【作者】 王伟明；

【导师】 盛国光；

【作者基本信息】 湖北中医药大学 ， 中医内科学， 2010， 博士

【摘要】 目的肝气郁结证的发生非常普遍,是常见的中医内科证型,据中医内科临床住院病例的抽样统计中,大约有21%左右都有肝气郁结证侯的表现。近些年来,对于肝气郁结证本质生物学基础的研究较多,基本上都是单个系统的研究,主要是自主神经功能,分子生物学,微量元素,血液流变学,血浆生化,性激素,脏器损伤与量表测试等方面,至今还少有从多系统方面来研究肝气郁结证生物学基础的,对肝气郁结证目前仍未能够全面整体地阐明其生物学基础,代谢组学是现今生物与医学界的研究热点之一,它可以对各种常见生物体内几乎所有代谢产物进行定量分析,并描述生物体内代谢物动态变化,来发现代谢物变化与生理病理相关的改变关系,通过生物整体、系统或器官代谢途径及内源性代谢物质所受的不同影响因素,来研究机体的整体与全局。应用代谢组学研究肝气郁结证候,正是中医整体观的具体科学表现。在本研究中,我们一方面利用情志刺激法制作大鼠肝气郁结证模型,并用柴胡疏肝散进行灌胃给药,观察造模及给药前后代谢组的差异变化,另一方面在临床上筛选肝气郁结证患者,通过尿液与血液中代谢物的变化,以期从小分子代谢物组学水平来整体、系统地探讨肝气郁结证的特征代谢表型与可能存在的生物学本质,为肝气郁结证的进一步防治提供相应的理论与实验依据。方法30只健康SD大鼠,体重220g,随机分为3组：正常组10只(A组)、模型组10只(B组)和给药组10只(C组),除正常组外,其余组采用夹尾法造模建立大鼠肝气郁结证模型,给药组造模后给予柴胡疏肝散灌胃(0.5g/ml),每天两次,各组分别在造模前一天、成模后、给药后收集尿液与血液,通过NMR波谱仪检测,运用代谢组学的方法,观察大鼠正常与肝气郁结模型以及柴胡疏肝散干预后,尿液与血液小分子代谢物组的差异性变化谱；并通过30例临床肝气郁结证患者与30例健康人的尿液与血液代谢组学观察,从整体性角度来探寻肝气郁结证的生物学基础和生理病理机制。结果一、动物实验研究(1)与正常组相比,模型组肝气郁结证大鼠尿液马尿酸3.97ppm,7.55ppm,7.85ppmα-酮戊二酸3.01ppm,2.45 ppm柠檬酸(citrate)2.57 ppm,4.10 ppm,2.88 ppm异柠檬酸(isocitrate) 3.99 ppm,2.51ppm,2.43和乌头酸(aconitate) 3.77 ppm,6.96 ppm,3.96 ppm的谱峰相对积分面积明显降低,在尿液中含量降低,肌酸酐3.05 ppm,4.07ppm,丙酮2.23 ppm,乙酸1.93 ppm,肌酸3.93 ppm,3.03 ppm,烟酸(nicotinate) 4.41 ppm,1.41 ppm和5-羟基吲哚-3-乙酸(5-hydroxyindole-3-acetate,5-HIAA) 3.03 ppm,2.91 ppm,6.63 ppm,7.15 ppm谱峰相对积分面积显著增高,在尿液中的含量升高。(2)肝气郁结证大鼠给予柴胡疏肝散干预后,给药组代谢表型为肌酸3.93,3.03,丙酮2.23肌酸酐3.05等含量明显下降,N-氧化三甲胺4.07, TMAO 3.27,牛磺酸3.43等含量明显增高。差异性表达代谢物结果显示,在造模后升高的差异表达代谢物在给予柴胡疏肝散干预后,大多数变化显著性降低或者已无显著性,其尿液代谢表型有向正常范围回归的趋势,呈现代谢网络修复的结果。这提示柴胡疏肝散对肝气郁结模型组大鼠有一定的恢复作用。(3)从正常组和模型组肝气郁结证大鼠血液的比较代谢主成分分析来看,肝气郁结证大鼠血液代谢表型为不饱和脂肪酸HDL(0.83),谷氨酸G1u,谷氨酰胺G1n(2.43),撷氨酸Va1(1.03,0.98),VLDL(1.29,0.87),LDL,(1.25,0.85)类脂,(1.27)等化合物含量降低,乳酸Lac(1.31,1.32,4.11),N-乙酰糖蛋白Nac(2.02,2.05,2.06,2.09),胆碱,饱和脂肪酸,B-葡萄糖(3.47,3.91)含量上升。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。(4)给药组肝气郁结证大鼠给予柴胡疏肝散干预后,对血液1H-NMR谱峰积分值进行PCA分析后,得到得分图(scoreplot),两组均不能区分开,说明没有找到模型组与给药组的血液代谢表型差异化合物。二、临床研究(1)从正常组和肝气郁结证患者组尿液的比较代谢PCA分析来看,肝气郁结证患者尿液代谢表型为柠檬酸(citrate) 2.57,2.71,2.66,苯丙氨酸(phenylalanine) 3.12,色氨酸(tryptophan) 3.32,乙酰基柠檬酸(acetyl citric acid) 1.28,1.26,酪胺(tyramine) 2.71,2.53,乌头酸(aconitic acid) 3.17 3.45,3.77等化合物含量降低,肌酐(creatinine)3.06,4.06,4.1上升。(2)对正常组与肝气郁结证患者组血液代谢组统计分析,两组血液的代谢组学PCA分析结果可以看出正常组与肝气郁结组之间PC没有区分开来,说明肝气郁结证患者血浆内源性代谢谱变化不明显。结论(1)肝气郁结证反应了能量代谢水平偏低,主要代谢途径是三羧酸循环,标志代谢物为柠檬酸和乌头酸的显著性降低。(2)肝气郁结发生机制“怒伤肝”的生物学基础为烟酸的代谢。(3)肝气郁结的抑郁症状与色氨酸代谢降低密切相关。(4)柴胡疏肝散能够部分调控平抑肝气郁结证中发生异常的代谢产物。(5)“肝主疏泄”对情志的调节作用通过酪氨酸代谢发挥作用。(6)在肝气郁结证候中氨基酸代谢表现明显。通过以上发现的肝气郁结证候代谢产物发生变化的生物基础,得到肝气郁结证生物体生理与病理发生的代谢组变化机制,对肝气郁结证的生物特征从代谢组学整体角度进行了成功的探索。意义：初步发现肝气郁结证生物体的生理与病理发生变化的代谢机制基础是具有整体特征的多个代谢途径。更多还原

【Abstract】 Objective:The stagnation of Liver-Qi syndrome is a very far-ranging and pervasive syndrome, and also a familiar TCM (Traditional Chinese Medicine) internal medicine syndrome. According to sample statistic of TCM internal medicine history cases, around 21% patient get the stagnation of Liver-Qi syndrome. Recent years, entitative biological study to the stagnation of Liver-Qi syndrome is basically study of single system, which covers aspects of Blood Biochemical, autonomic nerve function, Trace Element Level, Blood rheology study, molecular biology, sex hormone, Damage of Organs, and Parametric Testing, etc., but few study is based on multi-system aspect. There is no illustrated biological foundation for the stagnation of Liver-Qi syndrome. Metabonomics