【作者】 顾强；

【导师】 肖颖彬；

【作者基本信息】 第三军医大学 ， 外科学， 2011， 博士

【摘要】 背景:紫绀型先心病是临床常见的先天性畸形,心肌慢性缺氧是此类患者共同的病理生理过程。紫绀型先心病的手术时机的选择往往会对患者预后产生重要的作用。但相对而言,紫绀型先心病的手术死亡率仍较高。在我国,紫绀型先心病的手术死亡率约为10%,而且1岁以内及低体重紫绀型先心病患儿手术死亡率高已成为不可忽视的现象。基于以上思考,国家科技部已将先心病心肌缺氧适应机制研究纳入“十二五”医学科技发展的目标中,目的是更有效的降低我国先心病治疗的死亡率。心肌慢性缺氧适应是维持先心病患儿心肌细胞存活和生长的关键,但是心肌慢性缺氧适应的确切机制尚不清楚。因此深入研究紫绀型先心病的病理生理改变以及慢性缺氧条件下心肌细胞的自我调节和适应机制,将有望提高紫绀型复杂性先心病的临床治疗水平。对于心肌细胞缺氧适应,我们既往已做过系列性研究,分别从缺氧心肌细胞中线粒体合成改变、缺氧自噬体形成、内质网应激(ER stress)、未折叠蛋白反应(UPR)改变、促红细胞生成素受体(EPOR)表达改变以及腺苷酸活化蛋白激酶(AMPK)改变等方面阐述心肌细胞适应过程中产生的胞内相关结构蛋白、功能蛋白(酶)活性及代谢机能等等方面产生的适应性调整,进而重塑细胞的结构稳态,能量供求平衡,以增加氧气的释放,并减低能量的消耗,使心肌重新获得细胞稳态,保证细胞自身的存活和生长,对抗缺氧诱导的心肌细胞凋亡,实现心肌细胞对慢性缺氧的适应。作为最直接及最快速的细胞反应机制中,细胞信号通路的改变往往是一切适应性改变的开始环节,其活化或持续性活化必然会导致其下游的信号通路的级联反应,进而影响细胞接下来的适应性改变,如形态、代谢、凋亡等等方面,且一旦这种适应性调节稳态形成后,何种机制可以打断它的稳定运行,何时何种生物学指标可以提示缺氧适应平衡稳态已崩溃是目前紫绀型先心病病理生理机制研究的重点。因为对这些适应稳态的改变的掌握往往决定着紫绀型先心病患儿的疾病的转归,手术时机的选择及术后患者的预后。目前对于慢性缺氧心肌细胞内部信号分子系统产生怎样的适应性改变,如何产生这种适应调整的机制尚不清楚。细胞因子信号抑制家族(supressors of cytokine signaling , SOCS)有SOCS1- SOCS7和CIS共8种成员。SOCS3蛋白家族是Janus激酶/信号转导和转录激活子STAT( Janus kinase/signal transducer and activator)信号通路激活的靶基因产物,特异性地负反馈抑制的JAK/STAT通路活化。JAK/STAT信号通路受细胞因子(如IL-6及TNF-a)激活后,积极参与心肌自我适应性调整的各个环节如:早期缺血预处理的心肌保护、心肌重构及终末期扩张型心肌病的心肌功能维持,是心肌细胞中重要的内环境稳定因子。但正常的心肌细胞中SOCS3-JAK/STAT通路是不表达的,说明JAK/STAT通路在正常心肌中是不激活的,SOCS3基因作为胚胎时期表达的基因(ontogene)在非应激情况下不参与心肌细胞内的信号调控。JAK/STAT通路是否参与心肌慢性缺氧适应机制目前研究并不十分清楚,其下游调节因子的变化在心肌细胞达到缺氧适应平衡时的变化情况目前尚无报道。我们以前的研究发现心肌细胞缺氧复氧处理后NF-κB可以活化,且与心肌缺血预适应及一些药物预处理的保护作用有关。最新的研究表明:持续性表达的STAT3的磷酸化可以稳定的持续激活NF-κB活化进核,其活化的RelA的亚基的乙酰化形态(AC-RelA)表达同样受磷酸化的p-STAT3的活化调控。因此本课题拟阐明在慢性缺氧条件下SOCS3-IL-6/JAK2/STAT3信号系统与NF-κB信号系统相互交联参与调控心肌细胞适应的作用机制。目的:本研究的目的在于验证以下假说:在慢性缺氧心肌细胞中:1)IL-6- STAT3蛋白和NF -κB信号通路激活的持续稳定; 2)STAT3/NF-κB相互协调作用存在,共同介导心肌细胞抗凋亡作用;3)SOCS3的表达可以被缺氧刺激诱导,它会抑制STAT3的过度激活和调节NF -κB的激活,形成有序的信号表达稳态。研究方法:本研究符合医学道德伦理规范,共分三部分:第一部分:选取紫绀型(紫绀组,n=18)和非紫绀型(非紫绀组,n=22)先天性心脏病患儿,收集手术中切除的肥厚右室流出道心肌组织。Western blotting及免疫组化观察IL-6,NF-κB p65 (acetyl K310), SOCS3, STAT3, phosphor-Tyr-705-STAT3和phosphor-Ser-276-NF-κB p65在心肌组织中的表达情况;提取心肌组织的总RNA,采用Real time RT-PCR检测IL-6 mRNA,C-myc mRNA和SOCS3 mRNA表达水平; EMSA法检测心肌组织中NF-κB活化情况;ELISA检测先天性心脏病患儿外周血IL-6浓度。第二部分:体外培养H9c2大鼠心肌细胞株,将细胞暴露于1% O2、5% CO2的环境中建立缺氧细胞模型,培养不同时间段后(分为缺氧6h组, 12h组, 24h组, 48h组, and 72h组),Western blot检测NF-κB p65 (acetyl K310),SOCS3,STAT3,phosphor-Tyr-705-STAT3和phosphor-Ser-276-NF-κB p65在心肌组织中的表达情况;提取心肌组织的总RNA,采用Real time RT-PCR检测IL-6 mRNA, C-myc mRNA和SOCS3 mRNA表达水平;ELISA检测体外培养各组心肌细胞株上清液中IL-6浓度。第三部分:建立SOCS3稳定表达H9c2细胞株,给予转染SOCS3质粒的上述细胞缺氧处理,检测第二部分实验指标的变化;同时通过检测缺氧处理72h后转染SOCS3质粒组与未转染组间不同的生物活性:细胞凋亡检测(凋亡率(TUNEL)试验/Hoest活细胞染色);细胞活性检测(MTT试验);细胞培养基上清液中LDH (乳酸脱氢酶)浓度的差异。研究结果:主要结果如下:1、与非紫绀组相比,免疫组化检测提示:紫绀组心肌组织中信号分子IL-6蛋白表达明显高于非紫绀组,非紫绀组心肌组织中无明显IL-6表达;通过Western blot证实: NF-κB p65 (acetyl K310), SOCS3, phosphor-Tyr-705-STAT3和phosphor- Ser-276-NF-κB p65在紫绀组心肌组织中蛋白表达明显高于非紫绀组,免疫组化检测也证实以上结果。另外,通过Real time RT-PCR检测表明紫绀组心肌组织中NF-κB信号通路调节基因产物IL-6 mRNA;JAK2/STAT3信号通路调节基因产物C-myc mRNA和SOCS3 mRNA表达均明显高于非紫绀组。EMSA检测同样证实:紫绀组心肌组织的NF-κB的活化明显高于非紫绀组。但先天性心脏病患儿外周血IL-6浓度在紫绀组及非紫绀组间无差异。2、在常氧培养的H9c2心肌细胞中,Western blot及Real-time RT-PCR未检测到SOCS3蛋白和mRNA的表达。在缺氧培养心肌细胞中,随着缺氧时间的延长,SOCS3蛋白出现表达,且呈现先升高后稳定的蛋白表达趋势。