肝郁证大鼠模型血清代谢组学研究
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摘要
研究背景:
证候是中医学认识疾病作为一种功能状态变化的症候群,是中医学理论和临床实践的核心。辩证施治在几千年的临床实践中不断发展和验效,充分体现了整体观和个体化的治疗理念,是中医学的精髓。因此,证本质的研究对于中医理论与临床实践的发展和中医现代化具有极其重要的意义。
肝郁证是指由于肝的疏泄功能异常,疏泄不及而致气机郁滞所表现的证候,见于现代医学多种疾病,是中医脏腑病证中常见的证候之一,也是中医肝病发病学的基本证候。在不同疾病中,经中医辩证同属肝郁证的,施以疏肝理气相应方药,这样的“异病同治”确能取得较好疗效。因此,肝郁证被认为是多种疾病发展过程中的一种具有共性的“功能态”,研究肝郁证的实质,不仅是中医证本质研究的重要内容,更能为临床多种疾病的诊断和防治提供新的思路和方法;通过肝郁证这一典型证候的研究,探索证本质研究的合适方法,对闸释中医学的科学内涵有实际意义。
继基因组学、蛋白质组学发展起来的代谢组学,是研究生物体系受外部刺激所产生的所有代谢产物变化的科学方法,它迴避了复杂的生命调控过程,通过对生命体所有低分子量代谢产物进行定量、定性分析,给出最终的、整体的病理变化结果。同蛋白质组学一样,中医学两大特色——整体观和辨证论治观在代谢组学中也都有体现,通过代谢组学的方法有助于阐释中医“证”的本质。
研究目的:
比较慢性束缚肝郁证大鼠模型与正常大鼠血清差异表达的代谢物,确定肝郁证的“代谢组学特征”和小分子标志物;阐明肝郁证代谢物水平的物质基础并探讨其实质,为肝郁证本质的系统研究提供科学依据。建立证候研究的代谢组学技术平台,为从代谢物水平探讨中医证本质提供思路和方法。
研究方法:
1.慢性束缚肝郁大鼠模型的建立
动物分组:实验用清洁级雄性Wistar大鼠24只,鼠龄5周,体重(150±20)g,适应性饲养1天后随机分为模型组(E组)、正常对照组(F组)、逍遥散+模型组(A组)和逍遥散+空白组(B组),每组各6只。F组3只1笼,其余3组单笼孤养。
模型建立:参照文献,模拟中医情志抑郁的病因,将A、E组大鼠置于特制的束缚制动筒内,通过移动插条逐步缩小大鼠活动空间,调节到其不产生强烈反抗的最小空间。每日开始时间随机,每日束缚制动1次,持续时间从第1天的4h逐渐增至每天6h,连续21d。A、E组大鼠在束缚制动期间禁食、禁水。
药物干预:A、B组大鼠根据正常成人逍遥散每日用量,按体表面积换算成大鼠用药量为10g/kg.d,根据大鼠个体体重每日上午8时给A、B组大鼠经胃灌相应药量,E、F组大鼠同时以0.9%生理盐水2ml灌胃。
从行为学、实验室检查血浆皮质酮、逍遥散干预后疗效观察等方面评价模型的可靠性。
2.肝郁证代谢组学初步研究
用基于~1H NMR技术的代谢组学方法检测模型组(E组)和正常对照组(F组)血浆代谢组分差异,分析其差异物质与肝郁证可能存在的关系。
3.肝郁证方证的代谢组学研究
进一步比较A、B、E、F四组大鼠血清代谢组分差异,尤其关注逍遥散对正常大鼠(B组)和模型大鼠(A组)血清代谢组分的影响,并与E、F组间差异表达的代谢组分比较,从方证互参角度了解肝郁证中代谢组分的变化规律。
结果:
行为学观察发现E组大鼠开始表现为易激惹、挣扎,随后表现为躲避、活动减少和对外界刺激反应减少,安静时埋首、拱背,大便松散,皮色失去光泽;A组大鼠上述反应较轻,而B、F组未见异常行为状态。各组间体重增长存在显著性差异(P＝0.010),E组生长较为缓慢,A组服药后体重增长缓慢的情况得到改善。液体消耗实验提示各组间糖水偏好存在显著性差异(P＜0.001),E组大鼠对糖水的偏好率小于其余三组。第22天血浆皮质酮水平比较,各组间存在显著性差异(P＝0.0001);浓度从高到低依次为E＞A＞B＞F,且A、E组之间存在显著性差异(p＝0.005)。常规HE染色下,各组胃粘膜、肾上腺未见急性应激的病理改变,与E组比较无明显变化。四组间肾上腺体重指数比较存在显著性差异(P＝0.045),E组最高,A组有改善。分别从病因、证候、实验室病理改变和治疗后的反应四个方面验证慢性束缚肝郁证大鼠模型的可靠性。
