桔梗引经治疗慢性阻塞性肺疾病机理研究

【作者】 黄颖；

【导师】 李宇航；

【作者基本信息】 北京中医药大学 ， 中医临床基础， 2008， 博士

【摘要】 慢性阻塞性肺疾病(chronic obstructive pulmonary disease, COPD)是一种常见的呼吸系统疾病,以气道炎症及气流阻塞为发病特点。桔梗作为手太阴肺经的引经药,在肺系疾病的治疗中常常配伍使用。本课题在文献、理论研究基础上,从实验角度观察桔梗作为引经药与清热解毒药物配伍后对COPD的治疗作用及作用机理,进一步探讨桔梗引经的作用机制。论文共分三部分:第一部分文献综述。系统综述了COPD的现代研究、中医药治疗概况、桔梗在呼吸系统的应用以及归经、引经理论的研究进展。目前,COPD发病情况已在全球引起重视。全球许多学者围绕COPD病因学、发病机理、诊断、治疗等从实验和临床方面开展了大量研究工作,取得了一定的研究成果。大量研究显示,中医药通过其多靶点、多环节、多途径的调理作用在COPD的治疗中具有一定的优势。归经、引经理论是中医药理论体系的重要组成部分,对中医临床用药发挥指导作用。近年来,许多学者尝试用现代理论、技术和方法对归经、引经进行研究,并取得了一定的成果,但仍未有重大突破,尚需结合相关领域的最新研究进展,不断地探索。第二部分理论探讨。梳理历代医家对归经、引经理论的认识,对常用引经药的功效及临床应用进行分析,探讨张仲景《伤寒杂病论》中有关归经、引经的学术思想。中药归经、引经理论起于秦汉,在金元时代基本确立,明清时代渐趋完善。其中张仲景《伤寒杂病论》所创立的六经辨证与脏腑辨证体系为归经理论的形成奠定了基础。引经药在方剂中能够引导其它药物的药力直达脏腑、经络等病变部位,使药效集中,从而提高疗效。历代医家认为桔梗是手太阴肺经的引经药,可为“诸药舟楫”。第三部分实验研究。目的:从病理形态学角度观察清热解毒药中配伍桔梗后对COPD的治疗作用。从炎症细胞、细胞因子角度探讨清热解毒药中配伍桔梗对COPD治疗作用的机理。从肺内生物活性肽肠三叶因子(trefoil factor family3,TFF3)、血管活性肠肽(vasoactive intestinal peptide,VIP)角度探讨引经机制。方法:用熏香烟加气管注入脂多糖的联合造模方法建立大鼠COPD模型,将大鼠随机分为七组,即假手术组、模型组、桔梗组(桔梗)、桔甘组(桔梗、甘草)、银翘组(银花、连翘等)、桔银翘组(桔梗、银花、连翘等)、桔甘银翘组(桔梗、甘草、银花、连翘等)。假手术组、模型组灌胃生理盐水,各给药组灌胃相应中药,连续28天。取大鼠肺组织分别制作光镜、电镜标本。通过病理切片及半定量病理分析评价各给药组对COPD中的治疗效果。用ABC-ELISA法测定大鼠肺泡灌洗液(BALF)中IL-1β、TNF-α、TGF-β的含量并计数大鼠BALF中白细胞数量。用RT-PCR技术测定肺组织肠三叶因子(TFF3)mRNA表达。用放免法测定肺组织中血管活性肠肽(VIP)的含量。结果及分析:(1)模型组光镜下观察结果显示,大鼠肺泡结构破坏,局部肺泡萎陷,肺泡间隔明显增宽,甚至融合,肺泡壁和间质内有大量炎细胞浸润。支气管上皮明显增生,部分上皮脱落,管壁及其周围有明显炎细胞浸润。超微结构观察显示,大鼠肺泡Ⅱ型上皮细胞变性,细胞表面微绒毛减少、断裂甚或脱失,线粒体肿胀,嵴消失,甚至呈空泡样,肺泡腔内巨噬细胞次级溶酶体增多。半定量病理分析显示,肺平均内衬间隔(MLI)、平均肺泡数(MAN)较假手术组有显著性差异(P<0.01)。表明模型组的肺泡直径增大,单位面积上的肺泡数减少,肺泡结构破坏明显。提示: COPD模型组镜下改变与假手术组相比具有明显差别,肺组织结构严重受损,炎症反应增多。桔梗组、桔甘组、银翘组对COPD病理改变与模型组相比均无明显统计学差异。提示单味桔梗或单纯清热解毒药对COPD病变改善不明显。桔银翘组即在清热解毒药中配伍桔梗,光镜下观察显示,病变范围缩小,程度减轻。炎性浸润明显减少,大部分肺泡结构基本恢复。超微结构显示,肺泡上皮细胞变性不明显,细胞结构多完整,线粒体结构正常,中性粒细胞、巨噬细胞等炎症细胞减少。半定量病理分析显示,肺平均内衬间隔(MLI)较模型组有显著差异(P<0.05),平均肺泡数(MAN)虽无统计学差异,但有一定升高趋势。说明桔银翘组能明显改善肺泡直径,在一定程度上增加肺泡数。提示:桔银翘组与COPD模型组相比病理改变具有明显差异。清热解毒药中配伍桔梗对COPD的炎症反应、肺组织结构改变具有一定的改善作用,优于单味桔梗组和单纯清热解毒药组,说明桔梗在其中发挥了引经增效作用。桔甘银翘组即在清热解毒药中配伍桔梗、甘草,光镜下观察显示,病变范围缩小,程度减轻。炎性浸润明显减少,大部分肺泡结构基本恢复。超微结构显示,桔甘银翘组肺泡上皮细胞变性不明显,细胞结构多完整,线粒体结构正常,中性粒细胞、巨噬细胞等炎症细胞减少。肺平均内衬间隔(MLI)较模型组有降低趋势,平均肺泡数(MAN)较模型组显著升高(P<0.01)。提示桔甘银翘组能明显增加单位面积上的肺泡数。对COPD病理状态下肺组织结构有一定恢复作用。提示:桔甘银翘组与COPD模型组相比病理改变具有显著差异。清热解毒药中配伍桔梗、甘草对COPD的炎症反应、肺组织结构改变具有更明显的改善作用,说明桔梗、甘草配伍后发挥了配伍引经增效作用。(2)模型组与假手术组相比白细胞总数、IL-1β、TNF-α、TGF-β含量升高,具有显著统计学差异(P<0.01,P<0.01,P<0.05,P<0.05),提示COPD病理模型的具有明显的炎症征象,这与上述病理切片结果相一致。桔银翘组与模型组相比,白细胞总数、IL-1β有显著统计学差异(P<0.05,P<0.01),对TNF-α有一定降低趋势。说明桔银翘组能够在一定程度上减少COPD模型白细胞总数、IL-1β、TNF-α含量。提示桔银翘组改善COPD病理改变,发挥治疗作用可能与降低白细胞总数、IL-1β、TNF-α含量有关。桔甘银翘组与模型组相比,白细胞总数、IL-1β、TNF-α、TGF-β含量均有降低,具有显著统计学差异(P<0.01,P<0.01,P<0.05,P<0.05)。提示桔甘银翘组能够在一定程度上减少白细胞总数、IL-1β、TNF-α、TGF-β含量,与桔甘银翘组改善COPD病理改变,发挥治疗作用有关。(3)模型组TFF3基因表达、VIP含量较假手术组均呈降低趋势。表明COPD病理状态下这两种神经肽水平均有降低。各治疗组TFF3、VIP含量与模型组虽无统计学差异,但均有不同程度的上升趋势,其中以桔梗调节TFF3的变化趋势最大,推测桔梗的引经增效作用与调节TFF3有关,TFF3可能为桔梗的作用靶点之一。桔甘银翘组调节VIP变化趋势最大,推测桔梗、甘草的配伍引经效果与调节VIP有关。结论:清热解毒药物中配伍桔梗后,表现出一定的肺部引经增效作用,能够使清热解毒药物在肺部的治疗作用增加。这种引经增效作用机制可能与桔梗调节TFF3、VIP有关。配伍桔梗、甘草后表现出一定的配伍引经增效作用,其机制可能与调节VIP关系更为密切。为进一步确定TFF3、VIP是否为桔梗引经增效作用的靶点,仍需采用多种检测方法从不同角度证实。更多还原

