汪受传治疗肺炎喘嗽用药规律及金欣口服液、黄芩素治疗RSV肺炎的代谢调控机理

发布时间：2018-07-23 07:50

【摘要】：背景中医药治疗小儿肺炎喘嗽具有一定优势。汪受传教授治疗肺炎喘嗽,效果较显著,且安全性高、副作用少。因此,需要全面深入挖掘汪教授治疗肺炎喘嗽的用药规律。目的利用聚类和关联规则分析方法挖掘汪教授治疗儿童肺炎喘嗽的用药规律。方法以汪教授门诊治疗肺炎喘嗽的249份病案为基础,通过使用SPSS 19.0和Clementine 12.0软件对29味高频药物进行聚类和关联规则分析,探究其用药规律。结果1.聚类分析共分4个类别。C1:炙麻黄、苦杏仁、黄芩、虎杖、葶苈子、苏子及前胡;C2:桑叶、桑白皮、胆南星、枇杷叶、辛夷及重楼;C3:桔梗、枳壳、浙贝母、地龙、蝉蜕、瓜蒌、紫苑、百部、生石膏及丹参;C4:炙黄芪、白术、防风、煅龙骨、煅牡蛎。2.关联规则分析:从两药关联来看,前胡与炙麻黄、炙麻黄与杏仁、桑叶与桑白皮、前胡与杏仁、苏子与葶苈子、苏子与前胡、前胡与黄芩、虎杖与黄芩、胆南星与黄岑、胆南星与前胡、桑白皮与黄芩等两位常用药物组合;从三药关联来看,基本体现出治疗肺炎喘嗽当以宣肃肺气,清热化痰为治疗大法。宣肺类代表药物为炙麻黄、桑叶,肃降肺气类代表药物为杏仁、桑白皮、枇杷叶、前胡、葶苈子、苏子等,清肺泻热类代表药物为黄芩、虎杖等,化痰类代表药物为胆南星、杏仁等。结论运用聚类分析和关联规则分析可以初步揭示汪教授临证治疗肺炎的用药规律,即以炙麻黄、苦杏仁、桑叶、桑白皮、黄芩、虎杖、葶苈子、苏子、胆南星、前胡等为核心药物,配伍桔梗、枳壳、浙贝母、瓜萎、紫菀、百部、生石膏等清热化痰,肃肺止咳。背景肺炎是儿科常见的呼吸系统疾病,严重危害儿童健康,世界卫生组织将其列为全球3种重要儿科疾病之一。据统计,在儿童急性下呼吸道感染中,病毒性肺炎所占比例呈上升趋势,而呼吸道合胞病毒作为其中最重要的病原体之一,可以引起婴幼儿毛细支气管炎、支气管肺炎、间质性肺炎等。呼吸道合胞病毒(respiratory syncytial virus,RSV)感染的发病机制尚未完全明确,且目前缺乏安全有效的疫苗和药物进行针对性的预防和治疗。中医药因其辨证论治、作用柔和及副作用少等特点,在治疗儿童呼吸道病毒感染中具有一定的优势。汪教授已研究儿童病毒性肺炎二十余年,根据该病特点和关键病机,研制出金欣口服液,作为临证治疗病毒性肺炎的有效验方,具有宣肺开闭、清热化痰、止咳平喘的功用。前期大量临床和实验研究已表明其可显著抗RSV感染及调节免疫作用。黄芩素是金欣口服液组成药物黄芩的重要有效成分,研究表明具有广谱的抗病毒活性。但是,和许多中医药实验研究一样,上述研究可能存在诸多缺陷,如指标单一、独立、非同步等,难以全面反映药物的作用机制。因此,若深入探讨其治疗作用,应当引入系统化、整体化的研究技术方法以综合分析。代谢组学是系统生物学的重要组成部分,在生物体受到各种外界因素刺激时,能即时、灵敏、真实表现出生物体整体功能的应答与调节。作为一种系统的研究方法,其以机体的整体代谢为研究对象,动态研究生物体受刺激或者扰动之后,其内源性代谢物种类、数量的变化,可以从较为全面的视角发现药物真正的作用机理及其赖以发挥作用的物质基础。因此,本课题组采用代谢组学研究方法,尝试阐明金欣口服液和黄芩素治疗RSV感染的作用,以及其中的作用机制。目的研究RSV肺炎BALB/c小鼠血浆、尿液、肺泡灌洗液、脾脏组织及细胞的代谢特征,进而探讨金欣口服液以及黄芩素对RSV肺炎小鼠的干预作用以及其中可能涉及的代谢调控机制。方法RSV鼻腔吸入法感染BALB/C小鼠之后,分别予以金欣口服液以及黄芩素进行干预,并设正常对照组,分别采集小鼠血浆、尿液、肺泡灌洗液和脾脏组织,行肺部组织病理病理学分析以观察肺部病变情况。培养细胞,予RSV感染1.5-2h后,予黄芩素处理,24h后予以液氮淬灭,以水-甲醇-氯仿提取细胞内代谢物。将上述小鼠样本和细胞样本预处理后,运用气相色谱-质谱联用(Gas Chromatography-Mass Spectrometer,GC-MS)分析小鼠的血浆、尿液、脾脏组织和细胞的整体代谢变化,运用超高效液相色谱-离子阱质谱联用(Ultra Performance Liquid Chromatography-LTQ/Orbitrap-Mass Spectrometer,UPLC-LTQ/Orbitrap-MS)研究小鼠肺泡灌洗液中代谢谱变化,利用主成分分析(principal component analysis,PCA)和正交偏最小二乘法判别分析(Orthogonalpartial least squares-discriminant analysis,OPLS-DA)分析所采集的数据,筛选潜在的血浆、尿液、脾脏、肺泡灌洗液和细胞内的生物标记物,分析相关代谢通路,阐释金欣口服液和黄芩素对RSV肺炎的可能的代谢调控机制。结果1.RSV感染BALB/c小鼠的肺组织病变主要位于肺间质部位,可见肺脏可见间质多灶性炎细胞浸润(++),以单核细胞、淋巴细胞为主;肺泡壁轻度充血(+),肺泡壁明显增厚。分别运用金欣口服液和黄芩素治疗后,肺部充血、水肿、炎性浸润均有不同程度减轻。2.金欣口服液的代谢组学研究(1)血浆:正常组、RSV肺炎模型组与金欣口服液治疗组小鼠的血浆的OPLS-DA分析显示,三组间能完全分离,无交叉和重叠,模型参数R2Y=0.934,Q2=0.889,鉴定并筛选了 6个潜在生物标记物,分别为乳酸、尿素、谷氨酰胺、1,5-脱水山梨醇、葡萄糖、花生四烯酸。(2)尿液:正常组、RSV肺炎模型组与金欣口服液治疗组小鼠的尿液的OPLS-DA分析显示,三组间能完全分离,无交叉和重叠,模型参数R2Y=0.85,Q2=0.746,鉴定并筛选了 4个潜在生物标记物,分别为乳酸、甘氨酸、硬脂酸、棕榈酸。(3)脾脏组织:正常组、RSV肺炎模型组与金欣口服液治疗组小鼠的脾脏样本的OPLS-DA分析显示,三组间能完全分离,无交叉和重叠,模型参数R2Y=0.959,Q2=0.895,鉴定并筛选了 6个潜在生物标记物,分别为L-脯氨酸、L-谷氨酸、缬氨酸、尿素、次黄嘌呤、葡萄糖,(4)肺泡灌洗液:正常组、RSV肺炎模型组与金欣口服液治疗组小鼠的肺泡灌洗液样本的OPLS-DA分析显示,三组间能完全分离,无交叉和重叠,模型参数R2Y=0.972,Q2=0.924,鉴定并筛选了 6个潜在生物标记物,分别为左旋肉碱、丙酰肉碱、腺苷、二氢神经鞘氨醇、白三烯D5和葡萄糖醛酸雌素酮。3.黄芩素的代谢组学研究(1)细胞:正常组、RSV肺炎模型组与金欣口服液治疗组小鼠的肺泡灌洗液样本的OPLS-DA分析显示,三组间能完全分离,无交叉和重叠,模型参数R2Y=0.974,Q2=0.917,鉴定并筛选了 6个潜在生物标记物,5种差异性代谢物,分别为丝氨酸、L-天冬氨酸、L-谷氨酸酸、柠檬酸与甘氨酸。