肝郁证代谢组学初步研究发现E组、F组之间血清代谢物存在差异,E组中含量升高的有血糖(Glucose)、肌酸(Cr),含量下降的有高密度脂蛋白(HDL)、3-羟基丁酸(3-HB)、谷氨酰胺(Gln)、谷氨酸(Glu)、乳酸(Lac)、N-乙酰糖蛋白(NAc)、O-乙酰糖蛋白(OAc)、不饱和脂肪酸(UFA)、极低/低密度脂蛋白(VLDL/LDL)、磷酸胆碱(PCho)和磷脂酰胆碱(PtdCho)。上述物质的集合共同构成了肝郁证大鼠模型的代谢组学特征。这些物质大多与机体能量代谢有关;此外,Gln、UFA、VLDL/LDL、PCho和PtdCho是维持细胞正常结构的基本物质,Gln转化生成的谷胱甘肽和PtdCho还有非常重要的抗氧化、抗损伤作用;UFA、PtdCho、Glu与神经系统高级功能相关;Gln、UFA、PCho、NAc、OAc是维持机体正常免疫功能的基本物质;NAc、OAc、UFA与血液流变学和凝血功能相关。这些物质的变化反映了肝郁证状态下能量代谢紊乱、神经内分泌失调、免疫功能减弱、微循环障碍、体细胞活性及抗损伤修复减退的病理机制,并进一步提示肝郁证存在肿瘤高发危险,能导致学习、记忆能力减退;提示可能会影响凝血功能。
进一步对肝郁证方证进行代谢组学检测、分析:逍遥敞对正常大鼠血清代谢组分有轻微影响,逍遥散干预后肝郁模型大鼠血清代谢谱有所变化,向正常组靠近。CPMG、LED实验得分图显示四组大鼠沿第一主成份明显分开,提示代谢组分的变化几乎是单向的。四组间两两比较得出代谢物质变化的方向和幅度,找出随证候模型和相应方剂治疗后变化一致并趋于正常的物质,剔除那些变化与方证无关的物质,推测Lac、Cr、Glucose、3-HB、Glu、VLDL/LDL、PtdCho、UFA这8种物质也许更能体现肝郁证模型大鼠血清代谢组学特征。
结论:
慢性束缚大鼠模型能够较好的模拟肝郁证证候。通过对肝郁证大鼠模型的代谢组学研究,尝试将中医证候病机与现代医学理论联系起来,其纽带就是存在差异的物质。中医证候的生物标志物很可能不是单一物质,而是差异物质的集合,即“生物标志物群”的概念,也许才能更好的用于疾病/证候的判别分析。从代谢组学去理解肝郁证:在涉及细胞新陈代谢的多种物质改变基础上,以神经内分泌失调、免疫功能减弱、微循环障碍、体细胞活性及抗损伤修复减退等非特异性表现的综合症;其对应的代谢物水平物质基础就是包含PtdCho、3-HB、Gln、Glucose、UFA、PCho等多种物质在内的生物标志物谱的改变;以上结果为肝郁证本质研究的系统生物学奠定基础。通过逍遥散干预的比较并结合药物本身对正常大鼠代谢组学的影响,找出随证候和药物治疗后变化规律的物质,推测它们更能代表肝郁证模型的代谢组学特征。本研究例证了代谢组学用于证候研究的可行性,根据代谢组学特点设置药物干预和单独药物组,可以进一步明确证候中发生相应变化的物质,为基于代谢组学技术的证候研究方法做了有益的探索。
Background
Syndrome in Chinese Traditional Medicine(TCM) is an abstraction of the collection of changes in functional status.It is a way in understanding diseases quite different from west medicine,and also the core of TCM theory and clinical practice. The Philosophy of diagnosis and treatment based on an overall analysis of the illness and tire patient's condition has always taken its effects and made progression in the years of practice.In its procedure the overall concept and individualization have been valued,and this idea is also accepted and praised highly in today's medical circles.But what is the essence of Syndrome in TCM? This question has interested so many scholars in these years.The answer wills surely impetus the development of TCM theory and clinical practice,as well as the modernization of TCM to some extend.
