發(fā)布時(shí)間：2018-07-23 07:50

【摘要】：背景中醫(yī)藥治療小兒肺炎喘嗽具有一定優(yōu)勢(shì)。汪受傳教授治療肺炎喘嗽,效果較顯著,且安全性高、副作用少。因此,需要全面深入挖掘汪教授治療肺炎喘嗽的用藥規(guī)律。目的利用聚類(lèi)和關(guān)聯(lián)規(guī)則分析方法挖掘汪教授治療兒童肺炎喘嗽的用藥規(guī)律。方法以汪教授門(mén)診治療肺炎喘嗽的249份病案為基礎(chǔ),通過(guò)使用SPSS 19.0和Clementine 12.0軟件對(duì)29味高頻藥物進(jìn)行聚類(lèi)和關(guān)聯(lián)規(guī)則分析,探究其用藥規(guī)律。結(jié)果1.聚類(lèi)分析共分4個(gè)類(lèi)別。C1:炙麻黃、苦杏仁、黃芩、虎杖、葶藶子、蘇子及前胡;C2:桑葉、桑白皮、膽南星、枇杷葉、辛夷及重樓;C3:桔梗、枳殼、浙貝母、地龍、蟬蛻、瓜蔞、紫苑、百部、生石膏及丹參;C4:炙黃芪、白術(shù)、防風(fēng)、煅龍骨、煅牡蠣。2.關(guān)聯(lián)規(guī)則分析:從兩藥關(guān)聯(lián)來(lái)看,前胡與炙麻黃、炙麻黃與杏仁、桑葉與桑白皮、前胡與杏仁、蘇子與葶藶子、蘇子與前胡、前胡與黃芩、虎杖與黃芩、膽南星與黃岑、膽南星與前胡、桑白皮與黃芩等兩位常用藥物組合;從三藥關(guān)聯(lián)來(lái)看,基本體現(xiàn)出治療肺炎喘嗽當(dāng)以宣肅肺氣,清熱化痰為治療大法。宣肺類(lèi)代表藥物為炙麻黃、桑葉,肅降肺氣類(lèi)代表藥物為杏仁、桑白皮、枇杷葉、前胡、葶藶子、蘇子等,清肺瀉熱類(lèi)代表藥物為黃芩、虎杖等,化痰類(lèi)代表藥物為膽南星、杏仁等。結(jié)論運(yùn)用聚類(lèi)分析和關(guān)聯(lián)規(guī)則分析可以初步揭示汪教授臨證治療肺炎的用藥規(guī)律,即以炙麻黃、苦杏仁、桑葉、桑白皮、黃芩、虎杖、葶藶子、蘇子、膽南星、前胡等為核心藥物,配伍桔梗、枳殼、浙貝母、瓜萎、紫菀、百部、生石膏等清熱化痰,肅肺止咳。背景肺炎是兒科常見(jiàn)的呼吸系統(tǒng)疾病,嚴(yán)重危害兒童健康,世界衛(wèi)生組織將其列為全球3種重要兒科疾病之一。據(jù)統(tǒng)計(jì),在兒童急性下呼吸道感染中,病毒性肺炎所占比例呈上升趨勢(shì),而呼吸道合胞病毒作為其中最重要的病原體之一,可以引起嬰幼兒毛細(xì)支氣管炎、支氣管肺炎、間質(zhì)性肺炎等。呼吸道合胞病毒(respiratory syncytial virus,RSV)感染的發(fā)病機(jī)制尚未完全明確,且目前缺乏安全有效的疫苗和藥物進(jìn)行針對(duì)性的預(yù)防和治療。中醫(yī)藥因其辨證論治、作用柔和及副作用少等特點(diǎn),在治療兒童呼吸道病毒感染中具有一定的優(yōu)勢(shì)。汪教授已研究?jī)和《拘苑窝锥嗄?根據(jù)該病特點(diǎn)和關(guān)鍵病機(jī),研制出金欣口服液,作為臨證治療病毒性肺炎的有效驗(yàn)方,具有宣肺開(kāi)閉、清熱化痰、止咳平喘的功用。前期大量臨床和實(shí)驗(yàn)研究已表明其可顯著抗RSV感染及調(diào)節(jié)免疫作用。黃芩素是金欣口服液組成藥物黃芩的重要有效成分,研究表明具有廣譜的抗病毒活性。但是,和許多中醫(yī)藥實(shí)驗(yàn)研究一樣,上述研究可能存在諸多缺陷,如指標(biāo)單一、獨(dú)立、非同步等,難以全面反映藥物的作用機(jī)制。因此,若深入探討其治療作用,應(yīng)當(dāng)引入系統(tǒng)化、整體化的研究技術(shù)方法以綜合分析。