【作者】 罗和古；

【导师】 陈家旭；

【作者基本信息】 北京中医药大学 ， 中医诊断学， 2007， 博士

【摘要】 选题依据:方证对应研究是中医药学研究的核心问题、也是热点和难点问题。目前多以经典方剂为切入点,应用现代技术和相关的分析方法进行尽可能的全方位评价。代谢组学研究机体内源性代谢产物谱的动态变化过程,可以反映出刺激或药物作用的发生、发展和结果的全过程。将其与基因组学和蛋白质组学的结果结合起来,就能够全面、客观地反映刺激或药物作用的情况,为疾病发生发展机理和药理作用机理的研究提供全方位的信息。基于代谢组学强调把人体作为一个完整的系统来研究,通过测定人体各种体液内代谢物的组成变化来认识和反映人体代谢网络在疾病和药物作用下的变化规律,对于揭示复杂性疾病的机理和药物的代谢模式具有独特的优势,也与中医学的整体观、系统观和辨证论治思维非常吻合,与中医重视从人与自然、人与社会和人体内在的普遍联系和动态变化去分析、认识把握疾病发生、发展、变化的客观规律的认识一致。笔者试图以慢性束缚应激大鼠模型(肝郁脾虚证)和逍遥散为切入点,运用代谢组学的技术与方法,探索出一种研究中医方证相应理论的新方法、新途径。目的:本研究基于代谢组学的研究方法,重点研究基于代谢组学的慢性束缚应激大鼠模型(肝郁脾虚证)的相关变化及逍遥散对其的作用。以NMR,生物信息学等现代技术,通过对血液代谢组的分析,研究慢性束缚和逍遥散干预引起的大鼠内源性代谢物的变化,获得慢性束缚应激(肝郁脾虚证)和逍遥散给药前后的代谢物指纹图谱;分析血液代谢指纹图谱变化的原因,确定与慢性束缚应激(肝郁脾虚证)发生和逍遥散作用机理密切相关的代谢组学特征和小分子标志化合物;阐明药物作用靶点及作用机理,为慢性束缚应激(肝郁脾虚证)和逍遥散作用机理的系统研究提供科学依据。也试图以逍遥散为切入点,运用代谢组学的技术与方法,探索出一种研究中医方证相应理论的新方法、新途径。方法:选用实验用二级雄性SD大鼠42只,质量200 20g,以慢性束缚方法制作应激大鼠模型,随机分为:7天正常对照组(A组,n=6)、21天正常对照组(B组,n=6)、7天模型组(C组,n=6)、21天模型组(D组,n=6)、7天逍遥散1号组(E组,n=6)、21天逍遥散1号组(F组,n=6)、21天逍遥散2号组(G组,n=6)7组。分别于第8天、第22天,麻醉心室取血。在VARIAN UNITYINOVA600MHz超导傅立叶变化核磁共振波谱仪上分别调用弛豫编辑脉冲序列(CPMG)、扩散编辑脉冲序列(LED)进行实验,采用预饱和方式抑制水峰,饱和时间为2s,谱宽8000Hz,采样点数32k,累加次数64次,预饱和频率和中心频率都在水峰位置。自由感应衰减(FID)信号经过32k点傅立叶变换得到一维NMR谱图。以TSP为化学位移参考峰的位置,设为0ppm。调用VNMR软件中的程序将1H谱中从4.5-0.5ppm (CPMG)以及6.0-0ppm(LED)范围内的谱峰,按每段为0.04ppm,进行分段积分。将积分数据归一化之后,以文本文件或Excel文件贮存,用于模式识别分析。将积分值进行中心化和定标(centeringandscaling),用SIMCA-P 10.0软件包(瑞典,Umetrics AB,Umea)进行主成分分析(PCA),必要时进行判别分析(PLS-DA)。结果:①A、B、C、D、E、F、G各组间进行的PCA分析结果显示,各样本基本集中分布于得分图的椭圆形(95%置信区内)的4个区域,虽然A、B、C、D、E、F、G各组动物组内样品存在一定的差异,表现为有些样品相对离散,但各对照组间基本仍能分开,说明不同组别之间存在代谢产物的差异。正常组(A组、B组)与模型组(C组、D组)之间代谢产物有明显的不同,说明动物模型造模基本成功,模型组与正常组比较,两者存在代谢网路的改变。模型组(C组、D组)与治疗组(E组、F组、G组)各对照组间基本能分开,说明不同组别之间代谢产物存在差异、代谢网路有所不同,给与逍遥散后可以干预代谢物或代谢途径而致代谢终产物的改变。②正常组与模型组组间代谢标志物比较发现:A、C两组:A组含量高于C组有乳酸(1.36、1.38、1.4、4.16、4.12)、丙氨酸(1.44)、脂类化合物(0.92、1.36)、血糖(3-4)、以及谷氨酸(2.4)、VLDL和LDL(0.87、0.91、0.95、0.99)、HDL(0.83)、胆碱(3.23、3.27)、NAC-1(2.07);C组含量高于A组的有NAC-1(2.08)、小分子化合物(1.12)、3.44ppm未知化合物、长链脂肪酸(1.31、1.23、1.27、1.36、1.39)、NAC-2(2.15)以及1.59ppm处未知化合物。B、D两组:B组含量高于D组的有LDL(0.87、0.91、1.27)、不饱和脂肪酸(4-5)、饱和脂肪酸(4-5);D组含量高于B组的有NAC-1(2.07)、NAC-2(2.16)、饱和脂肪酸(1-3)。③模型组与治疗组组间代谢标志物比较发现:C、E组:C组样本中磷脂酰胆碱(3.28)、脂类化合物(0.92、0.96、1.32)、NAC-2(2.15)、NAC-1(2.08、2.11)、小分子氨基酸(1)、VLDL(0.95、1.35、1.39、1.43)、甘油三酯(3.55、3.59)以及1.63ppm3.2和3.16ppm、3.44ppm等未知化合物含量较高;而E组中乳酸(1.4、4.16、4.2)、谷氨酸(2.44)、丙氨酸(1.52)、缬氨酸(1.08)、HDL(0.87、1.21)、不饱和脂肪酸(5.31、5.35)及小分子化合物(1-2)含量较高。与同时点的A组比较,其乳酸(1.4、4.16、4.2)、谷氨酸(2.44)、丙氨酸(1.52)、HDL(0.87、1.21)有所恢复,并有不饱和脂肪酸(5.31、5.35)及小分子化合物(1-2)含量的提升。D、F组:D组样本中乙酸(1.96)、乳酸(4.2、1.4、4.16)、缬氨酸(1.04)、NAC-1(2.08)、NAC-2(2.15)以及小分子化合物(1-2)、化合物(3-4)含量较高;F组样本中苏氨酸(0.92、0.96、1.32、1.36)、丙氨酸(1.48)、谷氨酸(2.44)以及3.44ppm、VLDL和LDL(0.87、0.91、1.27、1.31、1.35、)、胆碱(3.27)以及不饱和脂肪酸(5.31、5.35)等化合物含量较高。与同时点的B组比较,其VLDL和LDL(0.87、0.91、1.27、1.31、1.35、)及不饱和脂肪酸(5.31、5.35)等化合物有所恢复,并有苏氨酸(0.92、0.96、1.32、1.36)、丙氨酸(1.48)、谷氨酸(2.44)、胆碱(3.27)等化合物含量的增加。