(2)血浆:正常组、RSV肺炎模型组与金欣口服液治疗组小鼠的血浆样本的OPLS-DA分析显示,三组间能完全分离,无交叉和重叠,模型参数R2Y=0.912,Q2=0.84,鉴定并筛选了 6个潜在生物标记物,乳酸、尿素、甘氨酸、谷氨酰胺、1,5-脱水山梨醇、D-葡萄糖。(3)尿液:正常组、RSV肺炎模型组与金欣口服液治疗组小鼠的尿液样本的OPLS-DA分析显示,三组间能完全分离,无交叉和重叠,模型参数R2Y=0.917,Q2=0.767,鉴定并筛选了 4个潜在生物标记物,乳酸、苹果酸、棕榈酸、硬脂酸。(4)肺泡灌洗液:正常组、RSV肺炎模型组与金欣口服液治疗组小鼠的尿液样本的OPLS-DA分析显示,三组间能完全分离,无交叉和重叠,模型参数R2Y=0.954,Q2=0.828,鉴定并筛选了5种差异性代谢物,鉴定结果分别为谷氨酸、碳酰肉碱、腺苷、葡萄糖苷酰鞘氨醇、白三烯D5。结论1.运用UPLC/MS和GC-MS分析代谢组学可以从代谢网络的角度阐释金欣口服液和黄芩素治疗RSV感染的作用机制。2.RSV感染BALB/c后,血浆、尿液、肺泡灌洗液及脾脏内的内源性代谢物发生紊乱,主要为氨基酸、有机酸及脂肪酸。3.金欣口服液和黄芩素可以治疗RSV感染,减轻BALB/c小鼠的肺部炎症。4.金欣口服液和黄芩素能够部分调整RSV感染导致的代谢紊乱,其中金欣口服液涉及谷氨酰胺代谢,丙氨酸、天冬氨酸和谷氨酸代谢,缬氨酸、亮氨酸和异亮氨酸代谢,花生四烯酸代谢,甘氨酸、丝氨酸和苏氨酸代谢,精氨酸和脯氨酸代谢,以及丙酮酸代谢;黄芩素发挥治疗作用主要与谷氨酰胺代谢,丙氨酸、天冬氨酸和谷氨酸代谢,甘氨酸、丝氨酸和苏氨酸代谢相关。
[Abstract]:Background Chinese medicine has some advantages in the treatment of pneumonia and asthma in children. Wang Chuan professor has a significant effect, high safety and less side effects in the treatment of pneumonia and asthma. Therefore, it is necessary to excavate Professor Wang in the treatment of pneumonia and asthma. Methods based on 249 cases of pneumonia and asthma in Professor Wang's outpatient clinic, by using SPSS 19 and Clementine 12 software to cluster and analyze the association rules of 29 kinds of high frequency drugs, the results of the 1. cluster analysis were divided into 4 categories:.C1:, bitter almond, Scutellaria, Polygonum cuspidatum, drapine seed, soda and front. Hu; C2: mulberry leaf, mulberry, loquat leaf, loquat leaf, Magnolia and heavy building; C3: Platycodon, Fructus aurantii, Fritillaria, Trichosanthes, Trichosanthes, garden, hundred, raw gypsum and Salvia; C4:, Radix Astragali, Rhizoma Atractylodes, Alba, calcined oysters,.2. association rules analysis: from the two drug association, the forelaus and ephedra, sago ephedra and almonds, mulberry leaves and mulberry and mulberry bark, the forelahu and Almond, soda and Sophie, Soviet and forelahu, Radix Scutellariae, Scutellaria, Scutellaria, Scutellaria, Scutellaria, cen, Dan Nan star and Cen, Dan Nan star and forelahu, mulberry and scutellaria, and other two common drug combinations; from the correlation of three drugs, the basic manifestation of the treatment of pneumonia and asthma is to declare the lung qi and heat the phlegm as the treatment of the great law. The drugs represent apricot kernel, mulberry skin, loquat leaf, arbor, Sophia, Sophia and so on. Qingfei diarrhoea represents medicine for baicaleum Scutellaria, Polygonum cuspidatum and so on. Yellow, bitter almond, mulberry leaf, mulberry leaf, Scutellaria, Polygonum cuspidatum, sophion, santian, gendahu and Hubei are the core drugs. They are compatible with Platycodon grandiflorum, trifoliate orange, Fritillaria thunbergii, Melon Wilt, aster, 100, gypsum and so on. The background pneumonia is a common respiratory system disease in pediatrics, which seriously endangers children's health. The WHO lists it as 3 of the world. It is one of the most important