【作者】 孟晓明；

【导师】 李俊；

【作者基本信息】 安徽医科大学 ， 药理学， 2008， 硕士

【摘要】 慢性支气管炎（chronic bronchitis,CB）是一种常见的慢性非特异性呼吸系统疾病,主要临床特征为反复发作的咳嗽、咳痰或伴喘息。其发病率高,病情反复,可并发阻塞性肺气肿,甚至肺动脉高压、肺原性心脏病等。目前认为,大气污染、吸烟、病原体感染及过敏因素等与慢性支气管炎的发病密切相关,但发病后的机体免疫功能状态则影响了慢性支气管炎的发生和发展。因此,纠正或调节机体免疫功能紊乱可能是防治慢性支气管炎的重要手段之一。慢支的治疗,目前应用较多着眼于抗炎、镇咳、平喘作用的研究,但尚无疗效很满意的药物,而通过使用改善机体免疫功能状态的免疫调节药物,尤其是具有免疫调节作用的中药来减少细胞因子、炎症介质等促炎因素的产生,从而抑制慢支炎症进展,可能是慢支治疗的新思路。枇杷叶是蔷薇科枇杷属枇杷Eriobotryajaponica（Thunb）Lindl的叶,一直是我国传统医药治疗呼吸道炎症性疾病的常见中药之一,本课题组前期在国家自然科学基金资助下（NO:30371766）证实枇杷叶的有效活性部位为三萜酸。枇杷叶三萜酸（triterpene Acids of Loquat Leaf,TAL）有高效、低毒的作用特征,并具有明显的抗炎及免疫调节作用,对慢支具有较理想的防治作用。肺泡巨噬细胞（alveolar macrophages,AM）是肺部炎症反应细胞因子、生长因子、炎症介质、氧自由基的重要来源,是肺内重要的防御、保护和调节细胞。AM的激活是肺部炎症反应的重要始动因素。前期研究表明TAL灌胃给药可明显降低慢支大鼠肺组织匀浆中TNF-a、IL-8、IL-1水平,升高IL-10的表达,并对慢支大鼠气道粘膜上皮细胞NF-κB、ICAM-1的蛋白表达具有一定的抑制作用。此外TAL能明显抑制慢支大鼠AM自由基的产生,抑制炎症介质PGE₂及LTB₄的合成及释放。一氧化氮（NO）是一多功能分子,在呼吸系统的生理和病理过程中起重要作用,因此研究TAL对其是否具有药理作用也显得尤为关键。此外对于AM等细胞激活的分子机制的目前尚不明确。鉴于MAPK信号传导通路在炎症反应中的重要作用,本课题旨在前期研究的基础上,以AM为平台,MAPK信号通路为研究对象,研究慢支大鼠AM MAPK信号传导通路并探讨TAL对其信号传导通路的影响,从而进一步阐明TAL对慢性支气管炎防治作用的细胞分子机制,为将其开发为治疗慢支的新药提供试验依据。课题为国家自然科学基金资助项目（NO:30572355）。主要研究内容包括以下三个方面:1.枇杷叶三萜酸对慢性支气管炎大鼠肺泡巨噬细胞一氧化氮合酶及一氧化氮表达的影响复制大鼠慢支模型,通过TAL灌胃给药治疗,观察TAL对CB大鼠AM iNOS表达、活性及NO释放的影响。结果显示:TAL抑制CB大鼠AM iNOS的表达及其活性,从而减少NO的释放,这可能是其对慢支的治疗与防治作用机制之一。2.慢支大鼠肺泡巨噬细胞内MAPK信号传导通路及其他通路的关系分别应用MAPK通路上游信号蛋白（PTK,PI₃K,Akt,PKC）的特异性阻滞剂预处理AM,观察下游蛋白磷酸化水平,以确定转录前调节相关信号蛋白。结果显示:Genistein可以不同程度的抑制PKC、PI₃K/Akt、MAPK通路磷酸化的程度;LY294002可以抑制PI₃K/Akt磷酸化的水平,同时诱导p-p38、p-JNK的高表达,对PKC/ERK这条经典途径的磷酸化也有一定的激活作用:Calphostin C阻断PKC的磷酸化,对p-ERK水平有较大影响,而对p-p38和p-JNK则影响不大。推出大鼠慢支模型大鼠AM中LPS信号转导MAPK通路可能为PTK—PI₃K/Akt—JNK/p38或者PTK—PI₃K—PKC—ERK。3.枇杷叶三萜酸（TAL）治疗慢性支气管炎的作用位点研究基于第二部分的研究,进一步探讨TAL对MAPK通路的转录前信号蛋白的作用环节。发现TAL对p-p38,p-JNK,p-ERK,p-PKC及p-Akt皆有一定抑制作用（其中对p38和JNK磷酸化水平有显著的抑制效应）,而对p-PI₃K,其作用不甚明显;同时,TAL对模型组TLR4受体水平的增高也无明显的降低作用。提示TAL对MAPK调控可能发生在PI₃K下游某个环节,具体机制还有待于进一步研究。更多还原