【Abstract】 Chronic obstructive pulmonary disease (COPD) is a kind of familar respiratory disease, characterized by inflammation in air passage and obstruction of air flow.As a meridian guiding herb in Lung Meridian of Hand-Taiyin, Radix Platycodi is usually used in pulmonary disease.Base on the study of document and theory,we observe the therapeutic effect on COPD and its mechanism when Radix Platycodi as a meridian guiding herb,combined with heat-clearing and detoxicating herbs.Furthermore,we explore the mechanism of meridian guiding effect of Radix Platycodi.The thesis is totally divided into three parts:The first part is literature review.Summarize the modern research and therapeusis of Chinese medicine in COPD,the Radix Platycodi’application in respiratory system ,the researching progress in the theory of meridian tropism and guiding.At present,the incidence of COPD is attached importance by many researchers in the world.Many people do a lot of experimental and clinical study work with COPD about etiology, pathogenesy, diagnosis, therapeusis ect.And these studies acquire many achievements.A great deal of researchs show that chinese medicine have some advantages on the treatment of COPD.Because they have effects on many targets, elements and pathways.The theory of meridian tropism and guiding is one of the important part in Chinese medicine.It has directive effect on clinic.Recently years,many researchers attempt to study it using mordern theory, technique and methods.These studies obtained a certain progress, but have not grave breakthrough and still need explore it combining the latest researching progresses.The second part is theoretical study.Comb the understanding of Chinese doctors about the theory on meridian tropism and guiding in the history of Chinese medicine.Analyse the effect and clicical application of meridian guiding herbs.Explore academic ideology of Zhangzhongjing about the theory of meridian tropism and guiding in Treatise On Cold-Attack and Miscellaneous Disease.The theory of meridian tropism and guiding was originated from Qin and Han dynasties, established in Jin and Yan dynasties, perfected in Ming and Qing Dynasties.The six meridian syndrome differentiation and visceral syndrome differentiation,which founded by Zhang zhongjing,were the foundation of the theory .The meridian guiding herbs can lead other medicine to diseased region,concentrate drug action,improve the effect. Radix Platycodi is considered as a meridian guiding herb in Lung Meridian of Hand-Taiyin,possessing the floating effect. It is usually used in pulmonary disease. The third part is experimental study.Objective:Obsever the therapeutic effect of heat-clearing and detoxicating herbs compined with Radix Platycodi on COPD through pathomorphology.Explore the mechanism of the therapeutic effect through inflammatory cell and cytokine. Study the mechanism of meridian guiding through TFF3、VIP.Methods:COPD rat model was induced by cigarette smoking inhalation and intratracheal LPS exposure.Rats were divided into 7 groups,namely normal group,model group, Jiegeng group(Radix Platycodi),Jiegan group(Radix Platycodi, Glycyrrhiza),Yinqiao group(Flos Lonicerae, Forsythia suspense, ect),Jieyinqiao group(Radix Platycodi,Flos Lonicerae, Forsythia suspense,ect),Jieganyinqiao group(Radix Platycodi,Glycyrrhiza,Flos Lonicerae, Forsythia suspense,ect). Normal group and model group were given saline,other groups were given