三拗汤及其加味方功效物质靶点网络构建及干预PM2.5诱导加重哮喘的研究

发布时间：2018-05-18 05:32

本文选题：哮喘 + 三拗汤　；参考：《南京中医药大学》2017年博士论文

【摘要】：目的:1.本研究在梳理中医对支气管哮喘的认识和三拗汤(出《太平惠民和剂局方·卷二》)及加味治疗哮喘的基础上,以三拗汤及其加味方中的五拗汤(出《医学集要·卷二》,药用麻黄、杏仁、甘草、石膏、细茶)为研究对象,构建其配伍药物的功效成分多靶点网络药理学模型,并与哮喘的靶点通路进行比较研究,预测三拗汤及加味方防治哮喘的潜在靶标。2.结合临床哮喘发病特点,以空气污染物PM2.5作为刺激因素,建立OVA复合PM2.5诱导加重哮喘模型,探讨三拗汤及加味方干预PM2.5诱导加重哮喘的效应及效应机制。方法:1.整理历代医家对哮喘病因病机、辨证分型以及治法方药的认识,系统回顾三拗汤方证及其加味配伍,分析其证治特点,并对现代文献中哮喘方高频药物进行统计,对三拗汤临床疗效、功效物质、效应及效应机制的研究进行梳理。2.以三拗汤及其加味方为研究对象,采用网络药理学方法,构建其配伍药物的功效成分靶点通路网络,并与哮喘的靶点通路分析比较,预测三拗汤及加味方防治哮喘的潜在靶标。3.分别建立OVA复合空气污染物PM2.5诱导加重的28天和42天哮喘小鼠模型,比较两者在炎症反应、气道高反应、肺组织病变以及TRP通道表达方面的区别,获得效应确切且稳定的OVA复合空气污染物PM2.5诱导加重型哮喘小鼠模型。4.在潜在靶标筛选和PM2.5诱导加重哮喘模型研究的基础上,观察三拗汤及其加味方对PM2.5诱导加重哮喘在改善气道炎症、气道高反应和肺组织病变方面的作用,采用ELISA法观察小鼠BALF中IL-13、SP、PGD2、NGF含量的差异,q-PCR法测定肺组织TRPA1、TRPV1、TRPV4、TRPM8基因水平的表达,筛选对三拗汤及加味方效应敏感基因,以Western blot法进行相关蛋白水平测定,阐述两方干预PM2.5诱导加重哮喘的效应机制。结果:1.历代医家认为哮喘是病机复杂的疾病,根据其病证特点提出了宣降肺气、散寒祛痰法、散寒清肺法、清肺化痰法、引吐导痰法、降气豁痰法等治法。现代医家进一步提出了哮喘发作期标本兼治、从瘀论治、从风论治等治法。统计发现现代哮喘方中寒哮方和热哮方支持度较高的药物为麻黄、杏仁、甘草等。2.历代文献分析,三拗汤以咳嗽、哮喘为主证,其主要配伍为:加味解表药、化痰止咳平喘药、清热药、温里药、收涩药、理气药等。现代研究显示,三拗汤及加味方主要从抑制炎症、调节免疫、调控神经-受体和气道反应等方面干预哮喘。3.网络药理模型显示,三拗汤药物配伍后11个功效成分作用于74个靶点以及代谢-信号传导-免疫-细胞过程等47条通路,其中丝裂原活化蛋白激酶(MAPK)是其核心靶点群,与三拗汤宣肺平喘功效相关。三拗汤加味后功效成分和靶点通路均有增加。如五拗汤16个功效成分作用于98个靶点以及代谢-疾病-信号传导-免疫等71条通路,三磷酸鸟苷(GTP)蛋白酶和MAPK构成了五拗汤的核心靶点群,主要增加了代谢、疾病相关通路,这些改变与五拗汤宣肺清热的功效有关。哮喘82个靶点涉及疾病-免疫-信号传导-细胞过程等52条通路,肿瘤坏死因子(TNF)、核因子-1κB(NF-κB)家族是其核心靶点群,神经配体-受体相互作用通路靶点群是哮喘的潜在靶标。与哮喘靶点通路比较,三拗汤及其加味方的特点表现为主要通过酶调控多种活性物质的合成代谢以及免疫蛋白、炎症细胞因子等介质的参与,对哮喘气道炎症、信号传导等环节发挥作用。4.OVA复合PM2.5诱导加重小鼠哮喘反应主要表现在:增加血液和BALF中炎症细胞(尤其EOS)数量和比率,肺气道反应性增高,肺组织损伤严重,出现炎症渗出增加、支支气管黏膜层损坏以及气管平滑肌增厚。此外,哮喘小鼠出现了TRPA1、TRPV1蛋白及相关因子如IL-13、SP、PDG2和NGF的高表达。这些加重哮喘的效应以42天模型中施予PM2.5暴露的小鼠表现最为显著。5.三拗汤及其加味方五拗汤能改善PM2.5诱导加重哮喘小鼠(造模42天)的症状,尤其以两者高剂量效应更为显著,如减少血液和BALF中炎症细胞(尤其EOS)数量和比率,降低肺气道高反应性,改善肺组织损伤,其炎症渗出、支气管黏膜损害和支气管平滑肌增厚等病变程度均有所减轻。此外,三拗汤、五拗汤能够降低哮喘小鼠肺组织TRPA1、TRPV1基因和蛋白水平以及IL-13、SP、PDG2和NGF的高表达。与OVA+PM2.5组比较,三拗汤高剂量组显著降低TRPA1蛋白、IL-13、PDG2和NGF的高表达(P0.01),五拗汤高剂量组显著降低TRPV1蛋白、IL-13、SP、NGF(P0.01)和TRPA1蛋白、PDG2(P0.05)的高表达。结论:1.本研究系统梳理了历代文献对哮喘病因病机、辨证分型以及治法方药的认识,并对现代文献中哮喘方高频药物进行统计,其中寒哮方和热哮方支持度较高的药物为麻黄、杏仁、甘草等。系统回顾了三拗汤方证及加味应用,其主要配伍为:加味解表药、化痰止咳平喘药、清热药、温里药、收涩药、理气药等。现代研究显示,三拗汤及加味方主要从抑制炎症、调节免疫、调控神经-受体和气道反应等方面干预哮喘。2.构建了三拗汤、加味方五拗汤以及哮喘的网络药理学模型。三拗汤配伍药物11个功效成分作用于74个靶点以及代谢-信号传导-免疫-细胞过程等47条代谢通路,其中MAPK是其核心靶点群,与三拗汤宣肺平喘功效相关。三拗汤加味后功效成分和靶点通路均有增加。如五拗汤16个功效成分作用于98个靶点以及代谢-疾病-信号传导-免疫等71条通路,GTP蛋白酶和MAPK构成五拗汤的核心靶点群,主要增加了代谢、疾病相关通路,这些改变与五拗汤宣肺清热的功效有关。哮喘82个靶点涉及疾病-免疫-信号传导-细胞过程等52条通路,肿瘤坏死因子(TNF)、核因子-κB(NF-1κB)家族是其核心靶点群,神经配体-受体相互作用通路靶点群是哮喘的潜在靶标。与哮喘靶点通路比较,三拗汤及其加味方的特点表现为主要通过酶调控多种活性物质的合成代谢以及免疫蛋白、炎症细胞因子等多种介质的参与,在哮喘气道炎症、信号传导等环节发挥作用。3.成功建立OVA复合PM2.5诱导加重哮喘模型,结合三拗汤及其加味方网络靶标筛选结果,考察并比较三拗汤及加味方五拗汤的效应,揭示其潜在的神经-免疫学机制。研究证实三拗汤、五拗汤可改善哮喘症状,表现为减少炎症细胞浸润、降低气道反应性,减轻肺组织病理损伤,以两者高剂量改善效应最为显著。三拗汤、五拗汤可以通过神经调控干预TRP通道及神经递质释放对哮喘产生治疗作用,三拗汤调控TRPA1通道较为明显,五拗汤可显著调控TRPA1和TRPV1通道。
[Abstract]:Objective: 1. on the basis of combing the understanding of bronchial asthma in traditional Chinese medicine and on the basis of the three depression Decoction (Taiping Huimin and agent Bureau square.) and the treatment of asthma, the study object is to establish the work of the combination of three depression Decoction and the five depression soup of the Jiawei recipe (medicine jisayao, medicinal ephedra, almond, Gump, plaster and fine tea). A multi target network pharmacology model was used and compared with