Liver depression syndrome is a basic syndrome in TCM,which is characteristic of liver-Qi stagnation because of the dysfunction and insufficiency of abreaction of liver-Qi.This syndrome is also usual in many kinds of diseases defined by west medicine.It is really magic that the treatment of smoothing liver-Qi will do its work when the ill is diagnosed as liver depression syndrome even if it belongs to different diseases in west medicine.This is well-known as different diseases in same treatment. We may then deduce that liver depression syndrome is a functional status of the same characters in the development of different diseases.To explore the essence of this syndrome will,as a result,give some new inspiriation in the prevention and treatment of these diseases.Furthermore,by the study of this syndrome we may develop an appropriate method in the disclosing the nature of other syndromes in TCM.
Metabonomics,which will be referred to collectively as global metabolic profiling,is emerging as an exciting post-genomic science with application that span the scope of biotechnology and medicine.Metabonomics has been defined as the quantitative measurement of the dynamic multiparametric metabolic response of living systems to pathophysiological stimuli or genetic modification.Changes in cellular endogenous metabolite levels represent not only changes in gene function, but also environment influences.Biofluid samples for metabolic profiling studies, typically urine or serum,can be collected by minimally invasive methods and require little sample preparation prior to analysis.This enables temporal data to be generated rapidly.With the same concept of entirety and individualization as in TCM, metabonomics is supposed to be a promising method to be used in the interpretation of syndromes in TCM.
Objectives:
To compare the serum metabolites from rat model of liver depression syndrome with that from normal rats so as to determine the metabolic characters and small-molecular marker of liver depression syndrome.To clarify the material basis in this syndrome,therefore supply scientific basis in the following systematic research. By this research we hope to establish a tech platform based on metabonomics with MRN,so that a new method may be achieved in the interpretation of the essence of syndromes in TCM.
Methods:
Establishment of the model rats:Twenty-four male Wistar male rats(150±20g) were randomly divided into four groups with 6 rats in each after one-day's adaptation living,which are group A(model plus Xiaoyao Powder group),group B(blank plus Xiaoyao Powder group),group E(model group) and group F(blank group).The rats from the first 3 groups were housed individually in cages while rats from group F were living in two cages averagely.Rats from group A and E were bind in a specific bound frame,which may give chronic immobilization stress every day for 4hrs or 6hrs but at unpredictable time,to simulate the cause of depressed emotion.The upset continued for 21 days,and in the restraining moment the rats were deprived from diet or water.Rats from group A and B were also given Xiaoyao Powder by intragastric administration once a day at eight for consecutive 21 days,while the other two groups 2ml physiological saline with the same ways.Dose level for Xiaoyao Powder in the rats was set according to the day dose for a normal adult;it was 10g/kg body weight everyday.We may evaluate the model by the observation of behavior,laboratory examination and the curative effects.
Primary study using Metabonomics on rat model of liver depression syndrome: Serum samples were collected from rats in group E and F at day 22 for ~1H NMR spectroscopy.The NMR data were analyzed using principal component analysis.The differently expressed materials and their possible relations with this syndrome were further analyzed.
Further study using Metabonomics on rat model of liver depression syndrome by the intervention of Xiaoyao Powder:The serums from these four groups were compared altogether by ~1H NMR spectroscopy.More attention was paid to the effects of Xiaoyao Powder on the metabolite of group A and B.The effects were then used to make a further contrast with the previous deviation between group E and F.By this way the regularity of the metabolic changing in this syndrome was more determinate and clearer.
Results:
There were abnormal behaviors,such as dodge,slackness,looser stools,and matte fur were observed among rats from E.Rats from group A also showed some above abnormolrities,but they were not obvious.Rats in group B and F lived normally.In the altogether 22 days the rats' weight increase was significantly different between groups(p=0.010).It showed rats in group E grew slowest,while rats in group A the second lowest.Fluid consumption test revealed that group E lost appetite for sweet water during the whole procession.Statistics showed there were significant differences between these four groups(p<0.001).The plasma CORT was also quite different in the four groups(p＝0.0001).Group E had the highest concentration,and group A,group B lower sequentially,while group E the lowest. Even between group A and E there was significant deference(P＝0.045).By HE staining,no pathological changes were observed in gastric mucosa and adrenal gland of group E or group A rats.However,adrenal body mass index indicated an imbalance within the groups.Group E again had the biggest figure,and the next was group A,which was improved to some extend.Based on etiological,behavior, pathological observation and the reaction to the corresponding treatment,it can be confirmed that the rat model of Liver depression syndrome was successfully established by chronic restraining.
