| ObjectiveTo study the myocardial energy metabolic changes of blood stasis syndrome in Coronary Heart Disease(CHD) under the condition of acute and chronic pathogenic process as well as different factors that lead to blood stasis syndrome. And to explore the impact of different pathogenic process and different etiological factors on myocardial cell energy metabolism network model established based on the Flux Explorer platform of blood stasis syndrome in CHD.Methods(1) Theoretical research:Through the study on myocardial energy metabolism related literature, to theoretically investigate the characteristics and regulation of normal myocardial energy metabolism, and the changes in ischemic myocardial energy metabolism.Through studying on the ancient literature and modern literature related to CHD of Chinese Medcine,to explore the etiological factors and pathogenesis account for the formation of stasis obstruction syndrome in CHD.By the status analysis of research on biological metabolism network simulation, cardiovascular disease network model and syndromes network model of Chinese Medicine, to discover the characteristics and advantages of the metabolic network model.Eventually providing theoretical basis for introduction of systems biology to the syndrome essence research of Chinese Medicine. And then explore the impact of acute and chronic pathogenic process and different etiological factors including stasis obstruction, phlegm obstruction, congealing cold and qi stagnation on maocardial cell energy metabolism network model(2) Effects of different pathogenic process on myocardial cell energy metabolism and network model:(1)Through feeding high fat diet and intraperitoneal injection Vitamin D3 combined with coronary artery ligation, isoprenaline(ISO) repeatedly intraperitoneal injection to establish rat model of acute and chronic blood stasis syndrome in CHD. Extract the ischemic myocardium. Using gene chips test to investigate the gene expression profiling of acute and chronic blood stasis syndrome in CHD.Through GO enrichment analysis and pathway enrichment analysis of differential genes, finding out the differential genes and pathways involved in energy metabolism. And then select some of the energy metabolism related genes to be validated by fluorescence quantitative real-time PCR. Detecting the myocardial tissue Na+-K+-ATPase and Ca2+-Mg2+-ATPase activity and HK, LDH, CACT, ACS, CS content by enzyme-linked immuno sorbent assay(ELISA) method. Moreover, detecting the myocardial tissue metabolic profiling of acute and chronic blood stasis syndrome in CHD based on the GC/TOF-MS platform. Through multivariate statistical analysis and pathway enrichment analysis to find out differential metabolites and enriched metabolic pathways.(2)Establish myocardial energy metabolism network model of blood stasis syndrome in CHD based on the Flux Explorer platform. Obtain artery blood oxygen content of acute and chronic blood stasis syndrome in CHD by arterial blood gas analysis, along with metabolites detected from GC/TOF-MS such as the glucose and fatty acid content. Both the oxygen and the metabolites were served as energy metabolism substrates that were imported into the energy metabolism network model. Through flux balance analysis(FBA) and shadow price analysis(SPA) to explore the impact of acute and chronic pathogenic process on myocardial cell energy metabolism network model of blood stasis syndrome in CHD.(3) Effects of different factors inducing blood stasis syndrome on myocardial cell energy metabolism and network model:(1)Thrombin plus aminocaproic acid injection, atherosclerotic plaque formulation, freeze stress and tail clamp method combined with ISO intraperitoneal injection were used to establish the rat model of blood stasis syndrome in CHD caused by four different etiological factors including stasis obstruction, phlegm obstruction, congealing cold and qi stagnation. Extract the ischemic myocardium. Fluorescence quantitative real-time PCR was used to detect the expression of ME1、CPT2、ACSL1、CS、IDH3A genes involved in myocardial energy metabolism. Detecting the myocardial tissue Na+-K+-ATPase and Ca2+-Mg2+-ATPase activity and HK, LDH, CACT, ACS, CS content by enzyme-linked immuno sorbent assay(ELISA) method. Moreover, detecting the myocardial tissue metabolic profiling of different etiological factors induced blood stasis syndrome in CHD based on the GC/TOF-MS platform. Through multivariate statistical analysis and pathway enrichment analysis to find out differential metabolites and enriched metabolic pathways.(2)Establish myocardial energy metabolism network model of blood stasis syndrome in CHD based on the Flux Explorer platform. Obtain artery blood oxygen content of different etiological factors induced blood stasis syndrome in CHD by arterial blood gas analysis, along with metabolites detected from GC/TOF-MS such as the glucose and fatty acid content. Both the oxygen and the metabolites were served as energy metabolism substrates that were imported into the energy metabolism network model. Through flux balance FBA and SPA to explore the impact of four different etiological factors including stasis obstruction, phlegm obstruction, congealing cold and qi stagnation on myocardial cell energy metabolism network model of blood stasis syndrome in CHD.Results(1) Theoretical research:(1)Ischemia induced myocardial energy metabolism remodeling, causing changes in metabolic pathways refer to glucose, fatty acid, glycogen, lactic acid, etc.(2)The basic pathology of blood stasis syndrome in CHD is myocardial ischemia. And the pathogenesis of blood stasis syndrome in CHD is closely related to the myocardial energy metabolism disturbance. The pathogenesis of blood stasis syndrome is complex. However, the basic pathogenesis is "heart vessel blockage stasis", which usually induced by stasis obstruction, phlegm obstruction, congealing cold and qi stagnation, etc.