基于UPLC-MS技术的呼吸道合胞病毒肺炎痰热闭肺证代谢模式及金欣口服液干预研究

发布时间：2018-07-01 11:40

本文选题：金欣口服液 + 呼吸道合胞病毒　；参考：《南京中医药大学》2015年博士论文

【摘要】：背景：肺炎是常见的小儿肺系疾病,在综合性医院儿科住院病例单病种统计中居首位,WHO列为全球3种重要儿科疾病之一。在我国小儿急性下呼吸道感染中,病毒约占1/3,并呈上升趋势,其中以呼吸道合胞病毒(Respiratory Syncytial Virus; RSV)最多。RSV肺炎以热证为多见,其中痰热闭肺证是RSV肺炎最为常见和重要的一种证候。一个证候包含了多脏器多指标间的复杂联系,很难用单一指标或某几个指标的简单叠加来阐释证候物质基础。如何科学、全面地阐述中医证候的实质和内涵是现代中医学亟需解决的一个重要问题。国内外研究表明,对于小儿病毒性肺炎的治疗,西药尚缺乏理想药物,唯一被美国儿科协会推荐为“也许可以使用”的药物是利巴韦林。中医药治疗有一定的优势,且已经为大量临床和实验研究所证实。南京中医药大学汪受传教授领衔的本课题组从事中医药治疗小儿病毒性肺炎研究近20年,研制了有效中药方剂金欣口服液,具有宣肺开闭、清热解毒、化痰止咳等功效。课题组前期已经对金欣口服液在RSV感染方面的作用进行了大量研究,然而,由于金欣口服液活性化学成分的复杂性,目前金欣口服液对RSV肺炎的整体代谢网络调控作用仍不清楚。代谢组学(Metabonomics)是关于生物体内源性代谢物种类、数量及其变化规律的一门新兴学科,是系统生物学技术的重要组成部分。由于代谢物是生物体所有基因、蛋白功能活动的终点,因而被视作生物体整体功能状态的“生化表型”,能够即时、灵敏、真实的表现在各种外界因素的刺激下生物体整体功能状态的应答与调节,避免了既往采用单一或少数几个指标来研究某种生理、病理变化的弊端。代谢组学技术在方法学上融整体、动态、综合分析于一体的特点,与中医学的整体观念相一致。因此本课题采用代谢组学方法,结合其他生化检测,全面表征RSV肺炎痰热闭肺证所导致的代谢物组变化,尝试阐明RSV肺炎痰热闭肺证的本质和金欣口服液对RSV感染小鼠的治疗作用。目的：研究RSV肺炎痰热闭肺证患儿血浆和尿液的代谢特征,探讨RSV肺炎痰热闭肺证的证候实质；研究RSV肺炎BALB/c小鼠血浆和肺组织的代谢特征,探讨金欣口服液对RSV肺炎BALB/c小鼠的干预作用及可能的代谢调控机制。方法：RSV肺炎痰热闭肺证代谢组学的临床实验：选择符合中西医诊断条件的RSV肺炎痰热闭肺证患儿30例,设为疾病组,同时设健康正常组30例。分别采集两组儿童的血浆和尿液,采用超高效液相色谱-二维线性离子阱质谱联用仪(Ultra-performance Liquid Chromatography coupled with Linear Ion Trap Quadrupole Orbitrap Mass Spectrometry, UPLC-LTQ/Orbitrap-MS)检测两组的血浆和尿液的代谢产物,建立小儿RSV肺炎痰热闭肺证的证候相关代谢谱,利用主成分分析(Principal Component Analysis, PCA)和正交偏最小二乘法(Orthogonal Partial Least Squares Discriminant Analysis, OPLS-DA)对检测的矩阵数据进行统计分析,筛选潜在的血浆、尿液生物标记物,分析相关代谢通路,探索RSV肺炎痰热闭肺证的代谢特征本质。金欣口服液干预RSV肺炎BALB/c小鼠的动物实验：RSV滴鼻感染BALB/c小鼠,金欣口服液进行干预,并设利巴韦林为阳性对照,6天后分别采集小鼠血浆、肺组织,行肺组织病理切片形态学分析来评价肺部病变情况,基于UPLC-LTQ/Orbitrap-MS研究各组小鼠血浆和肺组织整体代谢变化,利用PCA和OPLS-DA分析检测数据,筛选潜在的血浆、肺组织生物标记物,分析相关代谢通路,评价金欣口服液的药效,阐明金欣口服液的代谢调控机制。结果：RSV肺炎痰热闭肺证代谢组学的临床实验：1.血浆代谢组学模式识别分析结果显示,RSV肺炎痰热闭肺证患儿与健康正常组儿童主成分积分值大部分集中分布于散点图的椭圆形(95%置信区)区域内,虽有一定的分离趋势,但仍存在交叉重叠,进一步构建OPLS-DA模型,模型参数分别为R2Y=0.907, Q2=0.567(血浆上层样本)和R2Y=0.816, Q2=0.619 (血浆下层样本),正常组和RSV肺炎痰热闭肺证组沿第一主成份t[1]轴方向能完全分离,无交叉和重叠,说明两组在代谢特征方而具有明显差异：筛选了21个潜在生物标记物,并鉴定了其中的20个；其中SM(18：2/24：0)、SM(d18:2/24:1)、SM(d18:0/18:1)等鞘磷脂,TG(16:0/18:1/22:6)、TG(16:0/18:0/22:6)、 TG(16:0/20:4/22:5)、TG(18:1/18:3/20:4)、TG(16:0/20:3/22:4)等甘油三酯在RSV肺炎痰热闭肺证患儿血浆中呈上调趋势,PC(18:0/18:2)、PC(18:0/18:1)、PC(16:0/20:3)、PC(18:0/20:3)、 PC(16:0/22:5)、PC(18:2/20:0)、LysoPC(18:2)等磷脂酰胆碱,TG(14:0/16:0/18:2)、TG(14：0/16：0/16：1)等甘油三酯在RSV肺炎痰热闭肺证患儿血浆中呈下调趋势,此外,还发现了Bilirubin、Carnitine、Tryptophan等与RSV肺炎痰热闭肺证有关的物质,较正常儿显著下降。2.尿液代谢组学模式识别分析结果显示,采用PCA方法对RSV肺炎痰热闭肺证患儿与健康正常组儿童样本建模,大部分样本集中分布于散点图的椭圆形(95%置信区)区域内,虽有一定的分离趋势,但仍存在交叉重叠,进一步建立OPLS-DA模型,参数分别为R2Y=0.865, Q2=0.580 (a Q色谱柱所测尿液样本)和R2Y=0.902, Q2=0.593 (HILIC色谱柱所测尿液样本),正常组和RSV肺炎痰热闭肺证组完全分离,说明两组在代谢物方面具有明显差异；筛选了15个差异性生物标记物,并鉴定了其中11个；Acetylcarnitine. 3-Methylglutarylcarnitine、1-Pyrroline-4-hydroxy-2-carboxylate、Kynurenine、Tryptophan、 Acetylspermidine、Cytosine、Tryptamine等在RSV肺炎痰热闭肺证患儿尿液中呈上调趋势,Methyldopa、N-Methylhydantoin等在RSV肺炎痰热闭肺证患儿尿液中呈下调趋势。