the target pathway of asthma. The potential target.2. of three depression soup and Jiawei Fang was predicted to combine the characteristics of asthma. The air pollutant PM2.5 was used as a stimulating factor to establish a OVA compound PM2.5 induced heavy asthma model, and the intervention of PM2.5 and the decoction of adding flavour to PM2.5 were discussed. The effect and effect mechanism of inducing exacerbation of asthma. Methods: 1. collation of the understanding of asthma pathogenesis, syndrome differentiation and treatment of prescription medicine by the past generation of doctors, the systematic review of the prescription and combination of three depression decoction, and the analysis of the characteristics of the syndrome, and the statistics of the high frequency medicine of the asthma prescription in the modern literature, the clinical effect, the effect substance and the effect of the three depression decoction. The research on the effect mechanism of.2. and the three depression soup and its flavour prescription as the research object, the network pharmacology method was used to construct the function component target network of its compatibility drugs, and compared with the target pathway analysis of asthma, it was predicted that the potential target of the three depression soup and the flavour prescription for the prevention and treatment of asthma was to establish the OVA compound air pollutant P respectively. M2.5 induced exacerbation of 28 and 42 days of asthma mice model, compared both in the inflammatory response, airway hyperresponsiveness, lung tissue lesion and TRP channel expression, to obtain the exact and stable OVA compound air pollutants PM2.5 induced by heavy asthma mice model.4. in the potential target screening and PM2.5 induced exacerbation model of asthma model research On the basis of the study, we observed the effect of three depression soup and its flavour Recipe on PM2.5 induced asthma in improving airway inflammation, airway hyperresponsiveness and lung tissue lesions. ELISA was used to observe the difference of IL-13, SP, PGD2 and NGF in BALF of mice. The expression of TRPA1, TRPV1, TRPV4, TRPM8 gene level in lung tissue was determined by q-PCR method, and the three depression soup was screened by the method of q-PCR. Western blot method was used to determine the level of related protein, and the effect mechanism of two parties to interfere with the exacerbation of asthma was introduced. Results: 1. physicians in the past generations believed that asthma was a complicated disease. According to the characteristics of the disease, the lung qi, dispel cold and expectorant method, scattered cold and clear lung method, clearing lung and phlegm method, and spitting and guiding the phlegm method were put forward. The modern doctors further put forward the specimen and treatment of the asthma attack period, from the stasis treatment and the wind theory. The statistics found that the drugs with high support for the cold wheeze and the hot wheeze in the modern asthma are the.2. literature of ephedra, almond and licorice, and the three depression soup is coughed and asthma is the main syndrome, and its main compatibility is: add The modern study shows that three depression Decoction and Jiawei prescription mainly interfere with the.3. network pharmacological model of asthma, such as inhibiting inflammation, regulating immunity, regulating nerve receptor and airway reaction, and 11 efficacy components of three depression decoction are used to act on 74 targets. 