在缺氧培养各组心肌细胞中,p-STAT3由胞浆转位入核活化情况也明显增强。NF-κB蛋白各亚单位(p-RelA;AC- RelA)在常氧培养心肌细胞中均未检测到,在缺氧各组心肌细胞中,随着缺氧时间的延长,核内NF-κB蛋白各亚单入核表达位均呈现先升高后稳定的趋势。对mRNA水平检测发现IL-6 mRNA和C-myc mRNA的表达也随缺氧时间的增加呈现先升高后稳定的趋势。3、成功建立SOCS3过表达H9c2心肌细胞株。对过表达SOCS3质粒心肌细胞组及未转染心肌细胞组同时给予体外缺氧培养处理后发现,同未转染SOCS3质粒心肌细胞组相比,转染组中的p-STAT3入核活化及NF -κB蛋白各亚单位(p-RelA;AC- RelA)核内表达均明显受到抑制。IL-6 mRNA和C-myc mRNA表达也明显受到抑制而转录减少。通过TUNEL及Hoest活细胞染色检测发现,同非转染SOCS3质粒组相比,转染组缺氧72h时心肌细胞凋亡率显著增加[43.5%(未转染组):14.5%(转染组),p<0.001]。相对应的各组细胞培养基上清液中的LDH (乳酸脱氢酶)浓度也明显增加;MTT检测发现转染组各缺氧时间段细胞活性明显减低;各缺氧时间段细胞培养基上清液中的IL-6明显减低。结论:1、在紫绀型先心病患者心肌组织中观察到STAT3及NF-κB信号系统均活化,其正性调节因子IL-6及负性调控因子SOCS3均处于高表达状态,这种高表达的信号通路对心肌细胞慢性缺氧适应可能具有重要的作用;2、慢性缺氧培养心肌细胞中,p-Ser-276-NF-κB p65、p-Tyr-705-STAT3、STAT3、SOCS3、NF-κB p65(acetyl K310)蛋白表达均随缺氧时间的延长,逐渐升高,后呈稳定表达状态,维持在高水平。随着缺氧时间的延长,IL-6/JAK2/STAT3信号通路的靶基因SOCS3和C-myc以及NF-κB信号通路的靶基因IL-6 mRNA转录水平也同样表现出先逐渐升高后稳定在高活性水平的趋势,说明随缺氧时间的延长,以上信号通路的改变是细胞为适应缺氧刺激的而做出的适应性改变;3.外源性的SOCS3明显减低慢性缺氧培养心肌细胞中p-Ser-276-NF-κB p65、p-Tyr-705-STAT3、NF-κB p65(acetyl K310)蛋白在核内的表达水平;同样过表达的SOCS3明显减低IL-6/JAK2/STAT3信号通路的靶基因C-myc mRNA以及NF-κB信号通路的靶基因IL-6 mRNA转录,说明外源性过表达的SOCS3明显抑制STAT3信号通路与NF-κB信号在细胞核内存在信号交联。外源性的SOCS3对两种信号通路抑制的效果使心肌细胞面对缺氧刺激出现细胞凋亡明显增高(TUNEL检测),细胞膜稳定(LDH分泌增加)明显降低,细胞活性明显减低,失去了对缺氧的适应,说明打破信号稳态对心肌细胞的存活及活性有明显的不利影响。综上所述,紫绀型先心病患者心肌组织中及缺氧培养心肌细胞中STAT3及NF-κB信号系统活性增强,存在核内相互协调作用,维持心肌细胞对缺氧刺激的适应,作为正反馈因子IL-6的旁分泌作用有效的维持信号系统的稳态表达,而作为重要的负反馈因子SOCS3的稳态表达,有效有序的维持心肌细胞内抗凋亡信号稳态的表达,起到对信号通路微调的作用,这些细胞内的信号系统自我调整对缺氧心肌细胞的存活和功能调节有重要意义。更多还原

【Abstract】 Background and Objective:Cytokines related to interleukin-6 (IL-6) comprise a family of substances known to play cytoprotective and growth-promoting roles in different cell types. IL-6 related cytokines potently promote tyrosine-phosphorylation of Janus kinases (JAKs) and cytoplasmic latent transcription factors of the signal transducers and activators (STATs) family. STAT proteins regulate the expression of genes encoding proteins involved in angiogenesis, inflammation, apoptosis, extracellular matrix composition and cellular signaling. The JAK-STAT signaling pathway mediates cytoprotective effects in cardiomyocytes. Eight members have been identified as suppressors of the cytokine signaling (SOCS) family (CIS and SOCS1 to SOCS7), and not only act as direct negative feedback regulators of JAK-STAT signaling, but are also involved in the fine tuning of the myocardial adaptation response and in crosstalk of the complicated cytokine signal network in myocytes.Accumulating evidence indicates that activation and expression of the IL-6-JAK-STAT signaling pathway are facilitated by hypoxia. Among these signaling molecules, STAT3 can be activated by hypoxia/reoxygenation stress and this signaling pathway exerts cardioprotection in the ischemic heart. Blockade of the STAT3 pathway aggravates myocardial injury after infarction. Evidence from cardiomyocyte-restricted ablation of STAT3 mice further indicates that STAT3 protects the heart from ischemic injury by suppressing cardiomyocyte apoptosis, inducing local growth factor production. As a product of the STAT3-inducible gene, SOCS3 is a major feedback regulator of STAT3, and it blocks STAT3 activation by the