胞内正常的基因转录调控，从而导致一系列基因／蛋白质表达异常。蛋白质的异常表达是心血管疾病发生发展及功能损害的基础，而各种致病因素刺激所导致的细胞氧化应激则是触发基因／蛋白质异常表达的“起始信号”。因此找到能直接被氧化应激所激活，并且具有多基因转录调节能力的核蛋白／转录因子将为心血管疾病的治疗探索提供一条新途径。Ⅰ型多聚ADP核糖合成酶(poly(ADP-ribose)polymerase 1，PARP-1，EC 2．4．2．30)是一种存在于除酵母外所有真核细胞内、分子量为113-116KD的核蛋白酶；在细胞维持其染色体稳定、DNA复制、细胞凋亡、死亡以及基因转录等活动中均起着重要的作用。作为一种蛋白酶，PARP-1将NAD~+中的ADP核糖转移到受体蛋白(包括多种转录因子以及PARP-1自身等)上形成多聚ADP核糖链，从而改变了受体蛋白的生物学活性，影响其生物学效应的发挥。新近的研究发现PARP-1在心肌缺血再灌注损伤、心肌病、心力衰竭、高血压、动脉粥样硬化等多种心血管疾病发生发展过程中发挥了重要的致病作用。在这些疾病中，PARP-1主要是通过对转录因子的调节作用调控相关基因／蛋白质的表达。本项研究围绕PARP-1与不同转录因子的相互作用机制深入探讨了PARP-1调控心血管系统相关基因转录的分子机制，所得结果为心血管疾病的发病机制研究提供了新的理论和实验依据。第一部分血管紧张素Ⅱ促进心脏成纤维细胞中c-Jun／c-Fos多聚ADP核糖化的机制研究目的：c-Jun／c-Fos(活化因子1，AP1)通过调节心脏成纤维细胞中细胞外基质蛋白的过度表达在血管紧张素Ⅱ诱导的心脏纤维化过程中发挥了重要作用。本部分主要目的是研究在培养的心脏成纤维细胞中血管紧张素Ⅱ促进c-Jun／c-Fos转录激活的机制。方法及结果：通过western blot和免疫共沉淀的方法，我们证实在培养的心脏成纤维细胞中c-Jun和c-Fos可以被PARP-1多聚ADP核糖化修饰。Southwestern blot和EMSA研究显示：将细胞核抽提物与NAD~+和活化的DNA共孵育能显著增加c-Jun和AP1的DNA结合能力；同时将c-Fos和／或c-Jun纯蛋白与PARP-1纯蛋白、NAD~+和活化的DNA共孵育也能显著增加c-Jun和AP1的DNA结合能力。血管紧张素Ⅱ处理激活了细胞内的PARP-1，从而增加了c-Fos和c-Jun的多聚ADP核糖化水平，进而促进了c-Jun和AP1的DNA结合。PARP-1抑制剂PJ34或PARP-1 siRNA能显著抑制血管紧张素Ⅱ诱导的c-Jun和AP1的DNA结合能力的增加，减少AP1靶基因(包括胶原Iα1和ⅢIα1，基质金属蛋白酶(MMP)-9和其组织抑制因子TIMP-1)的表达。结论：本研究显示c-Jun和c-Fos可以被PARP-1多聚ADP核糖化修饰，而这种修饰能明显促进c-Jun和AP1的DNA结合。在心脏成纤维细胞中，血管紧张素Ⅱ通过激活PARP-1增加了c-Jun和c-Fos的多聚ADP核糖化水平，从而增强了AP1下游靶基因的转录。第二部分PARP-1在心脏成纤维细胞中通过对PPAR-γ的多聚ADP核糖化作用抑制PPAR-γ转录激活和脂连素表达的机制研究目的：本部分主要目的是研究大鼠心脏成纤维细胞中PARP-1与过氧化物酶体增生物激活受体γ(PPAR-γ)相互作用调控脂连素及其受体转录的机制。方法及结果：使用western blot和实时定量RT-PCR的方法，我们检测了培养的心脏成纤维细胞中脂连素及其受体的表达情况。Southwestern blot和EMSA实验用于研究PPARγ的DNA结合能力。结果显示，心脏成纤维细胞中表达有脂连素和脂连素受体1。PARP抑制剂3AB或PJ34，或PARP-1 siRNA不仅能显著增加心脏成纤维细胞中脂连素和脂连素受体1的表达，还能增加其在3T3-L1脂肪细胞、心肌组织和白色脂肪组织中的表达。研究表明，PPARγ能够被PARP-1多聚ADP核糖化修饰，这种修饰作用显著抑制了PPARγ的DNA结合能力。抑制PARP-1能够增强PPARγ的DNA结合和转录激活，从而促进脂连素及PPARγ其他下游基因(如CD36，lipoprotein lipase和leptin)的转录水平。结论：在心脏成纤维细胞中，PARP-1通过对PPARγ的多聚ADP核糖化修饰作用抑制了PPARγ的转录激活和其下游基因脂连素及其受体的表达。第三部分不稳定心绞痛患者外周血单个核细胞中PARP-1的激活和过度表达促进炎性细胞因子TNF-α和IL-6表达的机制研究目的：炎性细胞因子的大量表达在不稳定心绞痛的发病过程中发挥了重要作用。研究表明PARP-1参与了对炎性细胞因子的转录调控。本部分的主要目的是研究不稳定心绞痛患者外周血单个核细胞中PARP-1的激活与患者血浆中炎性细胞因子肿瘤坏死因子(TNF)-α和白介素(IL)-6释放水平的关系，并阐明PARP-1通过NF-κB途径促进炎性细胞因子表达的机制。方法及结果：26名BraunwaldⅢB级不稳定心绞痛患者，25名稳定型心绞痛患者及25名健康志愿者被纳入本研究。使用ELISA及实时定量RT-PCR法检测血浆及外周血单个核细胞中TNF-α和IL-6的表达水平。使用western blot、EMSA等方法检测外周血单个核细胞中PARP-1的活性和表达，以及NF-κB的DNA结合能力。分离健康志愿者外周血单个核细胞进行体外细胞培养，研究PARP-1促进NF-κB转录激活的机制。结果显示，不稳定心绞痛患者外周血单个核细胞中PARP-1的活性和表达明显高于稳定型心绞痛患者和健康对照组，并且PARP-1的活性和表达水平与患者TNF-α和IL-6的表达水平呈显著正相关。同时不稳定心绞痛患者外周血单个核细胞中调控炎性因子转录的主要转录因子NF-κB的DNA结合能力也显著增强。在体外培养的外周血单个核细胞中，PARP抑制剂或PARP-1 siRNA能够显著抑制炎性刺激物脂多糖(LPS)诱导产生的NF-κB的的激活和炎性细胞因子TNF-α和IL-6的表达。Supershift研究表明PARP-1参与了NF-κB与DNA的结合。在核抽提物中加入PARP-1纯蛋白能进一步增加NF-κB的DNA结合能力。结论：不稳定心绞痛患者外周血单个核细胞中PARP-1活性和表达显著增加。过度表达的PARP-1促进了PARP-1／NF-κB复合体的形成，增强了NF-κB的DNA结合能力，进而促进了NF-κB下游基因TNF-α和IL-6的转录。更多还原