金欣口服液干预RSV肺炎BALB/c小鼠的动物实验：1.RSV感染BALB/c小鼠后肺组织病理改变主要表现为间质性肺炎,肺泡壁充血增厚,间质有单核巨噬细胞及中性粒细胞浸润,支气管上皮细胞无明显变性、坏死,支气管管腔及肺泡腔无明显渗出物；金欣口服液干预后肺部充血及炎症有不同程度的减轻。病理评分结果显示治疗组较模型组显著下降。2.RSV感染6天后,BALB/c模型小鼠与正常小鼠血浆数据基于OPLS-DA模型能完全分离,模型参数分别为R2Y=0.917,Q2=0.835(血浆上层样本)和R2Y=0.967,Q2=0.936(血浆下层样本),表明造模成功,RSV感染引起了小鼠血浆代谢特征的变化；筛选并鉴定25个生物标记物；其中PC(18:1/22:6).PC(18:0/205)等磷脂酰胆碱和TG(18:1/18:2/18:2)、 TG(18:1/18:1/18:2)等甘油三酯在模型组小鼠血浆中较正常组显著增加,PC(18:1/18:3)、 PC(18:0/22:6)、PC(P-18:1/22:2)、PC(O-18:0/22:6)、PC(O-20:0/22:6). PC(16:0/20:4)、 PC(P-20:0/14:0)、LysoPC(16:0)、LysoPC(18:2)、LysoPC(18:0)、LysoPC(20:4)、LysoPC(18:1)、 LysoPC(22:6)等磷脂酰胆碱,SM(d18:1/24:1).SM(d18:0/18:1).SM(d18:2/24:1)、 Phytosphingosine等鞘脂质和TG(16:0/18:0/14:0).TG(16:0/16:0/18:0)等甘油三酯在模型组小鼠血浆中较正常组显著下‘降,此外,PE(18:3/19:0)、Phytanic acid等也呈下调趋势。基于所筛选出的生物标记物,我们评价了金欣口服液的药效,发现金欣口服液能不同程度的回调其中11个生物标记物,且主要通过调节甘油磷脂的代谢通路而发挥作用。3.RSV感染6天后,基于BALB/c模型小鼠与正常组小鼠肺组织数据构建OPLS-DA模型,两组完全分离,无交叉和重叠,模型参数分别为R2Y=0.879,Q2=0.789(肺组织上层样本)和R2Y=0.918,Q2=0.853(肺组织下层样本),表明造模成功,RSV感染引起了小鼠肺组织代谢网络的变化；筛选并鉴定25个生物标记物；其中PC(18:0/18:1)、 PC(18:0/18:2)、PC(22:6/18:2)、PC(18:2/20:4)、PC(P-20:0/14:0)、LysoPC(18:0)、LysoPC(16:0)、 LysoPC(18:1)、LysoPC(20:4)、LysoPC(18:2)等磷脂酰胆碱,SM(40:1)、SM(d18:1/20:0)、 Sphinganine、Phytosphingosine等鞘脂质和TG(18：1/18：1/18：1)、TG(18:1/16:0/20:1)、 TG(18:1/18:1/18:2)、TG(18:1/18:2/18:2)、TG(16:0/18:0/22:6)、TG(18:1/18:0/20:1)等甘油三酯在RSV肺炎BALB/c小鼠中含量较正常组显著下降,此外,Phytanic acid、Leucine、 Phenylalanine、Choline、LysoPE(16:0)等亦与RSV感染有关,在模型组小鼠中呈下调趋势。基于所筛选出的生物标记物评价金欣口服液的抗病毒药效,结果发现金欣口服液能不同程度的回调肺组织中16个生物标记物,主要通过调节苯丙氨酸、酪氨酸和色氨酸生物合成、苯丙氨酸代谢、缬氨酸、亮氨酸和异亮氨酸的生物合成、甘油磷脂代谢、鞘脂质代谢通路而发挥作用。结论：1. UPLC-LTQ/Orbitrap-MS分析代谢组学能够初步阐释并区分RSV肺炎痰热闭肺证与健康正常儿二者不同的代谢模式。2.RSV肺炎痰热闭肺证患儿血浆和尿液中主要以色氨酸、甘油磷脂代谢紊乱为标志。3.RSV肺炎BALB/c小鼠血浆和肺组织主要表现为苯丙氨酸、酪氨酸和色氨酸生物合成、苯丙氨酸代谢、缬氨酸、亮氨酸和异亮氨酸的生物合成、甘油磷脂代谢、鞘磷脂代谢发生紊乱。4.金欣口服液能够明显减轻RSV感染小鼠的肺部炎症。5.金欣口服液能够部分调整RSV感染引起的代谢紊乱,主要通过调节苯丙氨酸,酪氨酸和色氨酸生物合成、苯丙氨酸代谢、缬氨酸、亮氨酸和异亮氨酸的生物合成、甘油磷脂代谢、鞘脂质代谢通路而发挥作用。
[Abstract]:Background: pneumonia is a common disease of children's lung system. It ranks first in the statistics of single disease of pediatric cases in a comprehensive hospital. WHO is one of the 3 major diseases of Pediatrics in the world. In our country, the virus accounts for about 1/3 in acute lower respiratory tract infection in children and is on the rise, among which the Respiratory Syncytial Virus; RSV) is the most important. Multiple.RSV pneumonia is commonly seen with heat syndrome, of which phlegm heat closed lung syndrome is the most common and important syndrome of RSV pneumonia. A syndrome includes complex connections between multiple organs and multiple indicators. It is difficult to explain the basis of syndromes with a single index or a simple superposition of some indexes. Culvert is an important problem to be solved in modern Chinese