Application Of Serum Metabonomics On An Experimental Rat Model Of Liver Depression Syndrome

BackgroundSyndrome 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.