gp130 receptor. Hypoxia has profound effects on the expression of SOCS3 in pulmonary arterial smooth muscle cell (PASMC). SOCS3 can also be induced and can inhibit signaling by a wide-spectrum of growth factors and cytokines including Toll-like receptor (TLR) agonists (such as lipopolysaccharide and CpG-DNA), IL-10, IL-6 and other gp130 signaling cytokines, leptin and interferon-γ. These properties suggest that SOCS3 may broadly regulate cytokine signaling and might be a chief factor in these classical signal loops. Moreover, it seems that SOCS3 expression determines the character of the response of cardiac myocytes to the IL-6-type cytokines and also impacts on other signaling pathways of other agonists. While many studies have evaluated the protective effects of STAT3 and regulatory effects of SOCS3 for hypoxia/reoxygenation injury of heart, it is unknown whether SOCS3 can be directly induced by hypoxia in cardiac myocytes. Additionally, little is known about the expression, activation and regulation of these signaling molecules in myocytes exposed to chronic low ambient oxygen levels.NF-κB is a ubiquitous transcription factor and its activation involves the regulation of a large variety of genes. NF-κB consists of five Rel-related proteins such as p50, p52 (NF-κB2), p65 (RelA), c-Rel and RelB. The prototypical NF-κB complex is a RelA/p50 heterodimer, which is important for NF-κB-mediated antiapoptotic effects. Recent studies have demonstrated that the amplitude and half-life of nuclear NF-κB are influenced by acetylation of RelA , which requires prior RelA phosphorylation. In particular , endogenous RelA is acetylated in a signal-coupled manner following stimulation. Reversible acetylation of RelA is essential for the duration of NF-κB activity.Signaling pathways that mediate protection from apoptosis involve activation of the transcription factor NF-κB, which in turn induces the expression of proteins that possess cytoprotective roles. NF-κB is induced by hypoxic stimulation and it regulates the secretion of IL-6 in cardiac myocytes, and may be the primary positive regulator of transcriptional activation of the IL-6-JAK-STAT signaling pathway during hypoxia. STAT3 is a transcription factor that can promote oncogenesis, and it is commonly activated in cancer as well as in tumor-associated myeloid cells. STAT3 and NF-κB stimulate a highly overlapping repertoire of pro-survival, proliferative, and pro-angiogenic genes. Crosstalk between STAT3 and NF-κB has been demonstrated at multiple levels, including activation of STAT3 by NF-κB-regulated factors such as IL-6 and Cox-2, possible inhibition of IκB kinase (IKK) activity in normal immune cells by STAT3, and nuclear translocation of unphosphorylated NF-κB by unphosphorylated STAT3. A recent study further demonstrated that activated STAT3 prolongs NF-κB nuclear retention through acetyltransferase p300-mediated RelA acetylation , thereby interfering with NF-κB nuclear export. Therefore, the STAT3/NF-κB interaction is important for tumor cells to adapt to a hypoxic tumor microenvironment and to mediate