并随给药时间的延长,其F组样本中3.4ppm、3.8ppm、2.44ppm未知化合物、磷酯酰胆碱(3.28)、胆碱(3.27)以及LDL或HDL(0.87、0.91)含量更高。D、G组:D组样本中血糖(3-4)、乙酸(1.96)、胆碱(3.24)、NAC-2(2.15)、NAC-1(2.08)、3.2ppm、1.48ppm等处所表示的化合物含量较高、脂类化合物含量相对较低;而G组样本中脂类化合物(0.92、0.96)、乳酸(1.36、1.4、4.16)、苏氨酸(1.32)、甲硫氨酸(2.16)、VLDL和LDL(0.87、0.91、1.27、1.31、1.35、)、磷酯酰胆碱(3.23)以及不饱和脂肪酸(1.99、5.31、5.35)等化合物含量较高。④随给药时间的延长, 3.4ppm、3.8ppm、2.44ppm未知化合物、磷酯酰胆碱(3.28)、胆碱(3.27)以及HDL(0.87、0.91)含量更高。⑤F组(逍遥散1号组)与G组(逍遥散2号组)组间代谢标志物比较发现: F、G两组样本中成分类似。相对而言,G组中各化合物含量比F组要高,G组样本中VLDL和LDL(0.87、0.91、1.27、1.31、1.35)、胆碱(3.23)、不饱和脂肪酸(1.99、5.31、5.35)、乳酸(1.4、1.36、4.16)、丙氨酸(1.52)、苏氨酸(1.32)、脂类化合物(0.92、0.96)等化合物含量较高;F组样本中血糖(3-4)、谷氨酸(2.44)、NAC-1(2.08、2.12)、2.12ppm化合物含量较高。结论:①代谢组学方法是研究中医症候和中医方证相关机理的良好平台。②从代谢组学的角度来看,慢性束缚应激大鼠模型与中医肝郁脾虚证存在对应关联性。③慢性束缚应激大鼠(肝郁脾虚证)代谢表型为乳酸、胆碱、NAC、饱和脂肪酸、血糖上升降,不饱和脂肪酸、HDL、3.44ppm等化合物含量降低。其代谢产物标志物可能是乳酸、胆碱、NAC、饱和脂肪酸、血糖、不饱和脂肪酸、HDL、3.44ppm等化合物。④慢性束缚应激(肝郁脾虚证)发生的代谢终产物的改变以脂类物质更明显,其具体发生的代谢物、代谢终产物或代谢途径的改变还需进一步研究。⑤逍遥散的作用显示出多途径、多靶点、双向调节特点。从代谢组学角度看,逍遥散可使代谢终产物乳酸、胆碱、NAC、饱和脂肪酸、血糖下降,不饱和脂肪酸、HDL、3.44ppm等化合物含量升高,表现为明显的代谢终产物的调节效应。其作用的靶点包含肝、脑、肾、肌肉、胃肠、脂肪组织、红细胞等多脏器组织细胞,其调节主要干扰何种代谢物或代谢途径而改变代谢终产物还需进一步研究确认。而且21天疗程的疗效优于7天疗程。⑥逍遥散中挥发油成分对慢性束缚应激大鼠(肝郁脾虚证)的代谢终产物的调节可能有负面影响。⑦代谢组学方法还可能用于有效药物组份的鉴别和度量。创新点:①首次用代谢组学方法研究慢性束缚应激(肝郁脾虚证)及逍遥散的作用机理,是方法学的创新。②首次从代谢组学的角度证明慢性束缚应激大鼠模型与中医肝郁脾虚证的对应关联性。进一步发现慢性束缚应激大鼠(肝郁脾虚证)代谢表型为乳酸、胆碱、NAC、饱和脂肪酸、血糖上升降,不饱和脂肪酸、HDL、3.44ppm等化合物含量降低。其代谢产物标志物可能是乳酸、胆碱、NAC、饱和脂肪酸、血糖、不饱和脂肪酸、HDL、3.44ppm等化合物。慢性束缚应激(肝郁脾虚证)发生的代谢终产物的改变以脂类物质更明显。③首次从代谢组学的角度证明逍遥散的作用显示出多途径、多靶点、双向调节特点。逍遥散可使代谢终产物乳酸、胆碱、NAC、饱和脂肪酸、血糖下降,不饱和脂肪酸、HDL、3.44ppm等化合物含量升高,表现为明显的调节代谢终产物的效应。而且21天疗程的疗效优于7天疗程。因此从代谢组学的角度来研究慢性束缚应激(肝郁脾虚证)及逍遥散的作用机理,丰富了慢性束缚应激大鼠模型、中医肝郁脾虚证、中医肝主疏泄的生物学内涵,初步揭示了逍遥散的作用机理。④首次发现逍遥散中挥发油成分对慢性束缚应激大鼠(肝郁脾虚证)的代谢终产物的调节可能有负面影响。证明代谢组学方法还可能用于有效药物组份的鉴别和度量。更多还原

【Abstract】 RATIONALES: Studies of prescriptions corresponding to syndromes are the core ofresearches in Traditional Chinese Medicine (TCM). These are also hot but difficult researchpoints in this area. Based on traditional prescriptions, thorough evaluations of these studieshave been attempted by the use of modern technology and relevant analyses. Themetabolomic analysis can be used to determine active alterations of endogenous metabolicspectra and subsequently reveal changes in the whole process, including occurrence,development, and consequences in response to stimuli or drug treatment. In addition, incombination with genomic and protein methods, the metabolomic analysis can be used toexamine actions of stimuli or drugs completely and objectively, which provides entireinformation about processes of diseases and mechanisms of pharmacological actions. Inmetabonomics, the human body is studied as an integrated system, in which changes inmetabolic components of various body fluids are examined and used to reflect alterations ofmetabolic