【作者】 王丽静；

【导师】 罗百灵；

【作者基本信息】 中南大学 ， 内科学， 2009， 博士

【摘要】 研究背景COPD是一种慢性气道炎症性疾病,以炎症基因表达增高为特点。香烟烟雾是COPD的主要致病因素,它可以导致氧化应激,激活转录因子,诱导炎症基因转录。香烟烟雾诱导的炎症反应在COPD的发生发展中起着关键的作用。组蛋白乙酰化/去乙酰化是影响基因转录的关键的调节器。组蛋白乙酰化和去乙酰化作用的失平衡会导致肺内促炎症反应基因的过度转录,形成慢性炎症循环,可能对COPD的发生发展产生重要影响。研究表明,氧化应激和细胞核心组蛋白乙酰化/去乙酰化失平衡有关,可诱导组蛋白乙酰化。氧化应激可能需要依赖于信号转导途径的介导作用才能诱导组蛋白乙酰化。研究表明,氧化应激可以激活MAPK途径,MAPK途径的激活在气道炎症中起着重要作用。而且,在某些条件下,MAPK途径能够影响组蛋白结构,可使组蛋白发生磷酸化或乙酰化。因此,在COPD中,MAPK途径可能参与了组蛋白乙酰化的调控。本研究拟以COPD大鼠模型和正常人支气管上皮细胞为研究对象,探讨香烟烟雾诱导气道上皮组蛋白乙酰化的机制和其对IL-8表达影响以及MAPK途径在该过程中的调控作用。第一章乙酰化组蛋白H4及HDAC2在COPD肺组织中的表达及氨茶碱的抗炎作用目的观察乙酰化组蛋白H4和组蛋白去乙酰基酶2（HDAC2）在慢性阻塞性肺疾病（COPD）大鼠肺组织中的表达和它们对IL-8表达的影响,以及氨茶碱的干预作用,以期探讨COPD中组蛋白乙酰化状态及其对炎症因子IL-8表达的影响,同时说明氨茶碱的抗炎作用。方法36只Wistar大鼠随机均分为3组:正常对照组、COPD模型组和氨茶碱干预组。采用被动吸烟加气管内注射脂多糖法建立大鼠COPD模型,干预组给予腹腔注射氨茶碱(5mg·kg^-1·d)。造模完成后,测定各组大鼠的肺功能、观察肺组织病理学变化;用比色法测定各组大鼠肺组织HDAC活性;免疫组织化学法及western blot法观察乙酰化组蛋白H4及HDAC2在肺组织中的表达情况;应用ELISA法检测支气管肺泡灌洗液中IL-8的水平;RT-PCR方法检测肺组织中IL-8mRNA及HDAC2mRNA水平。结果与正常对照组比较,模型组动物的PEF、FEV_0.3、FEV_0.3/FVC均显著降低,氨茶碱干预能改善肺功能。HDAC活性在模型组最低,显著低于对照组,氨茶碱可使HDAC活性部分恢复。免疫组化及western blot的结果显示,和对照组相比,乙酰化组蛋白H4在模型组的表达明显增高;和模型组相比,氨茶碱干预后乙酰化组蛋白H4的表达显著降低;和对照组相比,HDAC2在模型组的表达降低,氨茶碱干预后其表达有所增高。与正常对照组相比,模型组大鼠支气管肺泡灌洗液中IL-8的浓度明显增高,大鼠肺组织中IL-8mRNA的水平也显著增高;和模型组比较,氨茶碱干预能够使支气管肺泡灌沈液中的IL-8表达和IL-8mRNA的水平均下降。而HDAC2mRNA水平在三组之间没有差异。结论COPD大鼠模型中组蛋白乙酰化增强,导致炎症因子IL-8的高表达,氨茶碱能够通过激活组蛋白的去乙酰化而减少IL-8的表达,起到抗炎作用。第二章香烟烟雾通过MAPK途径诱导气道上皮细胞组蛋白乙酰化上调IL-8的表达目的观察香烟烟雾刺激正常人支气管上皮细胞后MAPK途径蛋白和HDAC2蛋白的表达,进一步研究MAPK途径（p38、ERK1/2和JNK）抑制剂对香烟烟雾刺激后人支气管上皮细胞中HDAC活性、乙酰化组蛋白H4以及IL-8表达的影响,以期探讨香烟烟雾刺激正常人支气管上皮细胞后诱导组蛋白乙酰化致IL-8表达增高的机制以及MAPK途径在该过程中的调控作用方法培养正常人支气管上皮（NHBE）细胞,分别用5%和10%的香烟烟雾提取物（CSE）进行刺激,用免疫细胞化学和western blot的方法观察香烟烟雾刺激后MAPK途径蛋白的激活形式p-p38、p-ERK1/2和p-JNK以及HDAC2在NHBE中的表达。再分别用10%CSE、TSA（HDAC抑制剂）、氨茶碱+CSE、SB203580（p38抑制剂）+CSE、PD98059（ERK抑制剂）+CSE以及SP600125（JNK抑制剂）+CSE对支气管上皮细胞进行干预,用比色法检测HDAC活性,用免疫细胞化学和western blot的方法观察乙酰化组蛋白H4表达的变化,用RT-PCR和ELISA方法进一步观察不同刺激对IL-8mRNA和细胞培养上清液中IL-8表达的影响。结果CSE刺激能够使MAPK途径激活,p-p38、p-ERK1/2和p-JNK蛋白的表达在CSE刺激组明显高于对照组,而HDAC2的表达则显著低于对照组。CSE刺激具有和TSA类似的效应,能够使NHBE中HDAC活性显著下降,乙酰化组蛋白H4表达显著增高,伴随细胞培养上清液中IL-8表达的增高和细胞中IL-8mRNA水平的增加;氨茶碱干预能够激活HDAC活性,减少乙酰化组蛋白H4表达和IL-8表达;SB203580和PD98059能够下调CSE刺激诱导的乙酰化组蛋白H4的表达和随后的IL-8基因的表达,而SP600125干预对香烟烟雾诱导NHBE组蛋白乙酰化的过程不起作用,也不能下调IL-8的表达。SB203580、PD98059和SP600125均对HDAC活性没有影响。结论体外CSE刺激NHBE可诱导组蛋白乙酰化,上调IL-8基因表达;氨茶碱能够激活HDAC活性减少组蛋白乙酰化而发挥抗炎作用;MAPK途径中的p38和ERK1/2能下调CSE诱导的组蛋白乙酰化和IL-8基因转录。更多还原

【Abstract】 Background:Chronic obstructive pulmonary disease（COPD） is a chronic inflammatory disease of airway,characterized by excessive inflammatory gene expression.Cigarette smoke is the main etiological factor in the pathogenesis of COPD.It can cause oxidative stress,activate transcription factors nuclear factor and enhance proinflammatory gene transcription.Cigarette smoke-triggered inflammation is considered to play a central role in the development of COPD.Histone acetylation and deacetylation is a key regulator of the specificity and duration of gene transcription.Disruption in the nuclear