pediatric diseases. According to the statistics, the proportion of viral pneumonia is rising in children's acute lower respiratory tract infection. As one of the most important pathogens, respiratory syncytial virus can cause bronchiolitis, bronchopneumonia, interstitial pneumonia, and so on. Respiratory syncytial virus (respiratory syncytia). L virus, RSV) the pathogenesis of infection has not been completely clear, and the current lack of safe and effective vaccines and drugs for targeted prevention and treatment. Chinese medicine has a definite advantage in the treatment of children's respiratory virus infection because of its syndrome differentiation, soft effect and less side effects. Professor Wang has studied two children's viral pneumonia. For more than ten years, according to the characteristics of the disease and the key pathogenesis, Jinxin oral liquid was developed as an effective prescription for the treatment of viral pneumonia. It has the function of Xuan lung opening and closing, clearing heat and eliminating phlegm and relieving cough and asthma. A large number of clinical and experimental studies have shown that it can significantly resist RSV infection and regulate the immune function. The important effective component of Scutellaria baicalensis has a broad spectrum of antiviral activity. However, as with many experimental studies of traditional Chinese medicine, there may be many defects, such as single, independent and non synchronous indexes, which are difficult to fully reflect the mechanism of drug action. Therefore, it should be systematized and holistic if the therapeutic effect is discussed in depth. Metabonomics is an important part of system biology. When the organism is stimulated by various external factors, it can instantly, sensitively and truly express the response and regulation of the whole function of the organism. As a systematic research method, it takes the whole metabolism of the body as the research object and dynamic postgraduate. After an object is stimulated or disturbed, the variety and quantity of its endogenous metabolites can be found from a more comprehensive perspective to discover the real mechanism of the drug and the material basis for its function. Therefore, the research group adopts the metabonomics method to try to clarify the effect of Jinxin oral liquid and baicalein on the treatment of RSV infection. Objective to study the metabolic characteristics of RSV pneumonia BALB/c mice plasma, urine, alveolar lavage fluid, spleen tissue and cells, and then explore the intervention effect of Jinxin oral liquid and baicalein on RSV pneumonia mice and the possible metabolic regulation mechanism. Methods RSV nasal inhalation method infected BALB/C mice. Do not give the intervention of Jinxin oral liquid and baicalein, and set up normal control group, collect the mice plasma, urine, alveolar lavage fluid and spleen tissue, the