medicine. Research at home and abroad has shown that western medicine is still lack of ideal drugs for the treatment of viral pneumonia in children. The only drug recommended by the American Pediatrics Association as "may be used" is ribavirin. It has a definite advantage and has been a large number of clinical and experimental studies. It has been confirmed that the research group, led by Professor Wang received by Professor Wang of Nanjing University of Chinese Medicine, has been engaged in the study of Chinese medicine for the treatment of viral pneumonia in children for nearly 20 years, and has developed an effective oral liquid of Chinese medicine, Jinxin oral liquid, which has the effect of opening and closing lung, clearing heat and detoxifying, eliminating phlegm and relieving cough. However, due to the complexity of the active chemical components of Jinxin oral liquid, the role of Jinxin oral liquid in regulating the overall metabolic network of RSV pneumonia is still unclear. Metabolomics (Metabonomics) is a new subject about the species, quantity and variation of endogenous metabolites of organisms, and is the importance of systematic biological technology. Because the metabolite is the end of all the genes of the organism and the functional activity of the protein, the metabolite is regarded as the "biochemical phenotype" of the whole functional state of the organism, which can be immediately, sensitive and true in response to the response and regulation of the overall functional state of the organism under the stimuli of various external factors, avoiding the previous use of a single or a few. The metabolic histopathological technique combines the characteristics of the whole, the dynamic and the comprehensive analysis in a combination with the whole concept of traditional Chinese medicine. Therefore, this subject uses metabonomics and other biochemical tests to comprehensively characterize the metabolite changes caused by RSV pneumonia and phlegm heat closure. To try to clarify the essence of RSV pneumonia and phlegm heat closed lung syndrome and the therapeutic effect of Jinxin oral liquid on RSV infected mice. Objective: To study the metabolic characteristics of plasma and urine in children with RSV pneumonia phlegm heat closed syndrome, explore the syndrome essence of RSV pneumonia and phlegm heat closed syndrome, study the metabolic characteristics of plasma and lung tissue of RSV pneumonia BALB/c rats, and explore Jinxin The intervention effect of oral liquid on RSV pneumonia BALB/c mice and the possible metabolic regulation mechanism. Methods: the clinical experiment of the metabolic group of RSV pneumonia sputum heat closed lung syndrome: 30 children with RSV pneumonia and heat closed syndrome were selected in accordance with the