The primary study using metabonomics on model rats of liver depression syndrome showed the method can clearly discriminate model rats(group E) and blank rats(group F).There were obvious differences in the serum metabolite between two groups.The concentration of Glucose and creatine(Cr) was higher in group E than in group F,while 3-hydroxybutyric acid(3-HB),high density lipoproteins(HLD), glutaminate(Gin),glutamic acid(Glu),lactic acid(Lac),N-acetyl-glucoprotein (NAc),O-acetyl-glucoprotein(OAc),unsaturated fatty acid(UFA),very low density lipoprotein/low density lipoprotein(VLDL/LDL),phosphocholine(PCho) and Phosphatidylcholine(PtdCho) lower than in group F.All the above materials constitute the metabolic character of liver depression syndrome.Most of them are the substrates in the energy metabolism;furthermore,among these mutative substances, Gln,UFA,VLDL/LDL,PCho and PtdCho are the basic stuff maintaining normal structure of cells;Glu,which may transfer into glutathione(GSH),as well as PtdCho have an anti-oxidant effect that may resist damages to cellules;UFA,PtdCho and Glu have a close relationship with the advancing function of nervous system;Gln,UFA, PCho,NAc and OAc are the indispensable materials to keep appropriate immune function;while NAc,OAc and UFA have a correlation with haemorheology and coagulation.So the variances of above substances indicate a pathomechanism under the condition of liver depression syndrome that includes metabolic disorder, neuroendocrine disturbance,immunodeficiency,microcirculation disturbance, decrescence in cell activity,fall-off in anti-injury and impairment in restoration.The result further confirms liver depression syndrome has a positive relationship with the occurrence of cancer and it may impair the ability of study and memory.It also gives some clues that the syndrome may have an effect on the blood clotting. Further analysis was conducted on this syndrome and its corresponding prescriptions, that is to say Xiaoyao Powder.Metabonomics shows Xiaoyao Powder has moderate effect on the blank rats,while it can alleviate the variances in model rats and make them get close to blank rats.The Loadings plot of CPMG and LED can clearly discriminate the four groups according to the first principal component,which indicates the change of metabolites is nearly monodirectional.By comparisons one by one at a time in the four groups,the metamorphic direction and extent is obtained.In the comparisons some metabolites shows no relationship with interferes of restraining or prescription,while the others has a tendency toward the normal after the usage of Xiaoyao Powder.It may deduce that these normal-oriented metabolites,which include Lac,Cr,Glucose,3-HB,Glu,VLDL/LDL,PtdCho and UFA,can better represent the metabonic characteristics of rat model of liver depression syndrome.
Conclusions:
Rat model by chronic restraining can appropriately simulate liver depression syndrome in patients.By the study using metabnomics on rat model of liver depression syndrome,we may attempt to set up a linkage between the pathogenesis of syndrome in TCM and the theory of west medicine.The vinculum of this correlation is the differently expressed metabolites.The biological marker of syndrome in TCM perhaps is not a single material but a collection of such changed substances,which we may call biomarker group.Only by this biomarker group may we clearly discriminate and determinde TCM syndromes.From this study,we may in the light of metabonomics interpreter liver depression syndrome as a syndrome based on the change of several materials related to cell metabolism includes a series of non-specific pathological changes such as neuroendocrine disturbance, immunodeficiency,microcirculation disturbance,decrescence in cell activity,fall-off in anti-injury and impairment in restoration.In this pathogenesis the corresponding material bases at metabolism level are the alternation of biomarker group that includs PtdCho,3-HB,Gln,Glucose,UFA,PCho,and so on.The results establish groundwork for the further study on the essence of liver depression syndrome by systematic biological methods.By the comparisons between rat models with or without Xiaoyao Powder,as well as the blank rats with or without Xiaoyao Powder, some of the changed metabolites are confirmed to have a variation pattern after the interfere of restraining and prescription.These materials maybe better represent the metabonomic characteristics of rat model of liver-depression syndrome.The research illustrates the feasibility of using metabonomics in the study of TCM syndrome. Setting up two additional groups designed according to the characteristic of metabonomics,which are groups interfered with drug alone or with disposal plus drug,may further confirm whether the changed materials are really realated with the syndrome.This group-setting design may be a helpful exploration in the study on TCM syndromes by metabonomics.