antiapoptotic effects. The present study was undertaken to test the following hypotheses: in chronic hypoxia cardiac myocytes: 1) the IL-6-STAT3 and NF-κB signaling pathways are sustained and stably activated; 2) STAT3/NF-κB interaction occurs; and 3) expression of SOCS3 is induced by hypoxia, and it suppresses STAT3 phosphorylation and mediates NF-κB activation.We examined the expression and activation of IL-6, STAT3 and NF-κB in the myocardium of infants with cyanotic cardiac defects, as well as in cultured cardiac myocytes subjected to chronic hypoxia. We evaluated the interaction of SOCS3 with STAT3 and NF-κB by transfecting the SOCS3 plasmid to hypoxic cultured H9c2 cells. The purposes of this study were to determine the crosstalk between NF-κB and STAT3 signaling and the effect of SOCS3 on this interaction on myocardial adaptation to chronic hypoxia. Methods:Samples taken from the right ventricular outflow tract were collected from patients with cyanotic (n = 18) or acyanotic (n =22) congenital heart disease. The expression of IL-6, NF-κB p65 (acetyl K310), SOCS3, STAT3, phosphor-Tyr-705-STAT3 and phosphor-Ser-276-NF-κB p65 was examined by immunohistochemistry and western blotting, while IL-6, C-myc and SOCS3 mRNA were tested by Real time RT-PCR. The activity of NF-κB were tested by Gel electrophoretic mobility shift assay (EMSA). Serum levels of IL-6 were measured by ELISA set.To evaluated the effect of chronic hypoxia on expression and activation of IL-6, STAT3 and NF-κB signaling pathway, the cardiomyocytes in embryonic rat-heart-derived H9c2 cells were cultivated and exposed to 1.0% O2, 5.0% CO2 for different durations to establish the chronic hypoxic cell model. Control cells were cultivated in the same conditions except for 21% O2 concentration. After different duration of hypoxic exposure (6, 12, 24, 48, and 72h), cells were collected and subjected to RT-PCR and western blot to detecting the mRNA and protein expression of the two signaling pathways. The expression of NF-κB p65 (acetyl K310), SOCS3, STAT3, phosphor-Tyr-705-STAT3 and phosphor-Ser-276-NF-κB p65 was examined by western blotting, while IL-6, C-myc and SOCS3 mRNA were tested by Real time RT-PCR. IL-6 concentrations in cultured H9c2 cell medium were determined by ELISA. To address whether SOCS3 can inhibit hypoxia-induced STAT3 and NF-κB activation in hypoxic cultured H9c2 cells, we transfected them with the SOCS3 gene and examined changes in activated STAT3 and NF-κB. The expression of NF-κB p65 (acetyl K310), STAT3, phosphor-Tyr-705-STAT3 and phosphor-Ser-276-NF-κB p65 was examined by western blotting, while IL-6 and C-myc mRNA were tested by Real time RT-PCR. IL-6 concentrations in cultured H9c2 cell medium were determined by ELISA, too.To evaluate the effects of SOCS3 overexpression on cell survival , SOCS3 gene-transfected cardiac myocytes and non-transfected cardiac myocytes were submitted to a hypoxic environment for 6, 12, 24, 48, and 72h. Cell viability was assessed by the colorimetric 3-(4, 5-dimethylthia-zol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. LDH levels in the