diagnosis and treatment of Chinese and Western medicine. The disease group was set up and 30 cases of normal healthy group were set up. The plasma and urine of two groups of children were collected respectively. Liquid, using Ultra-performance Liquid Chromatography coupled with Linear Ion Trap Quadrupole Orbitrap Mass Spectrometry, detecting the metabolites of plasma and urine in two groups by super high performance liquid chromatography and two-dimensional linear ion trap mass spectrometry (coupled with Quadrupole Orbitrap Mass Spectrometry). Principal Component Analysis (PCA) and orthogonal partial least squares (Orthogonal Partial Least Squares Discriminant Analysis, OPLS-DA) were used to analyze the detected matrix data, screening potential plasma, urine biomarkers, analysis of related metabolic pathways, and exploring RSV pneumonia phlegm heat closed lung syndrome. The nature of metabolic characteristics. Jinxin oral liquid intervention in RSV pneumonia BALB/c mice: RSV infection BALB/c mice, Jinxin oral liquid intervention, and set Leigh Bhave Lin as the positive control, 6 days later, collect the mice plasma, lung tissue, pathological section of lung tissue morphologic analysis to evaluate the pulmonary pathological changes, based on UPLC-LTQ/Orb Itrap-MS studied the changes in the whole metabolism of plasma and lung tissue in each group. Using PCA and OPLS-DA to analyze the data, screening potential plasma, biomarkers of lung tissue, analyzing related metabolic pathways, evaluating the efficacy of Jinxin oral liquid and clarifying the metabolic regulation mechanism of Jinxin oral liquid. Results: the metabolic group of RSV pneumonia phlegm heat closed lung syndrome is the following. Bed experiment: 1. the results of pattern recognition analysis of plasma metabolomics show that the main components of the main components of the children with RSV pneumonia and phlegm heat closed syndrome and the healthy normal group are mostly distributed in the ellipse (95% confidence area) area of the scatter plot. Although there is a certain trend of separation, there are still overlapping overlaps, and the OPLS-DA model is further constructed and the model parameters are further constructed. R2Y=0.907, Q2=0.567 (plasma upper samples) and R2Y=0.816, Q2=0.619 (sample of lower plasma), normal group and RSV pneumonia sputum heat closed lung syndrome group can be completely separated along the direction of the first principal component t[1] axis, no cross and overlap, indicating that the two groups have distinct differences in metabolic characteristics: screening 21 potential biomarkers and identifying 20 of them are SM (18:2/24:0), SM (d18:2/24:1), SM (d18:0/18:1) and other sphingomyelin, TG (16:0/18:1/22:6), TG (16:0/18:0/22:6), TG (16:0/20:4/22:5), etc. 