证候是中医学认识疾病作为一种功能状态变化的症候群,是中医学理论和临床实践的核心。辩证施治在几千年的临床实践中不断发展和验效,充分体现了整体观和个体化的治疗理念,是中医学的精髓。因此,证本质的研究对于中医理论与临床实践的发展和中医现代化具有极其重要的意义。
肝郁证是指由于肝的疏泄功能异常,疏泄不及而致气机郁滞所表现的证候,见于现代医学多种疾病,是中医脏腑病证中常见的证候之一,也是中医肝病发病学的基本证候。在不同疾病中,经中医辩证同属肝郁证的,施以疏肝理气相应方药,这样的“异病同治”确能取得较好疗效。因此,肝郁证被认为是多种疾病发展过程中的一种具有共性的“功能态”,研究肝郁证的实质,不仅是中医证本质研究的重要内容,更能为临床多种疾病的诊断和防治提供新的思路和方法;通过肝郁证这一典型证候的研究,探索证本质研究的合适方法,对闸释中医学的科学内涵有实际意义。
继基因组学、蛋白质组学发展起来的代谢组学,是研究生物体系受外部刺激所产生的所有代谢产物变化的科学方法,它迴避了复杂的生命调控过程,通过对生命体所有低分子量代谢产物进行定量、定性分析,给出最终的、整体的病理变化结果。同蛋白质组学一样,中医学两大特色——整体观和辨证论治观在代谢组学中也都有体现,通过代谢组学的方法有助于阐释中医“证”的本质。
研究目的:
比较慢性束缚肝郁证大鼠模型与正常大鼠血清差异表达的代谢物,确定肝郁证的“代谢组学特征”和小分子标志物;阐明肝郁证代谢物水平的物质基础并探讨其实质,为肝郁证本质的系统研究提供科学依据。建立证候研究的代谢组学技术平台,为从代谢物水平探讨中医证本质提供思路和方法。
研究方法:
1.慢性束缚肝郁大鼠模型的建立
动物分组:实验用清洁级雄性Wistar大鼠24只,鼠龄5周,体重(150±20)g,适应性饲养1天后随机分为模型组(E组)、正常对照组(F组)、逍遥散+模型组(A组)和逍遥散+空白组(B组),每组各6只。F组3只1笼,其余3组单笼孤养。
模型建立:参照文献,模拟中医情志抑郁的病因,将A、E组大鼠置于特制的束缚制动筒内,通过移动插条逐步缩小大鼠活动空间,调节到其不产生强烈反抗的最小空间。每日开始时间随机,每日束缚制动1次,持续时间从第1天的4h逐渐增至每天6h,连续21d。A、E组大鼠在束缚制动期间禁食、禁水。
药物干预:A、B组大鼠根据正常成人逍遥散每日用量,按体表面积换算成大鼠用药量为10g/kg.d,根据大鼠个体体重每日上午8时给A、B组大鼠经胃灌相应药量,E、F组大鼠同时以0.9%生理盐水2ml灌胃。
从行为学、实验室检查血浆皮质酮、逍遥散干预后疗效观察等方面评价模型的可靠性。
2.肝郁证代谢组学初步研究
用基于~1H NMR技术的代谢组学方法检测模型组(E组)和正常对照组(F组)血浆代谢组分差异,分析其差异物质与肝郁证可能存在的关系。
3.肝郁证方证的代谢组学研究
进一步比较A、B、E、F四组大鼠血清代谢组分差异,尤其关注逍遥散对正常大鼠(B组)和模型大鼠(A组)血清代谢组分的影响,并与E、F组间差异表达的代谢组分比较,从方证互参角度了解肝郁证中代谢组分的变化规律。
结果:
行为学观察发现E组大鼠开始表现为易激惹、挣扎,随后表现为躲避、活动减少和对外界刺激反应减少,安静时埋首、拱背,大便松散,皮色失去光泽;A组大鼠上述反应较轻,而B、F组未见异常行为状态。各组间体重增长存在显著性差异(P＝0.010),E组生长较为缓慢,A组服药后体重增长缓慢的情况得到改善。液体消耗实验提示各组间糖水偏好存在显著性差异(P＜0.001),E组大鼠对糖水的偏好率小于其余三组。第22天血浆皮质酮水平比较,各组间存在显著性差异(P＝0.0001);浓度从高到低依次为E＞A＞B＞F,且A、E组之间存在显著性差异(p＝0.005)。常规HE染色下,各组胃粘膜、肾上腺未见急性应激的病理改变,与E组比较无明显变化。四组间肾上腺体重指数比较存在显著性差异(P＝0.045),E组最高,A组有改善。分别从病因、证候、实验室病理改变和治疗后的反应四个方面验证慢性束缚肝郁证大鼠模型的可靠性。