networks in the body under conditions of diseases and treatments with drugs. Thus,the metabolomic analysis has unique advantages in studying mechanisms of complexdiseases and metabolic models of drugs. This is consistent with the views of integration,systems, and dialectical diagnosis and therapeutics in TCM. It is also in agreement with theopinions of understanding and analyzing objective profiles of diseases in their occurrence,development, and alterations in TCM, which emphasizes on the common links of human tonature and societies and connections within the human body. Using metabolomic techniquesand methods, the author found a novel method for studying prescriptions and syndromes inTCM based on the therapeutic effects of the Xiaoyaosan decoction (XYS) on changesinduced by chronic immobilization stress (CIS), which is a model of Liver Stagnation andSpleen Deficiency Syndromes (LSSDS) in rats.OBJECTIVES: To determine CIS (LSSDS)-induced changes in metabolisms with andwithout XYS treatment using metabolomic analyses. Specifically, we aimed to studyCIS-induced changes in endogenous metabolites and obtain“fingerprint spectra ofmetabolites”under CIS (LSSDS) in the absence and presence of XYS by analyzingmetabolisms in the rat blood using modern techniques such as NMR and bioinformatics; todetermine small molecule marking compounds and“characteristics of metabonomics”,which were related to the mechanisms of CIS (LSSDS) and XYS actions, via analysis ofcauses of changes in blood“fingerprint spectra of metabolites”; to explore drug targets andelucidate mechanisms underlying drug actions, which were expected to provide scientificevidence for systemic studies of CIS (LSSDS) and mechanisms of XYS actions. In addition,novel methods and pathways were anticipated for studies of TCM prescriptions andsyndromes, based on the exploration of XYS using metabolomic techniques and methods.METHODS: Forty-two male Sprague-Dawley rats, weighing 200±20 g, were subjected to chronic immobilization to develop stress models. Rats were randomly dividedinto 7 groups with 6 rats each: (A) 7-day control, (B) 21-day control, (C) 7-day model(CIS), (D) 21-day model, (E) 7-day XYS-1, (F) 21-day XYS-1, (G) 21-day XYS-2. Bloodwas collected from the cardioventricle under anesthesia on the 8th (A, C and E) or 22nd day(B, D, F and G) and detected CPMG and LED using the Fourier variable superconductingnuclear magnetic resonance (NMR) spectrometer (VARIAN UNITYINOVA 600MHz).Saturated inhibition of the water peak was used, with the saturation time 2 s, spectral width8,000 Hz, sample collecting points 32 k, and accumulation times 64. Both pre-saturated andcentral frequencies were located in the water peak. Free induction decay (FID) signals weretransferred into one-dimensional NMR spectrogram via 32 k Fourier