【作者】 王雅娟；

【导师】 陈季强； 吴希美；

【作者基本信息】 浙江大学 ， 药理学， 2009， 博士

【摘要】 cAMP和cGMP能够介导多种生物反应,如调节细胞分泌、收缩、代谢和生长等重要的细胞功能,对PKA和PKG、鸟苷酸交换因子、环核苷酸门控的钠通道和钙通道有调节作用。调控细胞内的cAMP和cGMP水平能够对细胞生理产生重要影响。cAMP和cGMP在细胞内的水平主要通过腺苷酸环化酶和鸟苷酸环化酶的合成和多种磷酸二酯酶(phosphodiesterase,PDE)的水解来保持。PDE是细胞内存在的一种重要的酶类,分为11个家族,即PDE1-11,可以水解cAMP和cGMP中的磷酸二酯键,使其变为无活性的5’-AMP和5’-GMP。抑制磷酸二酯酶可以调节多种细胞功能,对呼吸系统疾病如哮喘和慢性阻塞性肺病有潜在的临床应用价值,其中PDE4抑制剂在呼吸系统疾病中的应用尤其受到重视,开发中的PDE4抑制剂主要包括roflumilast、cilomilast、piclomilast、tetomilst等,其中Roflumilast和Cilomilast最值得关注,已进入临床Ⅲ期研究,有望用于哮喘和COPD的治疗。分支杆菌是最强的Th1反应诱导剂之一,卡介苗是减毒活牛分支杆菌,最初用作结核疫苗,后来有研究发现卡介苗能够调节Th1/Th2平衡,可以用于预防或治疗哮喘,但是卡介苗防治哮喘的机制尚未完全阐明,本论文第一部分以PDE为中心,观察了卡介苗及其相关成分免疫对哮喘大鼠症状的改善作用,并对Th1/Th2平衡以及对肺组织中的PDE活性和表达的影响,同时对cAMP及其信号通路的变化进行了检测,从而进一步阐明卡介苗防治哮喘的可能机制。目的:观察卡介苗免疫对哮喘大鼠模型的治疗作用,并研究其可能的作用机制。方法:用OVA致敏并攻击复制大鼠哮喘模型,同时以卡介苗及其相关成分免疫大鼠,观察哮喘大鼠肺内的炎症变化,气道高反应性改变、Th1/Th2平衡、PDE活性及各PDE家族的表达、cAMP水平以及cAMP下游效应蛋白PKA和Epacl表达水平的变化;并用LPS刺激损伤A549细胞,模拟哮喘组织损伤的细胞模型,同时用卡介苗进行干预,观察卡介苗对细胞PDE以及PKA、Epacl表达水平的影响,从而推测卡介苗免疫治疗大鼠哮喘的可能机制。结果:卡介苗及其相关成分能够不同程度的减轻哮喘大鼠肺内的炎症,降低大鼠气道高反应性,抑制PDE活性和多种PDE的表达水平,降低cAMP和PKA水平,不同浓度的卡介苗也能够抑制A549细胞的PDE及PKA的表达。结论:卡介苗免疫能够改善哮喘大鼠的症状,而PDE抑制可能是其机制之一,卡介苗或其相关成分可能具有潜在的多重PDE抑制作用。PDE4抑制剂在COPD治疗中的应用也受到了广泛关注,有临床数据显示Roflumilast和Cilomilast这两个进入临床Ⅲ期研究的PDE4抑制剂能够改善COPD患者的呼吸功能和恶化率,并能够有效改善生活质量。目前治疗COPD的方法十分有限,迫切需要开展一些全新的疗法,而PDE4抑制剂由于其能够有效抗炎而成为一类较有前景的COPD治疗药物。本论文第二部分初步研究了一新型PDE4抑制剂Zl-n-91对大鼠COPD模型的治疗作用,提示了其可能存在的临床应用价值。目的:初步探讨新型PDE4抑制剂Zl-n-91对于大鼠COPD模型的治疗作用,为其将来的临床应用提供依据。方法:熏烟及气道滴入LPS复制大鼠COPD模型,并用0.03mg/kg、0.3mg/kg、3mg/kg的Zl-n-91进行治疗,观察大鼠肺内的炎症变化,PDE4活性变化,肺组织MPO活性和MMP-9水平以及大鼠肺顺应性和肺阻力的改变。结果:各浓度的Zl-n-91能够不同程度的减少COPD大鼠肺组织的炎症浸润,有效抑制PDE4活性,降低MPO活性和MMP-9水平,对COPD大鼠的肺功能也有明显的改善作用。结论:Zl-n-91能够有效抑制COPD大鼠肺内的炎症,改善COPD症状,可能具有COPD治疗的临床应用价值。更多还原