肝郁证代谢组学初步研究发现E组、F组之间血清代谢物存在差异,E组中含量升高的有血糖(Glucose)、肌酸(Cr),含量下降的有高密度脂蛋白(HDL)、3-羟基丁酸(3-HB)、谷氨酰胺(Gln)、谷氨酸(Glu)、乳酸(Lac)、N-乙酰糖蛋白(NAc)、O-乙酰糖蛋白(OAc)、不饱和脂肪酸(UFA)、极低/低密度脂蛋白(VLDL/LDL)、磷酸胆碱(PCho)和磷脂酰胆碱(PtdCho)。上述物质的集合共同构成了肝郁证大鼠模型的代谢组学特征。这些物质大多与机体能量代谢有关;此外,Gln、UFA、VLDL/LDL、PCho和PtdCho是维持细胞正常结构的基本物质,Gln转化生成的谷胱甘肽和PtdCho还有非常重要的抗氧化、抗损伤作用;UFA、PtdCho、Glu与神经系统高级功能相关;Gln、UFA、PCho、NAc、OAc是维持机体正常免疫功能的基本物质;NAc、OAc、UFA与血液流变学和凝血功能相关。这些物质的变化反映了肝郁证状态下能量代谢紊乱、神经内分泌失调、免疫功能减弱、微循环障碍、体细胞活性及抗损伤修复减退的病理机制,并进一步提示肝郁证存在肿瘤高发危险,能导致学习、记忆能力减退;提示可能会影响凝血功能。
进一步对肝郁证方证进行代谢组学检测、分析:逍遥敞对正常大鼠血清代谢组分有轻微影响,逍遥散干预后肝郁模型大鼠血清代谢谱有所变化,向正常组靠近。CPMG、LED实验得分图显示四组大鼠沿第一主成份明显分开,提示代谢组分的变化几乎是单向的。四组间两两比较得出代谢物质变化的方向和幅度,找出随证候模型和相应方剂治疗后变化一致并趋于正常的物质,剔除那些变化与方证无关的物质,推测Lac、Cr、Glucose、3-HB、Glu、VLDL/LDL、PtdCho、UFA这8种物质也许更能体现肝郁证模型大鼠血清代谢组学特征。
结论:
慢性束缚大鼠模型能够较好的模拟肝郁证证候。通过对肝郁证大鼠模型的代谢组学研究,尝试将中医证候病机与现代医学理论联系起来,其纽带就是存在差异的物质。中医证候的生物标志物很可能不是单一物质,而是差异物质的集合,即“生物标志物群”的概念,也许才能更好的用于疾病/证候的判别分析。从代谢组学去理解肝郁证:在涉及细胞新陈代谢的多种物质改变基础上,以神经内分泌失调、免疫功能减弱、微循环障碍、体细胞活性及抗损伤修复减退等非特异性表现的综合症;其对应的代谢物水平物质基础就是包含PtdCho、3-HB、Gln、Glucose、UFA、PCho等多种物质在内的生物标志物谱的改变;以上结果为肝郁证本质研究的系统生物学奠定基础。通过逍遥散干预的比较并结合药物本身对正常大鼠代谢组学的影响,找出随证候和药物治疗后变化规律的物质,推测它们更能代表肝郁证模型的代谢组学特征。本研究例证了代谢组学用于证候研究的可行性,根据代谢组学特点设置药物干预和单独药物组,可以进一步明确证候中发生相应变化的物质,为基于代谢组学技术的证候研究方法做了有益的探索。
Background
Syndrome in Chinese Traditional Medicine(TCM) is an abstraction of the collection of changes in functional status.It is a way in understanding diseases quite different from west medicine,and also the core of TCM theory and clinical practice. The Philosophy of diagnosis and treatment based on an overall analysis of the illness and tire patient's condition has always taken its effects and made progression in the years of practice.In its procedure the overall concept and individualization have been valued,and this idea is also accepted and praised highly in today's medical circles.But what is the essence of Syndrome in TCM? This question has interested so many scholars in these years.The answer wills surely impetus the development of TCM theory and clinical practice,as well as the modernization of TCM to some extend.
