Based On Metabonomics Technology To Find The Potential Biomarkers And Metabolic Pathways Of Three Syndromes In Rats With Chest Obstruction

Chest painful impediment(Xiongbi),a traditional Chinese medicine(TCM)disease name,is closely related to coronary atherosclerotic heart disease(myocardial ischemia and angina pectoris)in modern medicine.Modern "myocardial ischemia" and"coronary heart disease" belong to the chest painful impediment for traditional Chinese medicine.According to" Guidance for new drugs of traditional Chinese material medica 2002",it is divided into eight major syndrome types:Qi Stagnation and Blood Stasis syndrome(QSBS),Qi Deficiency and Blood Stasis syndrome(QDBS),Cold Obstruction and Qi Stagnation(COQS),Cardio-blood Stasis syndrome,Cardio-kidney Yin Deficiency syndrome,phlegm and heart pulse syndrome,Yang deficiency and Qi and Yin Deficiency syndrome.The clinical morbidity of QSBS,QDBS,and COQS syndrome are high,and they are very concerned about by research scholars.At present,most scholars from the relationship between single or several biochemical indicators to study different syndromes.Syndromes have features such as integrity,dynamics,the same syndromes of different diseases and the same disease of different syndromes.Each TCM syndrome is a complex phenotype composed of many etiological factors and difficult to elucidate though a single physiological and biochemical data.Metabonomics is an important part of systems biology.It can improve the scientific and quantitative diagnosis of TCM syndromes,and obtain biochemically expressed terminal information.It has the analysis function of "group","cluster",and"spectrum" to solve the syndrome problem.Metabolomics can systematically analyze the overall state of the body and build a data model based on the massive information obtained from different individual samples.Metabonomics can be used for multi relational,multi angle measurement,classification and prediction.Following the principle of entirety and dynamic,we get the syndrome related potential biomarkers and metabolic pathways.In our study,an ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry(UPLC-Q-TOF/MS)was used to classify QSBS,QDBS,and COQS syndromes as well as finding potential biomarkers for the corresponding syndromes and related metabolic pathways.The research progress of QSBS,QDBS,and COQS syndrome based on metabonomics technology:We reviewed domestic and foreign literatures in recent years,combing summary QSBS,QDBS,and COQS model preparation method.Multi-long-term restraint stimulation combined with subcutaneous epinephrine injection and epithelial subcutaneous injection of isoproterenol to replicate QSBS animal models;long-term forced exhausted swimming and superimposed subcutaneous injection of isoproterenol QDBS animal model;using external factors of cold interference and superimposed subcutaneous injection of isoproterenol can better reproduce COQS animal model.We summarized the research reports of metabonomics on three syndromes.Metabonomics technology was widely used in many fields such as pharmacodynamics research,toxicity assessment,and disease diagnosis of new drugs.As a systematic research method,it can play a role in the identification of pharmacological and disease models.It can systematic and comprehensive reflect changes in metabolites of biological organisms.Metabonomics methods were used to find the abnormalities of metabolites among different TCM syndromes in patients with chest painful impediment.Metabonomics technology is the basis of this research.Therefore,it can be summarized in detail to better understand how metabonomics technology is applied to our research.By reviewing the research of other scholars on the use of metabonomics for the three syndromes,it is can provide systematic documentation and reference for the follow-up work of the experiment.The main research contents and results of this article are as follows:1.Establishment of QSBS,QDBS,and COQS syndrome model and basic index detectionThe rats with QDBS,QSBS and COQS syndromes were used as study subjects to compare the blood viscosity,blood lipids,myocardial enzyme,electrocardiogram,pathological section of heart and blood microcirculation of different TCM syndrome.For QSBS,QDBS and COQS,the corresponding Guan-xin-dan-shen formula,Tong-xin-luo formula and Guan-xin-su-he formula were selected for drug intervention.Comparing changes in serum lipids,myocardial enzymes and pathological sections after drug intervention.Based on the myocardial ischemia model,combined restraint,forced exhaustion swimming and freezing modeling techniques to establish the three syndrome models(QSBS,QDBS,and COQS).QSBS model:After feeding the basal diet for a week to adapt to the animal room environment,the rats were transferred to feed with high fat diet for two week.Rats based on the traditional depression,liver Qi stagnation of depression,by the chronic binding up(4 h once a day above for total of 14 d)with adrenaline subcutaneous injection(0.8 mg·kg-1)to simulate QSBS model rats.QDBS model:After feeding the basal diet for a week to adapt to the animal room environment,the rats were transferred to feed with high fat diet for two week.Thus,the model of QDBS was established based on the Chinese medicine theory of overwork depleting Qi,which treated with exhaustive swimming exercising once a day above for total of 14 d,so that they were in a chronic state analogous to QDBS.COQS model:After feeding the basal diet for a week to adapt to the animal room environment,the rats were transferred to feed with high fat diet for two week.The model of COQS was duplicated in rats by whole body freezing method and continuous freezing(4 h once a day above for total of 7 d)in the cold environment of-20℃.Myocardial ischemia(MI)model was established by repeated subcutaneous injections of small dose ISO for three consecutive days(2,4,4 mg·kg-1,i.h.).Finally,QSBS QDBS,and COQS were received the same dose of ISO as the MI group.Then the rats with QSBS QDBS,and COQS were used to determine the blood viscosity,blood lipids,myocardial enzyme,electrocardiogram,pathological section of heart and blood microcirculation of TCM syndrome type.The results of electrocardiogram,myocardial enzymes,HE staining and pathological sections showed that the ischemic model of each group was successful.The results of blood lipids indicated that the three syndromes of blood showed thick and sticky state.The increase rate of high,middle and low shear rates in the QSBS and QDBS group were greater than the COQS