transformation. Thechemical migration reference peak was set to 0 ppm based on TSP. Segmental integralcalculus (0.04 ppm per segment) was performed from 4.5-0.5 ppm (CPMG) and 6.0-0 ppm(LED) within the peak ranges in 1H spectra using the VNMR software. Data were saved astext or Excel files after normalization and then used for pattern recognition analyses.Values by calculus were centering and scaling before PCA or PLS-DA, if necessary, usingthe SIMCA-P10.0 software (Umetrics AB, Umea, Sweden).RESULTS: (1) Based on the results from PCA in the groups of A-G, the samples wereprimarily distributed in four regions of the scoring oval (95% confidence intervals). Whilevariability existed within each of the groups, as evidenced by relative scatters in somesamples, control groups were basically separated, suggesting differences of metabolitesbetween groups. The metabolites of the model groups (C and D) were significantly differentfrom those of controls (A and B), suggesting that the animal models were successfullyestablished and that they displayed changes in the network of metabolisms relative tocontrols. Models (C and D) and XYS treatment (E, F, and G) were separated from therespective controls, suggesting differences of metabolites and metabolic networks betweengroups; treatment with XYS results in changes in final metabolites via interruptingmetabolites or the pathway of metabolisms. (2) By comparison of metabolic markersbetween controls and models, it was found that, relative to the 7-day CIS model rats (C),non-stress rats (A) displayed higher concentrations of lactic acid (1.36, 1.38, 1.4, 4.16, 4.12),alanine(1.44), lipids (0.92, 1.36), blood sugar(3-4), glutamate(2.4), VLDL, LDL(0.87, 0.91, 0.95, 0.99), HDL(0.83), choline(3.23, 3.27), and NAC-1(2.07)and lowerconcentrations of NAC-1(2.08), small molecule compounds(1.12), long fatty acid(1.31,1.23, 1.27, 1.36, 1.39), NAC-2(2.15), and 1.59 and 3.44 ppm unknown compounds.Similarly, compared to 21-day model rats (D), 21-day stress rats (B) displayed higher levelsof LDL(0.87, 0.91, 1.27), unsaturated fatty acid(4-5), saturated fatty acid (4-5), but lowerlevels of NAC-1 (2.07), NAC-2 (2.16), saturated fatty acid (1-3). (3) By comparison ofmetabolic markers between models and XYS treatment, it was found that CIS rats withoutXYS (C) displayed higher levels of phosphatidylcholine(3.28), lipids(0.92, 0.96, 1.32),NAC-2(2.15), NAC-1(2.08, 2.11), small molecular amino acid (1), VLDL(0.95, 1.35,1.39, 1.43), triglyceride(3.55、3.59), and several unknown compounds (1.63, 3.2, 3.16, and3.44 ppm). By contrast, XYS rats (E) showed higher levels of lactic acid(1.4, 4.16, 4.2), glutamate(2.44), alanine(1.52), valine(1.08), HDL(0.87, 1.21), unsaturated fatty acid(5.31, 5.35), and small molecular compounds (1-2). Compared to the non-stress