pathological pathological analysis of lung tissue to observe the pathological changes of lung. After RSV infection 1.5-2h, baicalein treatment, 24h after liquid nitrogen quenching, with water methanol and chlorine Gas Chromatography-Mass Spectrometer (GC-MS) was used to analyze the overall metabolic changes in the plasma, urine, spleen tissues and cells of mice after preprocessing of the above mice and cell samples. The ultra high performance liquid chromatography ion trap mass spectrometry (Ultra Performance Liquid) was used. Chromatography-LTQ/Orbitrap-Mass Spectrometer, UPLC-LTQ/Orbitrap-MS) study the changes of metabolic spectrum in mouse alveolar lavage fluid, using the data collected by principal component analysis (principal component analysis, PCA) and orthogonal partial least squares discriminant analysis (Orthogonalpartial least squares-discriminant analysis, OPLS-DA) analysis Select potential plasma, urine, spleen, alveolar lavage fluid and biomarkers in cell, analyze related metabolic pathways, explain the possible metabolic regulation mechanism of Jinxin oral liquid and baicalein on RSV pneumonia. Results the lung tissue lesion of 1.RSV infected BALB/c mice is mainly located in the interstitial area of the lung, and the pulmonary interstitial multifocal inflammatory cells can be seen in the lungs. Infiltration (+ +), mainly monocyte, lymphocyte, alveolar wall mild hyperemia (+), and alveolar wall thickening. After the treatment of Jinxin oral liquid and baicalein, the pulmonary congestion, edema, and inflammatory infiltration of.2. Jinxin oral liquid were alleviated in different degrees (1) plasma: normal group, RSV pneumonia model group and Jinxin oral liquid treatment OPLS-DA analysis in the plasma of the mice showed that the three groups could be completely separated, without cross and overlap, the model parameters R2Y=0.934, Q2=0.889, identified and screened 6 potential biomarkers, respectively, lactic acid, urea, glutamine, 1,5- dehydrated sorbitol, glucose, peanut four eNIC acid. (2) urine: normal group, RSV pneumonia model group and Jinxin oral liquid OPLS-DA analysis of the urine of the mice in the treatment group showed that the three groups could be completely separated, without cross and overlap, the model parameters R2Y=0.85, Q2=0.746, identified and screened 4 potential biomarkers, respectively, lactic acid, glycine, stearic acid, palmitic acid. (3) spleen tissues: normal group, RSV pneumonia model group and Jinxin oral liquid treatment group of mice spleen OPLS-DA analysis showed that the three groups could be completely separated, no cross and overlap, model parameters R2Y=0.959, Q2=0.895, identified and screened 6 potential biomarkers, respectively, L- proline, L- glutamic acid, valine, urea, hypoxanthine, glucose, and (4) alveolar lavage fluid: normal group, RSV pneumonia model group and Jinxin oral liquid treatment group small OPLS-DA analysis