代謝組學(xué)是系統(tǒng)生物學(xué)的重要組成部分,在生物體受到各種外界因素刺激時(shí),能即時(shí)、靈敏、真實(shí)表現(xiàn)出生物體整體功能的應(yīng)答與調(diào)節(jié)。作為一種系統(tǒng)的研究方法,其以機(jī)體的整體代謝為研究對(duì)象,動(dòng)態(tài)研究生物體受刺激或者擾動(dòng)之后,其內(nèi)源性代謝物種類(lèi)、數(shù)量的變化,可以從較為全面的視角發(fā)現(xiàn)藥物真正的作用機(jī)理及其賴(lài)以發(fā)揮作用的物質(zhì)基礎(chǔ)。因此,本課題組采用代謝組學(xué)研究方法,嘗試闡明金欣口服液和黃芩素治療RSV感染的作用,以及其中的作用機(jī)制。目的研究RSV肺炎BALB/c小鼠血漿、尿液、肺泡灌洗液、脾臟組織及細(xì)胞的代謝特征,進(jìn)而探討金欣口服液以及黃芩素對(duì)RSV肺炎小鼠的干預(yù)作用以及其中可能涉及的代謝調(diào)控機(jī)制。方法RSV鼻腔吸入法感染BALB/C小鼠之后,分別予以金欣口服液以及黃芩素進(jìn)行干預(yù),并設(shè)正常對(duì)照組,分別采集小鼠血漿、尿液、肺泡灌洗液和脾臟組織,行肺部組織病理病理學(xué)分析以觀察肺部病變情況。培養(yǎng)細(xì)胞,予RSV感染1.5-2h后,予黃芩素處理,24h后予以液氮淬滅,以水-甲醇-氯仿提取細(xì)胞內(nèi)代謝物。將上述小鼠樣本和細(xì)胞樣本預(yù)處理后,運(yùn)用氣相色譜-質(zhì)譜聯(lián)用(Gas Chromatography-Mass Spectrometer,GC-MS)分析小鼠的血漿、尿液、脾臟組織和細(xì)胞的整體代謝變化,運(yùn)用超高效液相色譜-離子阱質(zhì)譜聯(lián)用(Ultra Performance Liquid Chromatography-LTQ/Orbitrap-Mass Spectrometer,UPLC-LTQ/Orbitrap-MS)研究小鼠肺泡灌洗液中代謝譜變化,利用主成分分析(principal component analysis,PCA)和正交偏最小二乘法判別分析(Orthogonalpartial least squares-discriminant analysis,OPLS-DA)分析所采集的數(shù)據(jù),篩選潛在的血漿、尿液、脾臟、肺泡灌洗液和細(xì)胞內(nèi)的生物標(biāo)記物,分析相關(guān)代謝通路,闡釋金欣口服液和黃芩素對(duì)RSV肺炎的可能的代謝調(diào)控機(jī)制。結(jié)果1.RSV感染BALB/c小鼠的肺組織病變主要位于肺間質(zhì)部位,可見(jiàn)肺臟可見(jiàn)間質(zhì)多灶性炎細(xì)胞浸潤(rùn)(++),以單核細(xì)胞、淋巴細(xì)胞為主;肺泡壁輕度充血(+),肺泡壁明顯增厚。分別運(yùn)用金欣口服液和黃芩素治療后,肺部充血、水腫、炎性浸潤(rùn)均有不同程度減輕。2.金欣口服液的代謝組學(xué)研究(1)血漿:正常組、RSV肺炎模型組與金欣口服液治療組小鼠的血漿的OPLS-DA分析顯示,三組間能完全分離,無(wú)交叉和重疊,模型參數(shù)R2Y=0.934,Q2=0.889,鑒定并篩選了 6個(gè)潛在生物標(biāo)記物,分別為乳酸、尿素、谷氨酰胺、1,5-脫水山梨醇、葡萄糖、花生四烯酸。(2)尿液:正常組、RSV肺炎模型組與金欣口服液治療組小鼠的尿液的OPLS-DA分析顯示,三組間能完全分離,無(wú)交叉和重疊,模型參數(shù)R2Y=0.85,Q2=0.746,鑒定并篩選了 4個(gè)潛在生物標(biāo)記物,分別為乳酸、甘氨酸、硬脂酸、棕櫚酸。