histone acetylation/deacetylation balance may result in excessive transcription of specific proinflammatory genes in the lungs,finally,chronic inflammatory cycle will develop,and it may have great influence on COPD.It was indicated that oxidative stress has direct relationship with the disruption of histone acetylation/ deacetylation balance and it can enhance the acetylation of histone protein.Oxidative stress has been suggested to influence histone acetylation depending on some signal transduction pathway.It has been reported that oxidative stress can activate MAPK（Mitogen-activated protein kinases） pathway,which play an important role in the inflammation of airway.Furthermore,MAPK pathway can influence the structure of histone,it maybe induce histone phosphorylation or acetylation in some condition.SO,MAPK pathway may participate in the regulation of histone acetylation in COPD.Based on these results,we explored the histone acetylation regulating IL-8 release mediated by cigarette smoke and the regulative effect of MAPK pathway in COPD rat and bronchial epithelail cell. Chapter One Expression of acetylated histone H4 and HDAC2 in the lung tissues of COPD rat and the anti-inflammatory effect of AminophyllineObjective:To study the expression of acetylated histone H4 and histone deacetylase（HDAC2） in the lung tissues of COPD rat model and its influence on IL-8 expression.At the same time,to explore the anti-inflammatory effect of Aminophylline.Methods:36 Wister rats were divided into three groups at random:control group、COPD group and Aminophylline group.Rat COPD model was established by intratracheal instillation of lipopolysaccharide（LPS） twice and exposure to cigarette smoke daily. Aminophylline group received Aminophylline(5 mg·kg^-1·d ) daily via the abdominal cavity injection.The spirometry was conducted and the pathological changes were observed after the model was established.HDAC activity was examined by colorimetric method.The expression of protein of acetylated H4 and HDAC2 in the lung tissues was examined by using immunohistochemistry and western blot.The concentrations of IL-8 in bronchoalveolar lavage fluid（BALF） was measured by enzyme-linked inmunosorbent assay（ELISA）.And the mRNA of IL-8 and HDAC2 in the lung tissues was detected by reverse transcription-polymerase chain reaction（RT-PCR） respectively.Results:Significant decrease of PEF、FEV0.3、FEV_0.3/FVC were found in the COPD rat model group compared with the control group. Aminophylline could improve lung function.The activity of HDAC was lower in the COPD group than in the control group,and Aminophylline could restore HDAC activity partly.In the COPD group,the expression of acetylated H4 protein was increased markedly compared with the control group,and Aminophylline could decrease acetylated H4 protein.The expression of HDAC2 protein was decreased in the COPD group and Aminophylline could enhance the expression of HDAC2 protein.The