of the rats' alveolar lavage samples showed that the three groups could be completely separated, without cross and overlap, the model parameters R2Y=0.972, Q2=0.924, identified and screened 6 potential biomarkers, respectively, L-carnitine, malonyl carnitine, adenosine, two hydrogen neurinosine, leukotriene D5 and glucuronic.3. baicalein. Study (1) cells: normal group, RSV pneumonia model group and Jinxin oral liquid treatment group mice lung alveolar lavage samples OPLS-DA analysis showed that the three groups can be completely separated, no cross and overlap, model parameters R2Y=0.974, Q2=0.917, identification and screening of 6 potential biomarkers, 5 different metabolites, serine, L- aspartate, respectively Acid, L- glutamic acid, citric acid and glycine. (2) plasma: normal group, RSV pneumonia model group and Jin Xin oral liquid treatment group mice plasma samples OPLS-DA analysis showed that three groups can be completely separated, no cross and overlap, model parameters R2Y=0.912, Q2=0.84, identification and screening of 6 potential biomarkers, lactic acid, urea, glycine, glutamine, Amides, 1,5- dehydrated sorbitol, D- glucose. (3) urine: OPLS-DA analysis of urine samples from the normal group, the RSV pneumonia model group and the Jin Xin oral liquid treatment group showed that the three groups could be completely separated, no cross and overlap, the model parameters R2Y=0.917, Q2=0.767, identified and screened 4 potential biomarkers, lactic acid, malic acid, palmitic acid, hard. Lipoic acid. (4) alveolar lavage fluid: OPLS-DA analysis of urine samples from the normal group, RSV pneumonia model group and Jinxin oral liquid treatment group showed that the three groups could be completely separated, no cross and overlap, the model parameters R2Y=0.954, Q2=0.828, identification and screening of 5 different types of metabolites, the identification results were respectively glutamic acid, carnitine, adenosine, grapes, Glucosidyl sphingosine, leukotriene D5. conclusion 1. using UPLC/MS and GC-MS analysis metabolomics can explain the mechanism of Jinxin oral liquid and baicalein in the treatment of RSV infection from the angle of metabolic network.2.RSV infection BALB/c, the endogenous metabolites in plasma, urine, alveolar lavage fluid and spleen are disorder, mainly amino acids, organic acids and Fatty acid.3. Jinxin oral liquid and baicalein can treat RSV infection and reduce the pulmonary inflammation in BALB/c mice,.4. Jinxin oral liquid and baicalein can partly adjust the metabolic disorder caused by RSV infection. Metabolites of four arachidic acids, glycine, serine and threonine metabolism, arginine and proline metabolism, and pyruvate metabolism; baicalein plays a major role in the metabolism of glutamine, alanine, aspartate and glutamate metabolism, glycine, serine, and serine metabolism.
【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】：R272

【相似文献】