【Abstract】 Chronic Bronchitis（CB） is a common respiratory system disease whose clinical features are recurrent attacks of cough,expectoration or gasp.It has a very high attack rate and it can also lead to obstructive emphysema or even pulmonary artery hypertension as well as pulmonary heart disease.Currently,it is widely accepted that atmospheric contamination,infection and factors of hypersusceptibility have close relationships with the Chronic Bronchitis while the conditions of immune function play a more important role in the process of CB.Therefore,to regulate the disorder of neuroendocrine-immuno-function may the key method to prevent and cure the Chronic Bronchitis.At present,people pay more attention to anti-inflammatory,relieving cough on curing the disease,however,it is hard to find an effective medicine.On the other hand, medicines which improve the immune function of body,especially the traditional Chinese medicine,can prevent the course of aggravation through reducing the cytokines and mediators of inflammation.The leaf of Eriobotrya japonica（Thunb.）Lindl has a long history of medicinal use in China in a variety of inflammatory conditions especially CB therapy.According to our previous study,the triterpene acids extracted from Eriobotrya japonica（Thunb.）Lindl Leaf was the effective component and had therapy effect on chronic bronchitis.In addition,the characters of this medicine are the high performance and low toxicity.As a major effector of the innate immune system of the body,alveolar macrophage （AM） is the main source of cytokine,growth factor and oxygen free radical.Meanwhile, it is the principle cell whose functions are protection and adjusting in our lung.In our study,AM acts as the target on which we discuss the molecular mechanism of TAL on curing the CB.In this case it can provide the basement for the exploitation of new medicine on this field.Our previous research shows that intragastric administration of TAL can reduce the level of TNF-a,IL-8,IL-1 in tissue homogenate of lung and enhance the expression of IL-10,at the same time,the level of NF-κB and ICAM-1 in endothelial cell of air passage is inhibited.In addition,TAL can suppress free radical in AM of CB rats dramatically as well as the synthesizing and releasing of PGE2,LTB4 NO is a kind of multifunctional molecule which plays a significant role in the process of physiology and pathology in the respiratory system.Therefore it is important to research whether TAL has an effect on it.What’s more,the molecular mechanism in cell-stimulating of AM is not very clear.As widely known that MAPK play a key role in inflammatory reaction,we use the AM and MAPK,as the researching platform and target respectively,to study the MAPK signal transduction pathway in AM and discuss the effect of TAL.It can help us to have a deep view in the molecular mechanism of the curing effect of TAL,meanwhile,we can get the basement for the research of the new medicine.The main content is divided into three sections as follows: 1 The effect of triterpene acids of Eriobotrya japonica（Thunb.）lindl.leaf on the expression of iNOS and production of NO in alveolar macrophage of chronic bronchitis rats.Establishing the model of CB rats by using BCG plus LPS,a week later,the intragastric administration of TAL was operated.Consequently,we investigate the effect of Triterpene Acids of Eriobotrya japonica（Thunb.）Lindl.Leaf（TAL） on the iNOS and the production of NO in alveolar macrophages（AM） of chronic bronchitis （CB） rats and the mechanism related to anti-inflammatory action.The results show that TAL can reduce the expression of iNOS and release of NO in AM of CB rats,it may be one of the mechanism of the preventive and therapeutic effect in chronic bronchitis. 2 MAPK signal transfluction pathway in AM of CB ratsInhibitors of PTK,PI₃K,Akt and PKC were used to detect correlated signal protein of MAPK before transcription in AM of CB rats.PKC,PI₃K/Akt,phosphorylation were reduced after treatment with inhibitors of PTK.LY294002,the inhibitor of PI₃K suppressed PI₃K and Akt phosphorylation but increased p38 and JNK phosphorylation. ERK phosphorylation was decreased by Calphostin C,an inhibitor of PKC,However,it has no effect on the phosphorylation of p38 and JNK.According to our results,the LPS-induced MAPK signal pathway in AM of CB rats may be PTK---- PI₃K/Akt----JNK/p38 or PTK----PI₃K----PKC----ERK.3 The effect of TAL on MAPK activation.Further research suggested that TAL can reduce the phosphorylation of PKC,p38, JNK and ERK while it almost has no effects on PI₃K as well as Akt.TLR4 receptor expression in AM of CB rats was highly increased when stimulated by LPS,however, TAL can not decrease its production.In conclusion,action of TAL on MAPK in AM may be happened on downstream of PI₃K.更多还原