Liver depression syndrome is a basic syndrome in TCM,which is characteristic of liver-Qi stagnation because of the dysfunction and insufficiency of abreaction of liver-Qi.This syndrome is also usual in many kinds of diseases defined by west medicine.It is really magic that the treatment of smoothing liver-Qi will do its work when the ill is diagnosed as liver depression syndrome even if it belongs to different diseases in west medicine.This is well-known as different diseases in same treatment. We may then deduce that liver depression syndrome is a functional status of the same characters in the development of different diseases.To explore the essence of this syndrome will,as a result,give some new inspiriation in the prevention and treatment of these diseases.Furthermore,by the study of this syndrome we may develop an appropriate method in the disclosing the nature of other syndromes in TCM.
Metabonomics,which will be referred to collectively as global metabolic profiling,is emerging as an exciting post-genomic science with application that span the scope of biotechnology and medicine.Metabonomics has been defined as the quantitative measurement of the dynamic multiparametric metabolic response of living systems to pathophysiological stimuli or genetic modification.Changes in cellular endogenous metabolite levels represent not only changes in gene function, but also environment influences.Biofluid samples for metabolic profiling studies, typically urine or serum,can be collected by minimally invasive methods and require little sample preparation prior to analysis.This enables temporal data to be generated rapidly.With the same concept of entirety and individualization as in TCM, metabonomics is supposed to be a promising method to be used in the interpretation of syndromes in TCM.
Objectives:
To compare the serum metabolites from rat model of liver depression syndrome with that from normal rats so as to determine the metabolic characters and small-molecular marker of liver depression syndrome.To clarify the material basis in this syndrome,therefore supply scientific basis in the following systematic research. By this research we hope to establish a tech platform based on metabonomics with MRN,so that a new method may be achieved in the interpretation of the essence of syndromes in TCM.
Methods:
Establishment of the model rats:Twenty-four male Wistar male rats(150±20g) were randomly divided into four groups with 6 rats in each after one-day's adaptation living,which are group A(model plus Xiaoyao Powder group),group B(blank plus Xiaoyao Powder group),group E(model group) and group F(blank group).The rats from the first 3 groups were housed individually in cages while rats from group F were living in two cages averagely.Rats from group A and E were bind in a specific bound frame,which may give chronic immobilization stress every day for 4hrs or 6hrs but at unpredictable time,to simulate the cause of depressed emotion.The upset continued for 21 days,and in the restraining moment the rats were deprived from diet or water.Rats from group A and B were also given Xiaoyao Powder by intragastric administration once a day at eight for consecutive 21 days,while the other two groups 2ml physiological saline with the same ways.Dose level for Xiaoyao Powder in the rats was set according to the day dose for a normal adult;it was 10g/kg body weight everyday.We may evaluate the model by the observation of behavior,laboratory examination and the curative effects.
Primary study using Metabonomics on rat model of liver depression syndrome: Serum samples were collected from rats in group E and F at day 22 for ~1H NMR spectroscopy.The NMR data were analyzed using principal component analysis.The differently expressed materials and their possible relations with this syndrome were further analyzed.
Further study using Metabonomics on rat model of liver depression syndrome by the intervention of Xiaoyao Powder:The serums from these four groups were compared altogether by ~1H NMR spectroscopy.More attention was paid to the effects of Xiaoyao Powder on the metabolite of group A and B.The effects were then used to make a further contrast with the previous deviation between group E and F.By this way the regularity of the metabolic changing in this syndrome was more determinate and clearer.
Results:
There were abnormal behaviors,such as dodge,slackness,looser stools,and matte fur were observed among rats from E.Rats from group A also showed some above abnormolrities,but they were not obvious.Rats in group B and F lived normally.In the altogether 22 days the rats' weight increase was significantly different between groups(p=0.010).It showed rats in group E grew slowest,while rats in group A the second lowest.Fluid consumption test revealed that group E lost appetite for sweet water during the whole procession.Statistics showed there were significant differences between these four groups(p<0.001).The plasma CORT was also quite different in the four groups(p＝0.0001).Group E had the highest concentration,and group A,group B lower sequentially,while group E the lowest. Even between group A and E there was significant deference(P＝0.045).By HE staining,no pathological changes were observed in gastric mucosa and adrenal gland of group E or group A rats.However,adrenal body mass index indicated an imbalance within the groups.Group E again had the biggest figure,and the next was group A,which was improved to some extend.Based on etiological,behavior, pathological observation and the reaction to the corresponding treatment,it can be confirmed that the rat model of Liver depression syndrome was successfully established by chronic restraining.