【Abstract】 cAMP and cGMP can mediate many biological responses(e.g.,regulation of important cell functions such as secretion,contraction,metabolism,and growth),they have regulatory effects on protein kinase A(PKA) and protein kinase G(PKG),the guanine-nucleotide exchange factors(GEFs),and the cyclic-nucleotide gated(CNG) sodium and calcium channel.Manipulation of cAMP and cGMP levels in the cell represents a powerful nechanism for controlling cellular physiology.To maintain their intracellular levels,cAMP and cGMP are synthesized by adenylyl cyclases(ACs) and guanylate cyclases(GCs) whereas their degradation(hydrolysis) is mediated by a variety of phosphodiesterases(PDEs) present in the cells.PDEs are a group of 11 families(PDE1-11) which is essential important in cells and hydrolyze the phosphodiester bond of cAMP and cGMP to provide the inactive products,e.g.,5’-AMP and 5’-GMR To inhibit PDEs can regulate a variety of cell functions and PDE inhibitors have potential value in therapy of lung diseases such as asthma and chronic obstructive pulmonary disease(COPD).Particular emphasis will be placed on PDE4 inhibitors which include roflumilast,cilomilast,piclomilast,tetomilst in development.Roflumilast and cilomilast had reached the stageⅢof clinical trials and may become available for treating asthma and COPD.Mycobacteria are among the most potent inducers of T helper 1(Th1) type response, Bacillus Calmette-Guérin(BCG) is attenuated live Mycobacterium bovis and used as a vaccine for tuberculosis.There had studies shown that BCG infection regulates Th1/Th2 balance,and could be used in asthma,but the mechanism of which is not entirely clarified.The first part of this thesis studied the effect of BCG and its compositions on asthma in rats,and detected Th1/Th2,PDEs activity and mRNA expressions,cAMP and its signal pathways in asthma rats using PDEs as a center to further clarify the possible mechanism of it.Objective:To study the effect of BCG on asthma in rats and find it possible mechanism. Methods:male Sprague-Dawley rats are sensitized and challenged with ovalbumin (OVA) and infected with BCG and its compositions,the inflammation in lungs and airway hyperresponsivness(AHR),Th1/Th2,the activity and mRNA expressions of PDEs,cAMP and PKA,Epac1 were detected.A549 cells were used to further study the effect of BCG on PDEs mRNA expressions and PKA,Epacl expressions with or without LPS injury.Results:BCG and its compositions inhibited inflammation in asthma rats and decreased AHR,inhibited PDEs activity and mRNA expressions, decreased cAMP and PKA.Different contents of BCG could also inhibit PDEs mRNA and PKA expression in A549 cells.Conclusion:BCG infection is of some benefit on asthma improvement and PDE inhibition may be one of the mechanism,and BCG and/or its composition may have potential multiple PDE inhibition effect.PDE4 inhibitors is a group of significant importance among new therapeutic agents that are under development in COPD treatment.The two selective PDE4 inhibitors that are at PhaseⅢclinical trial stage are cilomilast and roflumilast.The studies have demonstrated that anti-inflammatory effects of cilomilast and roflumilast positively contribute to the respiratory function,frequency of exacerbations and quality of life of COPD patients.Treatment options for COPD are quite limited and there is an urgent need for new treatment strategies.PDE4 inhibitors is a group of potent COPD drugs with great prospect because of their anti-inflammatory actions.The second part of this thesis studied the effect of a new selective PDE4 inhibitor,Z1-n-91 on COPD rat,and suggested its potent value in clinical application.Objective:To investigate the therapeutic effect of a new selective PDE4 inhibitor, Z1-n-91 on COPD rats,provide some basis for its futher clinical application.Methods: Male Sprague-Dawley rats are exposed to cigarette smoking and intracheal lipopolysaccharide(LPS) instillation to induce COPD.0.03,0.3 or 3mg/kg Z1-n-91 were administered.Cells in bronchoalveolar lavage fluid(BALF) were counted, cAMP-PDE and PDE4 activities were assayed,MPO activity and MMP-9 level were assayed using assay kits,and lung function of rats were measured.Results:Z1-n-91 at 0.03,0.3 and 3 mg/kg inhibited inflammatory cells infiltration in lungs and significantly decreased total number of cells and numbers of eosinophils and neutrophils in BALF, inhibited PDE4 activity and MPO activity,and decreased MMP-9 level in lungs, fininally Z1-n-91 markedly improved lung function.Conclusion:Z1-n-91 could inhibit inflammation and improve the symptoms in COPD rats,and it may be of therapeutic potential as an alternative medicine in COPD therapy.更多还原