【Abstract】 Experimental evidence has demonstrated that reactive oxygen and nitrogen speciesare over-generated in cardiomyocytes,cardiac fibroblasts,smooth muscle cells,endothelial cells,and infiltrating inflammatory cells during a wide range of cardiovasculardiseases,such as circulatory shock,myocardial ischemic-reperfusion injury,myocardialhypertrophy and heart failure,cardiomyopathies,cardiovascular aging,atherosclerosis,and hypertension.Reactive oxygen and nitrogen species induce cellular DNA damage andconsequently activate poly (ADP-ribose) polymerase 1 (PARP-1),a multifunctionalnuclear enzyme present in eukaryotes.Upon binding to DNA damage breaks,PARP-1successively transfer ADP-ribose moiety of nicotinamide dinucleotide (oxidized,NAD~+)to a variety of target proteins.This process of poly(ADP-ribosyl)ation is an importantpost-translational modification of proteins.PARP-1 has been implicated in multiplecellular functions,including DNA repair,maintenance of genomic integrity,cellreplication and differentiation,and transcriptional regulation.A growing body of evidencesuggests that PARP-1 plays an important role in pathogenesis of various cardiovasculardiseases.In related animal models of these diseases,the activation of PARP-1 paticipatesin the transcriptional regulation of numerous genes,which eventually inducecardiovascular dysfunction associated with cardiovascular diseases.Although PARP-1 isimplicated in the transcriptional regulation of many genes through collaboration withtranscription factors,the mechanism underlying PARP-1 regulates transcription of genesinvolved in cardiovascular system,remains unclear.This study focuses on the mechanismthat PARP-1 interacts with several transcription factors AP1,PPARγand NF-κB,toregulate transcription of genes related with cardiovascular system. PartⅠAngiotensinⅡpromotes poly (ADP-ribosyl) ation ofc-Jun/c-Fos in cardiac fibroblastsAims:c-Jun/c-Fos (activator protein 1,AP1) contributes importantly to AngⅡ-inducedcardiac fibrosis through induction of extracellular matrix protein over-expression in cardiacfibroblasts.This study aims to explore the mechanism by which AngⅡpromotesc-Jun/c-Fos transactivation in cultured rat cardiac fibroblasts.Methods and results:In this study,we demonstrated that c-Fos and c-Jun werepoly(ADP-ribosyl)ated in cultured cardiac fibroblasts.Southwestern blot and EMSA assaysshowed that incubation of nuclear extracts with NAD~+ and active DNA increased the basalDNA binding activities of c-Jun (31.0±1.0%,P＜0.01) and AP1 (14.2±3.1%,P＜0.01);incubation of recombinant c-Fos or/and c-Jun with PARP-1,NAD~+ and active DNAincreased the basal DNA binding activities of c-Jun (48.3±4.2%,P＜0.01) and AP1(21.2±1.5%,P＜0.01).Treatment with Ang II promoted PARP-1 activation and enhancedpoly (ADP-ribosyl)ation of c-Fos (14.1+1.1%,P＜0.01) and c-Jun (15.5±5.6%,P＜0.01).AngⅡalso increased the basal DNA binding activities of c-Jun (13.5±2.4%,P＜0.01) andAP1 (18.7±3.5%,P＜0.01) in cultured cells.Inhibition of PARP-1 by PJ34 or siRNAeffectively prevented Ang II-induced increases in the DNA binding of c-Jun and AP1,anddecreased AP1-driven transcription (including collagenⅠα1 andⅢα1,MMP-9 andTIMP-1).Conclusions:This study illustrated that c-Jun and c-Fos were poly(ADP-ribosyl)ated byPARP-1,and poly(ADP-ribosyl)ation enhanced the DNA binding of AP1.Ang II promotedpoly(ADP-ribosyl)ation of c-Jun and c-Fos through activation of PARP-1 and,subsequently,enhanced AP1-driven transcription in cardiac fibroblasts. PartⅡPARP-1 suppresses adiponectin expression throughpoly(ADP-ribosyl)ation of PPARγin cardiac fibroblastsAims:Our