cultured supernatant were measured in duplicate following hypoxia using a commercially available kit. Terminal dUTP Nick End Labeling (TUNEL) and Hoescht staining were used for apoptosis analyses.Results:1. Immunohistochemistry revealed that all of these signaling molecules (IL-6, STAT3, p-STAT3, SOCS3, p-RelA and AC-RelA) were increased in myocardium of patients with cyanotic cardiac defects, in either the cytoplasm or the nuclei. Western blot analysis revealed that the protein levels of these signaling molecules were significantly elevated (p < 0.001) in patients with cyanotic compared to acyanotic congenital heart disease. Also, the expression of IL-6, C-myc and SOCS3 mRNA were markedly increased in cyanotic patients (p < 0.05). While there were no significant differences in serum levels of IL-6 between the acyanotic group and the cyanotic group (p >0.05).2. SOCS3 protein and mRNA were not detected in cardiomyocytes under normoxic conditions.While in hypoxia-exposed cells, the protein expression of STAT3-SOCS3 as well as NF-κB together with the mRNA of IL-6, SOCS3 and C-myc in hypoxia-exposed cells were significantly increased in a duration-dependent manner compared with the normoxia group (p < 0.001).3. The level of p-STAT3, p-RelA and AC-RelA did not increase in SOCS3 gene-transfected cells with prolonging time of hypoxia compared with the level of p-STAT3 in non-transfected cells (p < 0.001). Similarily, the mRNA of IL-6 and C-myc showed the same results(p < 0.001).In contrast to control cells, a greater proportion of SOCS3 gene-transfected cardiac myocytes subjected to hypoxia for 72 h exhibited pyknotic, TUNEL-positive nuclei.The non-transfected group subjected to hypoxia for 72 h showed significantly less hypoxia-mediated apoptosis of myocardial cells compared with the transfected group (p<0.001). Elevated LDH release was also observed in SOCS3 gene-transfected cardiac myocytes after hypoxia at each time interval. SOCS3 overexpression resulted in more LDH leakage from injured myocardium compared with non-transfected cardiac myocytes under hypoxic stress as well as SOCS3 gene-transfected group and control group in normoxia conditions (p < 0.001).Conclusions:1. The IL-6-STAT3-SOCS3 and NF-κB signaling pathways as well as the IL-6, SOCS3 and C-myc mRNA expression increase in cyanotic patients.2. In cardiac myocytes exposed to hypoxia, the activation of Ac-RelA, p-RelA, and p-STAT3 in the nucleus and the expression of SOCS3 and STAT3 in the cytoplasm are significantly increased in a duration-dependent manner compared with the normoxia group.The expression of levels of SOCS3,IL-6 and C-myc mRNA in the cytoplasm also increase in cardiac myocytes exposed to hypoxia.3. Overexpression of SOCS3 shows down-regulation of the IL-6 and C-myc genes. Transfected overexpression of SOCS3 suppresses hypoxia-induced STAT3 phosphorylation and attenuate the activation of Ac-RelA and p-RelA in cardiac myocytes exposed to hypoxia.And overexpression of SOCS3 inhibits the cytoprotective effect of the STAT3 and NF-κB signaling pathways in H9c2 cells exposed to hypoxia.更多还原