group,and the blood stasis effect was significant.The results of microcirculation indicated that cold stimulation had a significant effect on the establishment of model,and the modeling process of QSBS and QDBS produced microcirculation obstacles.After intervention with corresponding drugs,indicators such as blood lipids,myocardial enzymes,and cardiac pathology were adjusted back.Explain that the model was successfully established from the aspects of simulated etiology,symptom performance,testing of basic indicators.2.Study on three potential biomarkers of chest painful impediment syndrome based on metabonomicsPlasma metabonomics based on ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry(UPLC-Q-TOF/MS)were developed to explore the metabolic changes associated with TCM syndrome.The processed UPLC-MS data list was transferred to SIMCA-P software package.Principal component analysis(PCA)and partial least-squares-discriminant analysis(PLS-DA)were used to perform the separation by visualizing the score plot.The orthogonal partial least squares discriminant analysis(OPLS-DA)model was used to screen metabolites that contributed to the discrimination of different groups.In the univariate and multivariate analysis of statistical significance,P<0.05 and variable importance for projection(VIP)>1,respectively,were set as the screening criteria for potential markers responsible for the discrimination of different groups.28 differential metabolites were identified as potential markers associated with QSBS syndrome;21 differential metabolites were identified as potential biomarkers associated with QDBS syndrome;23 differential metabolites were identified as potential biomarkers associated with COQS.The potential biomarkers of three TCM syndromes overlapped in the Venn diagram and identified three characteristic TCM biomarkers.Based on the identified characteristic biomarkers,a metabolic pathway closely related to the corresponding syndrome was found.Three potential biomarkers of TCM syndromes are metabolically disordered in amino acid metabolism,fatty acid synthesis,fatty acid β oxidative metabolism,bile acid metabolism,glycerophospholipid metabolism,arachidonic acid metabolism,fatty acid amide metabolism,and vitamin B6 metabolic pathways.Cysteine and methionine metabolism,tryptophan metabolism,and alanine,aspartate and glutamate metabolism are the main pathways for metabolic disorders of QSBS.A series of amino acid metabolic disorders are associated with QSBS syndrome.Arachidonic acid metabolism,fatty acid metabolism,fatty acid β oxidative metabolism and vitamin B6 metabolism are the main pathways for metabolic disorders with QDBS.The synthesis and degradation of lysine,the metabolism of fatty acids,and the metabolism of glycerophospholipids are the main pathways for the COQS metabolic disorder.Amino acid metabolites can provide a substrate for energy metabolism.Energy metabolism disorders may lead to poor blood run,blood stasis into stasis.Energy metabolism disorders seriously affect the occurrence and development of QSBS.The inflammatory reaction caused by arachidonic acid,the disorder of energy metabolism caused by fatty acid amides and vitamin B6 metabolism affects the disorder of energy metabolism,resulting in blood stasis due to deficiency of blood and impediments to the development of QDBS.Both amino acid metabolism and fatty acid metabolism seriously affect energy metabolism.The two parts of the energy metabolism disorder affect the occurrence and development of COQS.Lysophosphatidylcholine involved in the metabolism of glycerophospholipids plays an important role in inflammatory diseases,indicating serious disorder of energy metabolism and inflammatory response are closely associated with the occurrence of COQS.3.Reverse verification of the three chest painful impediment syndrome models using the "certificate corresponding to prescription" formulaMetabonomics based on UPLC-Q-TOF/MS were carried to conduct "the corresponding prescription" drug intervention research for QSBS,QDBS and COQS syndromes.In order to reveal the characteristics of the corresponding metabolic pathways,three animal models of the TCM syndrome were validated using the "anti-evidence" model.On this basis,the unique and common potential biomarkers of the three types of TCM syndromes were analyzed in depth.The three kinds of chest painful impediment syndromes were characterized from the perspective of metabonomics,and their internal rule were explored.Three kinds of chest painful impediment syndrome models were induced by different compound factors.For QSBS,QDBS and COQS,the corresponding Guan-xin-dan-shen formula,Tong-xin-luo formula and Guan-xin-su-he formula were selected for drug intervention.Plasma metabonomics based on UPLC-Q-TOF/MS was used to analyze the plasma samples.Identifing the clustering trends of the possible abnormal samples and samples with dimensionality reduction model.The discriminant analysis method were used to classify and reverse the animal models.Compared with the model group,the metabolite profile of the drug administration group was adjusted back to the blank control group.The drug counter-evidence model was effective,and the corresponding biomarkers for the identified TCM syndrome had a trend of recovery.After the drug intervention,the level of most potential biomarkers affected by the three syndromes identified can be corrected,and the disorder of the corresponding metabolic pathways involved in the biomarker is improved.The disorder of amino acid metabolism in the QSBS is improved.The deficiency of ATP synthesis due to disease causes the disturbance of energy metabolism to be alleviated,and the poorly running of blood due to energy running disorders is alleviated.QSBS syndrome due to energy running disorders caused by the poor performance of the blood is relieved.The disorder of fatty acid metabolism in QDBS is improved.The obstacles to energy function are relieved,and the symptoms of blood stasis caused by qi deficiency and inability to promote blood flow obstacles are alleviated.The lysine degradation and anabolism in COQS are improved.The energy metabolism disorder is improved after drug intervention,and the cold stimulation causes energy metabolism disorder,resulting in impaired energy function after drug intervention.The blood stagnation symptoms are relieved.The successful establishment of the model was verified by the"certificate of correspondence" counter-proof method,and the reliability of the identification of biomarkers was verified at the same time.