47 pathways, such as metabolic signal transduction and immuno cellular process, in which mitogen activated protein kinase (MAPK) is the core target group, which is related to the efficacy of three depression soup to promote lung antiasthmatic effect. The effect components and target pathways of the three depression soup are increased after adding flavour. For example, the 16 functional components of five depression soup are used in 98 targets and metabolic disease signal transduction. Immune and other 71 pathways, GTP protease and MAPK constitute the core target group of five depression soup, which mainly increases metabolism and disease related pathways. These changes are related to the efficacy of five depression soup lung clearing heat. 82 targets of asthma involve 52 pathways, such as disease immuno signal conduction cell process, tumor necrosis factor (TNF), and nuclear factor -1 Kappa B (NF- kappa B) family is the core target group, and the target group of the nerve ligand receptor interaction pathway is a potential target for asthma. Compared with the target pathway of asthma, the characteristics of the three depression soup and its flavour formula are mainly through the enzyme regulating the synthesis and metabolism of various active substances and the involvement of immunoglobulin, inflammatory cytokines and other mediators. Airway inflammation, signal transduction and other links play a role in.4.OVA compound PM2.5 induced exacerbation of asthma response in mice: increasing the number and ratio of inflammatory cells (especially EOS) in blood and BALF, increased pulmonary airway reactivity, severe lung tissue damage, increased inflammatory exudation, bronchoalveolar mucosa damage and thickening of tracheal smooth muscle. In addition, asthma mice showed high expression of TRPA1, TRPV1 protein and related factors such as IL-13, SP, PDG2 and NGF. These effects of exacerbating asthma in mice exposed to PM2.5 in the 42 day model were the most significant of.5. three depression soup and flavored prescription five depression soup to improve the symptoms of PM2.5 induced exacerbation of asthma mice (42 days of modeling), especially both high and high. The dose effect is more significant, such as reducing the number and ratio of inflammatory cells (especially EOS) in blood and BALF, reducing pulmonary airway hyperresponsiveness, improving lung tissue damage, inflammatory exudation, bronchial mucosa damage and bronchial smooth muscle thickening. In addition, three depression soup, five depression soup can reduce lung tissue TRPA1 in asthmatic mice. TRPV1 gene and protein levels as well as the high expression of IL-13, SP, PDG2 and NGF. Compared with the OVA+PM2.5 group, the high dose group of the three depression soup significantly reduced the high expression of TRPA1 protein, IL-13, PDG2 and NGF (P0.01). The high dose group of the five depression soup significantly reduced the expression of TRPV1 protein. The history of the literature on asthma pathogenesis, syndrome differentiation and treatment of prescription medicine, and the modern literature of asthma prescription high