0/20:3), PC (16:0/22:5), PC (18:2/20:0), LysoPC (18:2) and other phosphatidylcholine, TG (14:0/16:0/18:2), TG (14:0/16:0/16:1) and other triglycerides were downregulated in the plasma of children with phlegm heat closed lung syndrome of RSV pneumonia. Besides, the substances related to the syndrome of sputum heat closure were also found to be more significant than those of normal children. .2. urine metabolomics model identification analysis results showed that the PCA method was used to model children with RSV pneumonia and phlegm heat closed lung syndrome and healthy normal group. Most of the samples were distributed in the ellipse (95% confidence area) area of the scatter plot. Although there was a certain trend of separation, there were still overlapping overlaps, and the OPLS-DA model was further established. The parameters were R2Y=0.865, Q2=0.580 (urine samples measured by a Q column) and R2Y=0.902, Q2=0.593 (urine samples from HILIC chromatograph), the normal group and RSV pneumonia phlegm heat closed lung syndrome group were completely separated, indicating that the two groups were significantly different in the metabolites, and 15 differential biomarkers were screened and 11 of them were identified; Acety Lcarnitine. 3-Methylglutarylcarnitine, 1-Pyrroline-4-hydroxy-2-carboxylate, Kynurenine, Tryptophan, Acetylspermidine, Cytosine, Tryptamine and so on were up-regulated in the urine of children with pneumonia and phlegm heat closed syndrome. Methyldopa, N-Methylhydantoin and so on were downregulated in the urine of children with phlegm heat closed lung syndrome. The intervention of RSV pneumonia BALB/c mice: the pathological changes of lung tissue in 1.RSV infected BALB/c mice were mainly interstitial pneumonia, alveolar wall hyperemia and thickening, interstitial infiltration of mononuclear macrophages and neutrophils, no obvious degeneration and necrosis of bronchial epithelial cells, no obvious exudation in bronchiolus tracheal cavity and alveolar cavity; Jinxin mouth. The results of pathological score showed that the treatment group was significantly lower than the model group for 6 days after.2.RSV infection. The plasma data of BALB/c model mice and normal mice were completely separated based on the OPLS-DA model, and the model parameters were R2Y=0.917, Q2=0.835 (plasma upper samples) and R2Y=0.967, Q2=0.936 (Q2=0.936). RSV infection caused the changes of plasma metabolic characteristics in mice, and 25 biomarkers were screened and identified, including PC (18:1/22:6).PC (18:0/205) and TG (18:1/18:2/18:2), TG (18:1/18:1/18:2) and other glycerol three esters in the model mice plasma were significantly increased, PC (18:1/18). 