control(A), XYS resumed lactic acid(1.4, 4.16, 4.2), glutamate(2.44), alanine(1.52), and HDL(0.87, 1.21)to a certain extent; XYS also increased, unsaturated fatty acid(5.31, 5.35), andsmall molecular compounds (1-2). In addition, 21-day model rats (D) exhibited higher levelsof acetic acid (1.9), lactic acid (4.2, 1.4, 4.16), valine(1.04), NAC-1(2.08), NAC-2(2.15)small molecular compounds (1-2), and compounds (3-4). In the presence of XYS (F), the21-day model rats displayed higher levels of threonine(0.92, 0.96, 1.32, 1.36), alanine(1.48), glutamate(2.44), 3.44ppm, VLDL and LDL(0.87, 0.91, 1.27, 1.31, 1.35), choline(3.27), and unsaturated fatty acid(5.31, 5.35). Compared to the 21-day control, XYS-1 inthe F group resumed VLDL and LDL(0.87, 0.91, 1.27, 1.31, 1.35)as well as unsaturatedfatty acid(5.31, 5.35); it also increased threonine(0.92, 0.96, 1.32, 1.36), alanine(1.48),glutamat(e2.44), cholin(e3.27). The levels of unknown compounds (3.4, 3.8, and 2.44ppm),phosphatidylcholine(3.28), choline(3.27), and LDL or HDL(0.87, 0.91)were furtherincreased in the presence of XYS over time. While the 21-day model rats (D) displayedhigher concentrations of blood sugar(3-4), acetic acid(1.96), choline(3.24), NAC-2(2.15)、NAC-1(2.08), and unknown compounds 3.2ppm and1.48ppm, the concentraionof lipids was relatively low. By contrast, the presence of XYS-2 (G) increased lipids(0.92,0.96), lactic acid(1.36, 1.4, 4.16), threonine(1.32), methionine(2.16), VLDL and LDL(0.87, 0.91, 1.27, 1.31, 1.35、), phosphatidylcholin(e3.23), and unsaturated fatty acid(1.99,5.31, 5.35). (4) The contents of the unknown compounds (3.4, 3.8, and 2.44 ppm),phosphatidylcholine(3.28), choline(3.27), and HDL(0.87, 0.91)were further increasedin the presence of XYS over time. (5) Comparison of the metabolic markers between XYS-1(F) and XYS-2 (G) revealed that both treatments affect the components similarly, althoughthe latter increased the contents of compounds to a greater extent relative to the former.Specifically, the XYS-2 rats (G) displayed higher contents of VLDL and LDL(0.87, 0.91,1.27, 1.31, 1.35), choline(3.23), unsaturated fatty acid(1.99, 5.31, 5.35), lactic acid(1.4,1.36, 4.16), alanine(1.52), threonine(1.32), and lipids (0.92, 0.96), whereas the XYS-1rats (F) displayed higher levels of blood suga(r3-4), glutamate(2.44), NAC-1(2.08, 2.12),and the unknown compound 2.12ppm.CONCLUSIONS: (1) Metabonomics is a useful platform for studying mechanisms ofsymptoms, syndromes, diagnoses, and prescriptions or treatments in TCM. (2) Inmetabolomics, the CIS model has a corresponsive connection with LSSDS in TCM. (3) Themetabolic phenotype of CIS (LSSDS) includes increases in the levels of lactic acid, choline,NAC, saturated fatty acid, and blood sugar and decreases in the levels of unsaturated fattyacid, HDL, and