frequency medicine statistics, of which cold wheech and hot wheeze prescription high support medicine for ephedra, almond, licorice and so on. Systematic review of the three depression soup prescription and flavored application, the main compatibility of the combination of flavored solution medicine, chemical Phlegm antitussive antiasthmatic medicine, antipyretic medicine, warm medicine, astringent medicine, physical Qi medicine and so on. Modern studies show that the three depression soup and Jiawei prescription mainly from the inhibition of inflammation, regulating immunity, regulating nerve receptor and airway reaction and other aspects of the intervention of asthma.2. construction of three depression soup, Jiafang five depression soup and Asthma Network pharmacological model. Three depression decoction combined with 11 drugs. 74 targets and 47 metabolic pathways, such as 74 targets and metabolic signal transduction and immuno cellular process, are the core target groups, which are related to the efficacy of three depression soup to promote lung antiasthmatic effect. Three depression soup has increased effect components and target pathways after adding flavour. For example, 16 components of five depression soup act on 98 targets and metabolic disease signal 71 pathways of conduction and immunization, GTP protease and MAPK constitute the core target group of the five depression soup, which mainly increases metabolism and disease related pathways. These changes are related to the efficacy of the five depression soup lung clearing heat. 82 targets of asthma involve 52 routes of disease immuno signal conduction cell process, tumor necrosis factor (TNF), nuclear factor kappa B (NF-1 kappa B). The family is the core target group, and the target group of the nerve ligand receptor interaction pathway is the potential target of asthma. Compared with the target pathway of asthma, the characteristics of the three depression soup and its flavour formula are mainly through the enzyme regulating the synthesis and metabolism of a variety of active substances and the involvement of various media, such as immunoglobulin, inflammatory cytokines, and asthma. Inflammation, signal transduction and other links play a role.3. successfully establish OVA compound PM2.5 induced aggravated asthma model, combined with the three depression soup and its Jiawei prescription network target screening results, investigate and compare the effect of three depression soup and Jiawei Fang five depression soup, reveal its potential neuroimmunological mechanism. The study confirmed that three depression soup, five depression soup can improve asthma. The expression is to reduce the infiltration of inflammatory cells, reduce airway responsiveness and reduce the pathological damage of lung tissue. The effect of high dose improvement is the most significant. Three depression soup, five depression soup can interfere with the TRP channel and the release of neurotransmitter in the treatment of asthma through neural regulation. The three depression soup is more obvious in the regulation of the TRPA1 channel, and the five depression soup can be significantly adjusted. Control TRPA1 and TRPV1 channels.
【学位授予单位】：南京中医药大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R289.5

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