The primary study using metabonomics on model rats of liver depression syndrome showed the method can clearly discriminate model rats(group E) and blank rats(group F).There were obvious differences in the serum metabolite between two groups.The concentration of Glucose and creatine(Cr) was higher in group E than in group F,while 3-hydroxybutyric acid(3-HB),high density lipoproteins(HLD), glutaminate(Gin),glutamic acid(Glu),lactic acid(Lac),N-acetyl-glucoprotein (NAc),O-acetyl-glucoprotein(OAc),unsaturated fatty acid(UFA),very low density lipoprotein/low density lipoprotein(VLDL/LDL),phosphocholine(PCho) and Phosphatidylcholine(PtdCho) lower than in group F.All the above materials constitute the metabolic character of liver depression syndrome.Most of them are the substrates in the energy metabolism;furthermore,among these mutative substances, Gln,UFA,VLDL/LDL,PCho and PtdCho are the basic stuff maintaining normal structure of cells;Glu,which may transfer into glutathione(GSH),as well as PtdCho have an anti-oxidant effect that may resist damages to cellules;UFA,PtdCho and Glu have a close relationship with the advancing function of nervous system;Gln,UFA, PCho,NAc and OAc are the indispensable materials to keep appropriate immune function;while NAc,OAc and UFA have a correlation with haemorheology and coagulation.So the variances of above substances indicate a pathomechanism under the condition of liver depression syndrome that includes metabolic disorder, neuroendocrine disturbance,immunodeficiency,microcirculation disturbance, decrescence in cell activity,fall-off in anti-injury and impairment in restoration.The result further confirms liver depression syndrome has a positive relationship with the occurrence of cancer and it may impair the ability of study and memory.It also gives some clues that the syndrome may have an effect on the blood clotting. Further analysis was conducted on this syndrome and its corresponding prescriptions, that is to say Xiaoyao Powder.Metabonomics shows Xiaoyao Powder has moderate effect on the blank rats,while it can alleviate the variances in model rats and make them get close to blank rats.The Loadings plot of CPMG and LED can clearly discriminate the four groups according to the first principal component,which indicates the change of metabolites is nearly monodirectional.By comparisons one by one at a time in the four groups,the metamorphic direction and extent is obtained.In the comparisons some metabolites shows no relationship with interferes of restraining or prescription,while the others has a tendency toward the normal after the usage of Xiaoyao Powder.It may deduce that these normal-oriented metabolites,which include Lac,Cr,Glucose,3-HB,Glu,VLDL/LDL,PtdCho and UFA,can better represent the metabonic characteristics of rat model of liver depression syndrome.
Conclusions:
Rat model by chronic restraining can appropriately simulate liver depression syndrome in patients.By the study using metabnomics on rat model of liver depression syndrome,we may attempt to set up a linkage between the pathogenesis of syndrome in TCM and the theory of west medicine.The vinculum of this correlation is the differently expressed metabolites.The biological marker of syndrome in TCM perhaps is not a single material but a collection of such changed substances,which we may call biomarker group.Only by this biomarker group may we clearly discriminate and determinde TCM syndromes.From this study,we may in the light of metabonomics interpreter liver depression syndrome as a syndrome based on the change of several materials related to cell metabolism includes a series of non-specific pathological changes such as neuroendocrine disturbance, immunodeficiency,microcirculation disturbance,decrescence in cell activity,fall-off in anti-injury and impairment in restoration.In this pathogenesis the corresponding material bases at metabolism level are the alternation of biomarker group that includs PtdCho,3-HB,Gln,Glucose,UFA,PCho,and so on.The results establish groundwork for the further study on the essence of liver depression syndrome by systematic biological methods.By the comparisons between rat models with or without Xiaoyao Powder,as well as the blank rats with or without Xiaoyao Powder, some of the changed metabolites are confirmed to have a variation pattern after the interfere of restraining and prescription.These materials maybe better represent the metabonomic characteristics of rat model of liver-depression syndrome.The research illustrates the feasibility of using metabonomics in the study of TCM syndrome. Setting up two additional groups designed according to the characteristic of metabonomics,which are groups interfered with drug alone or with disposal plus drug,may further confirm whether the changed materials are really realated with the syndrome.This group-setting design may be a helpful exploration in the study on TCM syndromes by metabonomics.
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