corresponding herbal medicine, continuously for 28 days. Takeing lung tissue of rats, making specimen for optical microscope and electron microscope.Measures the content of TNF-α,TGF-,IL-1βwith the ABC-ELISA method and count the quantity of white blood cell in BALF. Measures trefoil Factor Family3 (TFF3) mRNA expression on the lung with RT-PCR technique. Measures the content of vasoactive intestinal peptide (VIP) in the lung with radio-immunity methods.Results and analysis:The light-microscope observation show that in the model group ,the structure of bronchoalveolus is destructive,detelectasis and the interval is broaden, coalesce. There have many inflammatory cells infiltration in alveolar wall and interalveolar septum. Bronchial epithelium proliferate obviously.Some parts of epithelium are deciduous. Inflammatory cell infiltrate into bronchial wall.The ultra-microstructure observation show that alveolar typeⅡepithelial cells degenerate, microvilli in the surface of cell deplete, fragmentation and deciduous. Mitochondria swelling, ridge disappearing. secondary lysosome of macrophagocyte increase.Pathological semiquantitative analysis show that MLI、MAN have significant difference comparing with normal group(P<0.01).It manifest that alveolar diameter increased,the quantity of bronchoalveolus reduced,the structure of bronchoalveolus is detoryed obviously.The results manifest that the changes in model group have significant difference comparing with normal group in microscope.The structure of lung tissue is distoryed severly,inflammatory reaction increased.Jiegen group, Jiegan group,Yinqiao group have not obviously different on pathological changes comparing with model group.It manifest that when Radix Platycodi and heat– clearing and detoxicating herbs use separately ,the meliorative effect on pathological changes is not obviously.Jieyinqiao group ,that is heat-clearing and detoxicating herbs combined with Radix Platycodi.The light-microscope observation show,the range of pathological changes reduce,the degree relieve.The infiltration of inflammatory cell decrease obviously.The structure of bronchoalveolus recover mostly. The ultra-microstructure observation show,the change of alveolar typeⅡepithelial cells is not obviously.The structure of cell is integrated. The structure of mitochondria is normal.The quantity of neutrophile granulocyte and macrophagocyte are reduced. Pathological semiquantitative analysis show that, MLI has significant difference comparing with model group(P<0.05). MAN has trend of increase. It manifest that Jieyinqiao group can meliorative alveolar diameter obviously, increase the quantity of bronchoalveolus on some degree.It manifest that Jieyinqiao group have significant difference comparing with model group on pathological changes. Heat-clearing and detoxicating herbs combined with Radix Platycodi have some meliorative effect on inflammatory reaction and structure of lung tissue.The effect is better than Jiegeng group, Yinqiao group.It reveal that Radix Platycodi exerts synergized action through meridian guiding.Jieganyinqiao group, that is heat-clearing and detoxicating herbs combined with Radix Platycodi, Glycyrrhiza.The light-microscope observation show,the range of