expression of IL-8 mRNA and the concentrations of IL-8 in BALF were higher in the COPD group than in the control group.Aminophylline could decrease IL-8 levels,and this was associated with increased HDAC activity and HDAC2 protein levels.But there was no difference in HDAC2 mRNA levels between these three groups.Conclusion:In COPD,histones were highly acetylated in the lung tissue.The acetylation of histones can induce increased expression of IL-8.While,Aminophylline can activate HDAC and decrease acetylation of histones as well as the expression of IL-8,So it has the effect of anti-inflammation.Chapter Two Cigarette smoke induces histone acetylation and up-regulates IL-8 expression in NHBE cells via MAPK pathwayObjective:To investigate the mechanism of histone acetylation mediated by cigarette smoke inducing bronchial epithelial IL-8 release and the regulative effect of MAPK pathway in this process.Methods:We treated normal human bronchial epithelial（NHBE）cells with 5%and 10%cigarette smoke extract （CSE）respectivly,then the expression of p-p38、p-ERK1/2、p-JNK（activated MAPK pathway protein）and HDAC2 in NHBE cells were detected by immunocytochemistry and western blot.After that,we treated NHBE cells with 10%CSE、TSA（HDAC inhibitor）、Aminophylline+CSE、SB203580（p38 inhibitor）+CSE、PD98059 （ERKinhibitor）+CSE and SP600125（JNK inhibitor）+CSE respectively. HDAC activity was examined by colorimetric method.The expression of protein of acetylated H4 in NHBE cells was examined by using immunocytochemistry and western blot.The concentrations of IL-8 in NHBE cells supernatant was measured by enzyme-linked inmunosorbent assay（ELISA）.And the level of IL-8mRNA in NHBE cells was detected by reverse transcription-polymerase chain reaction（RT-PCR） respectively.Results:CSE stimulation could activate MAPK pathway.In the presence of CSE,the expression of p-p38、p-ERK1/2 and p-JNK significantly increased.While,the the expression of HDAC2 decreased in the CSE group.In the CSE and TSA group,the activity of HDAC was lower than in the control group,but the expression of acetylated H4 protein was increased markedly compared with the control group,and the concentrations of IL-8 in supernatant and the expression of IL-8 mRNA were higher than in the control group.Aminophylline could restore HDAC activity partly,as a result,decreasing the expression of acetylated H4 and IL-8.The expression of acetylated H4 and IL-8 which were induced by CSE was inhibited by SB203580 and PD98059,but not by SP600125.SB203580、PD98059 and SP600125 had no effect on HDAC activity.Conclusion:In NHBE cells,CSE stimulation could induce histone acetylation associated with IL-8 expression subsequently.Aminophylline may fulfill the effect of anti-inflammation through activating HDAC and decreasing acetylation of histone.p38 and ERK1/2 MAPK pathway could down-regulate histone acetylation and inhibit the expression of IL-8 subsequently which were induced by CSE.更多还原