aim was to explore the mechanism underlying the transcriptional regulation ofadiponectin and its receptors (AdipoR) in cultured rat cardiac fibroblasts.Methods and results:Using western blot and real-time RT-PCR assays,the expression ofadiponectin and its receptors was determined.Using southwestern blot and electrophoreticmobility shift assays,the DNA binding activity of peroxisome proliferator activatedreceptorγ(PPARγ) was determined.The results showed that adiponectin and AdipoR1were highly expressed in cultured rat cardiac fibroblasts.Inhibition of poly(ADP-ribose)polymerase 1 (PARP-1) by 3-aminobenzamide,PJ34,or PARP-1 siRNA markedlyincreased the transcription of adiponectin and AdipoR1 in cultured fibroblasts,mature 3T3-L1 adipocytes,rat myocardium,and white adipose tissue.PPARγwaspoly(ADP-ribosyl)ated by PARP-1 in cardiac fibroblasts under basal conditions.Poly(ADP-ribosyl)ation of PPARγprevented its binding to DNA.Inhibition of PARP-1enhanced the DNA binding and transactivation of PPARγand increased the transcription ofPPARγ-target genes including CD36,lipoprotein lipase,and leptin in cultured fibroblasts.Conclusions:PARP-1 inhibits adiponectin and AdipoR1 expression as well as PPARγtransactivation through poly (ADP-ribosyl)ation of PPARγin cultured rat cardiacfibroblasts. PartⅢActivation and Overexpression of PARP-1 inCirculating Mononuclear Cells Promote TNF-a and IL-6Expression in Patients with Unstable AnginaAims:Proinflammatory cytokines are involved in the development of unstable angina (UA).Poly(ADP-ribose) polymerase-1 (PARP-1) contributes importantly to regulating the transcriptionof inflammatory cytokines.This study aims to investigate the relationship of PARP-1 in circulatingmononuclear cells (MNCs) and plasma TNF-αand IL-6 in UA patients and to elucidate themechanism that PARP-1 promotes TNF-αand IL-6 expression via NF-κB pathway.Methods and results:Twenty six Braunwald class IIIB UA patients,25 stable anginapatients and 25 healthy volunteers were enrolled in this study.Plasma TNF-αand IL-6were determined with ELISA.Circulating MNCs were analyzed for PARP activity,PARP-1expression and NF-κB DNA binding activity.MNCs from healthy subjects were cultured toinvestigate the direct effects of PARP-1 on NF-κB DNA binding activity and the expressionof TNF-αand IL-6.PARP activity and PARP-1 expression in circulating MNCs wereincreased and positively correlated with plasma TNF-αand IL-6,respectively,in UApatients.Spontaneous NF-κB activation in MNCs was demonstrated in UA patients.Incultured MNCs from healthy subjects,inhibition of PARP-1 preventedlipopolysaccharide-induced increase in DNA binding activity of NF-κB and the expressionof TNF-αand IL-6.Supershift assay demonstrated that PARP-1 was a component ofNF-κB/DNA complex.Addition of recombinant human PARP-1 protein to nuclear extractsof MNCs significantly increased the DNA binding activity of NF-κB.Conclusions:Activation and overexpression of PARP-1 are demonstrated in circulatingMNCs of UA patients.Overexpressed PARP-1 promotes PARP-1/NF-κB/DNA complexformation,thereby enhancing the expression of TNF-αand IL-6 in circulating MNCs ofUA patients.更多还原