3), PC (18:0/22:6), PC (P-18:1/22:2), PC (O-18:0/22:6), PC (O-20:0/22:6), PC (16:0/20:4), PC (P-20:0/14:0). /16:0/18:0) the triglyceride and other triglycerides in the model mice plasma were significantly lower than those in the normal group. In addition, PE (18:3/19:0) and Phytanic acid were also down downward. Based on the selected biomarkers, we evaluated the efficacy of Jinxin oral liquid and found that Jinxin oral liquid could adjust 11 biomarkers in varying degrees. After 6 days of regulating the metabolic pathway of glycerol phospholipid, the OPLS-DA model was constructed based on the lung tissue data of the BALB/c model mice and the normal group. The two groups were completely separated, without cross and overlap, the model parameters were R2Y=0.879, Q2=0.789 (upper lung tissue samples) and R2Y=0.918, Q2=0.853 (the lower lung tissue samples). RSV infection caused the changes in the metabolic network of lung tissue in mice; 25 biomarkers were screened and identified, including PC (18:0/18:1), PC (18:0/18:2), PC (22:6/18:2), PC (18:2/20:4), PC (P-20:0/14:0), LysoPC. Inganine, Phytosphingosine and other sheath lipids and TG (18:1/18:1/18:1), TG (18:1/16:0/20:1), TG (18:1/18:1/18:2), TG (18:1/18:2/18:2), TG (16:0/18:0/22:6), etc. It is also associated with RSV infection and down trend in the model mice. Based on the selected biomarkers to evaluate the antiviral effect of Jinxin oral liquid, it is found that Jinxin oral liquid can adjust 16 biomarkers in different degrees of lung tissue, mainly by regulating phenylalanine, tyrosine and tryptophan biosynthesis, phenylalanine. Metabolism, valine, leucine and isoleucine biosynthesis, glycerol phospholipid metabolism, sheath lipid metabolism pathway. Conclusion: 1. UPLC-LTQ/Orbitrap-MS analysis of metabolomics can preliminarily explain and distinguish between RSV pneumonia phlegm heat closed lung syndrome and healthy normal children with different metabolic patterns.2.RSV pneumonia phlegm heat closed lung syndrome children's plasma The plasma and lung tissue of.3.RSV pneumonia BALB/c mice were mainly characterized by tryptophan and glycerol phospholipid metabolism disorder as the main manifestation of phenylalanine, tyrosine and tryptophan biosynthesis, phenylalanine metabolism, valine, leucine and isoleucine biosynthesis, glycerin phospholipid metabolism, and sphingomyelin metabolism disorder.4. Jinxin orally The liquid can obviously reduce the lung inflammation in RSV infected mice.5. Jinxin oral liquid can partly adjust the metabolic disorder caused by RSV infection, mainly by regulating phenylalanine, tyrosine and tryptophan biosynthesis, phenylalanine metabolism, valine, leucine and isoleucine biosynthesis, glycerin phospholipid metabolism, sheath lipid metabolism pathway. Volatiles.
【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】：2015
【分类号】：R272

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