is nowdays hot topic in biology and iatrology, which is used as quantitative analysis for almost all metabolites of all kinds of familiar organism, and describe the parameter dynamic change of metabolites, in order to find the relationship of metabolites change and pathophysiology, so that to study the macrocosm and the all, through studying different effect factor of the whole-body system, system and Apparatus etabolism approach and Endogenous etabolism matter. It is a representation of Tradicitional Chinese Medicine Holism, that apply metabonomics to The stagnation of Liver-Qi syndrome. In this study, on the one hand we use emotion stimulating and pinching the tail method to make rat model of stagnation of Liver-Qi syndrome, and intervent by Bupleurum smoothing liver medicine, observe the modification before and after take the medicin. On the other hand, the cilinic aspect, we choose patients with Ganqi stagnation, observe the change of emiction and blood metabolite, from the point of metabonomics, to holistic expound the biological fundation of stagnation of Liver-Qi syndrome, in order to analysis the Phenotype Characteristics and possible biological fundation of the stagnation of Liver-Qi sydrome, from a systemic and holistic Small molecule metabonomics level, and also provide relavant theory and experiment support for further prevention for the stagnation of Liver-Qi syndrome.Method:30 healthy SD rat, body weight 220g, divide to three groups by chance:Group A:Normal 10; Group B:model group 10; Group C: medicine group 10. Except for group A, the other two groups was made into model of the stagnation of Liver-Qi syndrome. Group C was provided Bupleurum smoothing liver medicine,0.5g/mL, twice per day. All groups is to collect emiction and blood samples the day before modelling, after modeling, and after take the medicine, go through NMR Spectrometer examination, and use metabonomics method, after observe group A, B and C, the change of emiction and blood Small-molecule metabolites, and meanwhile observe emiction and blood metabonomics study of 30 clinical patients with Ganqi stagnation and 30 healthy people, and study the biological fundation and pathophysiology, from the point of metabonomics.Result:一、Animal experiment study(1) Comparing with the normal group, the model rat emiction Hippuric acid content 3.97.7.55,7.85, a-Ketoglutarate 3.01,2.45, citrate2.57,4.10,2.88, isocitrate 3.99 2.51 2.45and aconitate 3.77 6.96 3.96 the curve relative integral acreage obviously decrease, and decrease in emiction, Creatinine 3.05,4.07, acetone 2.23, acetic acid 1.93, reatine 3.93,3.03, nicotinate 4.41, 1.41 and acid5-hydroxyindole-3-acetate,5-HIAA 3.03,2.91,6.63, 7.15 the curve relative integral acreage obviously increase, and increase in emiction.(2) After intervene by Bupleurum smoothing liver medicine, the Metabolic phenotype of group C was reatine 3.93,3.03, acetone 2.23 Creatinine 3.05 the content was obviously decreased, N-TMAO 4.07, TMAO 3.27, aurine 3.43content was obviousaly increased.(3) From normal group and model group rat blood comparison PCA analysis, the metabolic phenotype of model group is Lactic acid (1.31,1.32,4.11),N-O-GlcNAc(Nac2.02,2.05,2.06,2.09), Choline, saturated fatty acid, B-dextrose(3.47,3.91) increase, unsaturated fatty acid, HDL (0.83), Glu, Gln(2.43) Val (1.03,0.98), VLDL (1.29,0.87), LDL, (1.25,0.85) Lipoid (1.27), content was decreased。