the unknown compound 3.55 ppm. These metabolic components also may bethe markers of metabolites. (4) The final metabolites changed by CIS (LSSDS) primarilyinclude lipids. Further studies are still needed to determine specific metabolites, final metabolites, and changes in metabolic pathways. (5) The roles of XYS are characteristic ofmulti-pathways, multi-targets, and bi-direction regulation. In the view of metabonomics,XYS markedly regulates the contents of final metabolites, as evidenced by decreased lacticacid, choline, NAC, saturated fatty acid, and blood sugar and increased unsaturated fatty acid,HDL, and the compound 3.44 ppm. The targets of XYS include various organs, tissues, andcells, such as the liver, brain, kidney, muscles, gastrointestine, fatty tissues, and red bloodcells. Further studies are needed to determine which metabolites or metabolic pathways XYSinterrupts to alter final metabolites. The efficacy of 21-day XYS is greater than that of 7-dayXYS. (6) The vapor oil in XYS may play a negative role in the regulation of finalmetabolites by CIS (LSSDS). (7) Metabonomics can also be used to distinct and quantifyeffective components of medicines.INNOVATIONS: (1) Metabolomic analyses were used for the first time to study themechanisms underlying CIS (LSSDS) and XYS actions; this was an innovation inmethodology. (2) It was demonstrated metabolomically for the first time that CIS wascorrespondingly associated with LSSDS. It was found for the first time that the metabolicphenotype of CIS (LSSDS) includes increases in lactic acid, choline, NAC, saturated fattyacid, and blood sugar and decreases in unsaturated fatty acid, HDL, and the unknowncompound 3.55 ppm. These metabolic components also may be the markers of metabolites.CIS (LSSDS)-induced alterations of final metabolites primarily include lipids. (3) It wasdemonstrated metabolomically for the first time that the roles of XYS are characteristic ofmulti-pathways, multi-targets, and bi-direction regulation. XYS markedly regulates thecontents of final metabolites, as evidenced by decreased lactic acid, choline, NAC, saturatedfatty acid, and blood sugar and increased unsaturated fatty acid, HDL, and the compound3.44 ppm. The potency of 21-day XYS treatment was greater than that of 7-day XYS.Mechanisms underlying CIS (LSSDS) and XYS actions were examined metabolomically forthe first time. This enriched biological contents of CIS rat models, TCM LSSDS, andliver-dominated dispersion and discharge and revealed initial mechanisms of XYS actions.(4) It was found for the first time that the vapor oil in XYS might play a negative role in theregulation of final metabolites by CIS (LSSDS). It demonstrated that metabolomicalmethods could also be used to distinct and quantify effective components of medicines.更多还原