(3)脾臟組織:正常組、RSV肺炎模型組與金欣口服液治療組小鼠的脾臟樣本的OPLS-DA分析顯示,三組間能完全分離,無(wú)交叉和重疊,模型參數(shù)R2Y=0.959,Q2=0.895,鑒定并篩選了 6個(gè)潛在生物標(biāo)記物,分別為L(zhǎng)-脯氨酸、L-谷氨酸、纈氨酸、尿素、次黃嘌呤、葡萄糖,(4)肺泡灌洗液:正常組、RSV肺炎模型組與金欣口服液治療組小鼠的肺泡灌洗液樣本的OPLS-DA分析顯示,三組間能完全分離,無(wú)交叉和重疊,模型參數(shù)R2Y=0.972,Q2=0.924,鑒定并篩選了 6個(gè)潛在生物標(biāo)記物,分別為左旋肉堿、丙酰肉堿、腺苷、二氫神經(jīng)鞘氨醇、白三烯D5和葡萄糖醛酸雌素酮。3.黃芩素的代謝組學(xué)研究(1)細(xì)胞:正常組、RSV肺炎模型組與金欣口服液治療組小鼠的肺泡灌洗液樣本的OPLS-DA分析顯示,三組間能完全分離,無(wú)交叉和重疊,模型參數(shù)R2Y=0.974,Q2=0.917,鑒定并篩選了 6個(gè)潛在生物標(biāo)記物,5種差異性代謝物,分別為絲氨酸、L-天冬氨酸、L-谷氨酸酸、檸檬酸與甘氨酸。(2)血漿:正常組、RSV肺炎模型組與金欣口服液治療組小鼠的血漿樣本的OPLS-DA分析顯示,三組間能完全分離,無(wú)交叉和重疊,模型參數(shù)R2Y=0.912,Q2=0.84,鑒定并篩選了 6個(gè)潛在生物標(biāo)記物,乳酸、尿素、甘氨酸、谷氨酰胺、1,5-脫水山梨醇、D-葡萄糖。(3)尿液:正常組、RSV肺炎模型組與金欣口服液治療組小鼠的尿液樣本的OPLS-DA分析顯示,三組間能完全分離,無(wú)交叉和重疊,模型參數(shù)R2Y=0.917,Q2=0.767,鑒定并篩選了 4個(gè)潛在生物標(biāo)記物,乳酸、蘋(píng)果酸、棕櫚酸、硬脂酸。(4)肺泡灌洗液:正常組、RSV肺炎模型組與金欣口服液治療組小鼠的尿液樣本的OPLS-DA分析顯示,三組間能完全分離,無(wú)交叉和重疊,模型參數(shù)R2Y=0.954,Q2=0.828,鑒定并篩選了5種差異性代謝物,鑒定結(jié)果分別為谷氨酸、碳酰肉堿、腺苷、葡萄糖苷酰鞘氨醇、白三烯D5。結(jié)論1.運(yùn)用UPLC/MS和GC-MS分析代謝組學(xué)可以從代謝網(wǎng)絡(luò)的角度闡釋金欣口服液和黃芩素治療RSV感染的作用機(jī)制。2.RSV感染BALB/c后,血漿、尿液、肺泡灌洗液及脾臟內(nèi)的內(nèi)源性代謝物發(fā)生紊亂,主要為氨基酸、有機(jī)酸及脂肪酸。3.金欣口服液和黃芩素可以治療RSV感染,減輕BALB/c小鼠的肺部炎癥。4.金欣口服液和黃芩素能夠部分調(diào)整RSV感染導(dǎo)致的代謝紊亂,其中金欣口服液涉及谷氨酰胺代謝,丙氨酸、天冬氨酸和谷氨酸代謝,纈氨酸、亮氨酸和異亮氨酸代謝,花生四烯酸代謝,甘氨酸、絲氨酸和蘇氨酸代謝,精氨酸和脯氨酸代謝,以及丙酮酸代謝;黃芩素發(fā)揮治療作用主要與谷氨酰胺代謝,丙氨酸、天冬氨酸和谷氨酸代謝,甘氨酸、絲氨酸和蘇氨酸代謝相關(guān)。
[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.
【學(xué)位授予單位】：南京中醫(yī)藥大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R272

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