pathological changes reduce,the degree relieve.The infiltration of inflammatory cell decrease obviously.The structure of bronchoalveolus recover mostly. The ultra-microstructure observation show,the change of alveolar typeⅡepithelial cells is not obviously.The structure of cell is integrate. The structure of mitochondria is normal.The quantity of neutrophile granulocyte and macrophagocyte are reduced. Pathological semiquantitative analysis show that, MLI has trend of decrease comparing with model group. MAN has significant increase comparing with model group(P<0.01) . It manifest that Jieganyinqiao group can increase the quantity of bronchoalveolus obviously,have meliorative effect on lung tissue structure.The result reveal that Jieganyinqiao group have significant difference comparing with model group on pathological changes. Heat-clearing and detoxicating herbs combined with Jiegeng ,Glycyrrhiza have obviously meliorative effect on inflammatory reaction and structure of lung tissue.It make out that Radix Platycodi combined with Glycyrrhiza exerts synergized action through compatibility meridian guiding.Compare with normal group,the amount of white blood cell,the content of IL-1β, TNF-α, TGF-βof model group increase significantly(P<0.01,P<0.01,P<0.05,P<0.05).It show that the model of COPD have obviously sign of inflammation.It is consist with pathological results.Compare with model group, the amount of white blood cell and the content of IL-1βof Jieyinqiao group have significant different(P<0.05,P<0.01), the content of TNF-αhas trend of decrease. It show that Jieyinqiao group can reduce the amount of white blood cell ,the content of IL-1βand TNF-α.The result reveal that meliorative effect of Jieyinqiao group is related with the decrease of white blood cell ,IL-1βand TNF-α.Compare with model group, the amount of white blood cell,the content of IL-1β, TNF-α, TGF-βof Jieganyinqiao group decrease significantly(P<0.01,P<0.01,P<0.05,P<0.05).It manifest that therapeutic effect of Jieganyinqiao group is related with the decrease of white blood cell ,IL-1β,TNF-αand TGF-β.Compare with normal group ,the expression of TFF3 mRNA , the content of VIP in model group have trend of decrease .It show that this two kinds of peptide decrease in pathological state.On other therapeutic groups, TFF3、VIP have increasing trend on some degree. Jiegeng group regulate TFF3 better than other groups.It can speculate that Radix Platycodi‘s synergized action of meridian guiding is related with TFF3.TFF3 may be the target of Radix Platycodi.Jieganyinqiao group regulate VIP better than other groups.It can speculate that Radix Platycodi combined with have synergized action of compatibility meridian guiding ,it is consist with VIP.Conclusion:Heat-clearing and detoxicating herbsherbs combined with Radix Platycodi have synergized action through lung meridian guiding.It can strength the effect of heat-clearing and detoxicating herbsherbs.The mechanism is concerned with TFF3、VIP. Radix Platycodi combined with Glycyrrhiza show some synergized action through compatibility meridian guiding.Its mechanism have relation with VIP.In order to ensure TFF3、VIP are target of Radix Platycodi,we need many different testing methods to confirm it.更多还原