The symbol of metabolite may be lactic acid, Choline, NAC, saturated fatty acid, blood sugar, unsaturated fatty acid, HDL。(4) The rats from Liver Qi Stagnation Syndrome group were treated by Chaihu Shugansan. After the PCA analysis on the blood-lH-NMR spectral peak integral values, the obtained score plot factor loading diagram (loadingplot), and PC integral values distributed on the oval-shaped scatter (95% confidence region) in four regions, but the two groups can not be distinguished, which indicated that the Phenotypic differences in metabolism of blood compounds of model group and treatment group could not be found.二、clinical study(1) It can be see from the results of comparative PCA analysis on the urine of normal group and Liver Qi Stagnation patients’group that, Liver Qi Stagnation group’s urine metabolic phenotype was: creatinine 3.06,4.06,4.1 rose, citrate 2.57,2.71,2.66, Phe (phenylalanine) 3.12, tryptophan (tryptophan) 3.32, acetyl citric acid (acetyl citric acid) 1.28,1.26, tyramine (tyramine) 2.71,2.53, aconitic acid (aconitic acid) 3.17 3.45,3.77 and other compounds decreased.(2) It can be seen from the statistical analysis results of the normal group and Liver Qi Stagnation patients’group that, the PCA analysis on two groups of blood plasma metabonomics showed that there is no distinction of PC between the normal group and the group of liver qi stagnation, which indicated that Liver Qi Stagnation Syndrome patients’ endogenous metabolism of plasma spectra did not change significantly.ConclusionIn Liver Qi Stagnation Syndrome, the performance of amino acid metabolism was apparent; citric acid content in urine decreased; creatinine increased; blood lactate concentration increased; glutamic acid, Gln and Screenshot acid declined. The amino acid metabolism differences may be related to liver qi stagnation. Liver Qi Stagnation syndrome is related to energy metabolism, and its compounds include creatinine (ereatinine), lactate (laetate), citric acid (eitrate) and alanine (alanine); Liver Qi Stagnation syndrome is related to neural System Regulation:it was found in the sample patients with the Liver Qi Stagnation Syndrome that a series of metabolites of phenylalanine and tyramine content related with tyrosine metabolism decreased. It was found that Tryptophan may be neural basis for regulating the biological material for Liver Qi Stagnation Syndrome; Liver Qi Stagnation Syndrome group also may have weakened immune function:the metabolites is L-tryptophan metabolism; tryptophan is the Synthesis of 5-HT precursor substance; decrease of tryptophan metabolism resulted in the reduction of 5-HT synthesis; the activation of the sympathetic nervous system regulated the immune system mainly based on suppression, which led to the release of glucocorticoids from the adrenal cortex and the inhibition of immune cells.From the findings above, the changes of biological foundation of Liver Qi Stagnation Syndrome metabolite were found; the mechanism changes of Liver Qi Stagnation organism’ s physiological and pathological basis was obtained. Therefore, the study was a successful exploration of the biological features of Liver Qi Stagnation from an overall perspective of metabolomics.更多还原