(3)The physical basis of syndrome was probably consist of functionally related gene clusters or abnormal expression of the protein groups and specific metabolic components. Introducing systems biology to the study of syndrome essence may stand a chance to reveal the dynamic mechanism of syndrome more comprehensively and precisely.(2) Effects of different pathogenic process on myocardial cell energy metabolism and network model:(1)Analysis of myocardial tissue genomic data showed that most of the differentially expressed genes involved in myocardial energy metabolism were down-regulated in the acute and chronic blood stasis syndrome in CHD. And the differential genes were mainly enriched in glucose metabolism, lipid metabolism, citric acid cycle, oxidative phosphorylation, amino acid metabolism pathways. The results of fluorescence quantitative real-time PCR showed consistent with the gene chip, validating that the results of gene chip were accurate and reliable.(2)Analysis of key enzymes involved in energy metabolism pathways showed that Na+-K+-ATPase activity, LDH, CACT, CS content in the acute and chronic blood stasis syndrome in CHD were lower than the healthy control group(P<0.01 or P<0.05), while ACS content were higher than the control group(P<0.01 or P<0.05). Besides, the HK in the acute blood stasis syndrome in CHD were higher than the healthy control group(P<0.01), while the HK in the chronic blood stasis syndrome in CHD were lower than the healthy control group(P<0.01).(3)Multivariate analysis of myocardial tissue metabonomic data showed different distributed areas between acute and chronic blood stasis syndrome in CHD and healthy control group. And each model group could be distinguished from the healthy control group clearly. Analysis of differential metabolites showed that most of the differential metabolites of the acute and chronic blood stasis syndrome in CHD involved in myocardial energy metabolism were down-regulated, including carbohydrates(e.g. glucose, glucose-6-phosphate, etc.), fatty acids(e.g. oleic acid, linoleic acid, etc.), organic acids(e.g. fumaric acid, succinic acid, etc.),and amino acids(e.g. glutamic acid, etc.).Moreover, creatine, L-malic acid and allose significantly up-regulated in the three groups of acute blood stasis syndrome in CHD, while these three metabolites in the chronic group had no significant difference. In addition, maltose and glucoheptose in the acute groups appeared up-regulated, while glucose in the chronic group appeared down-regulated. Pathway analysis showed that the differential pathways involved in both acute and chronic groups including: starch and sucrose metabolism, TCA cycle, Alanine, aspartate and glutamate metabolism, amino sugars and nucleotides glucose metabolism, arginine and proline metabolism. Besides, the differential pathways in acute groups include galactose metabolism and other amino acid metabolism pathways, while the differential pathways in chronic contain unsaturated fatty acid biosynthesis, fatty acid biosynthesis.(4)Analysis of FBA showed the same variation trend of energy metabolism substrates(Gluc, FA, O2), key enzymes in glycolysis(Hk, Pfk, Pk), fatty acid oxidation(FAO, FACo AT, FAcl), TCA cycle(Cs, Icdh, Od), oxidative phosphorylation(NADHd, Sdh2) and ATP generation. That is healthy control group>group of acute blood stasis syndrome in CHD>group of chronic blood stasis syndrome in CHD(P<0.05 or P<0.01).(5)Analysis of SPA showed the sensitivity variation trend of Gluc and O2 were healthy control group>group of acute blood stasis syndrome in CHD>group of chronic blood stasis syndrome in CHD(P<0.05 or P<0.01), while he sensitivity variation trend of FA were group of chronic blood stasis syndrome in CHD>group of acute blood stasis syndrome in CHD>healthy control group(P<0.05 or P<0.01).(3) Effects of different factors inducing blood stasis syndrome on myocardial cell energy metabolism and network model:(1)Analysis of myocardial energy metabolism related genes showed ME1, CPT2, ACSL1, CS, IDH3 A in syndrome of stasis obstructing the heart vessels group, syndrome of phlegm obstructing the heart vessels group, syndrome of qi stagnation the heart vessels group in CHD were were significantly lower than those in corresponding healthy control group( P<0.01 or P<0.05). And ME1, ACSL1, CS, IDH3 A in syndrome of cold congealing the heart vessels group were significantly lower than those in C3 control group(P<0.01), while there was no significant difference between the two groups on CPT2.(2)Analysis of key enzymes involved in energy metabolism pathways showed that Na+-K+-ATPase activity, CACT, CS content in four model groups of different etiological factors induced blood stasis syndrome in CHD were significantly lower than the corresponding healthy control group(P<0.01 or P<0.05), moreover, Na+-K+-ATPase activity and CS content in syndrome of cold congealing the heart vessels group were significantly lower than those in stasis obstruction, phlegm obstruction and qi stagnation groups(P<0.01). HK and ACS in syndrome of stasis obstructing the heart vessels group, syndrome of phlegm obstructing the heart vessels group, syndrome of qi stagnation the heart vessels group were significantly higher than those in corresponding control group(P<0.01), while HK and ACS in syndrome of cold congealing the heart vessels group was significantly lower than C3 control group(P<0.01) as well as the model group of stasis obstruction, phlegm obstruction and qi stagnation(P<0.01). LDH in syndrome of phlegm obstructing the heart vessels group and syndrome of qi stagnation the heart vessels group were significantly lower than those in C2 and C4 control group(P<0.01), and LDH in syndrome of cold congealing the heart vessels group was significantly higher than those in stasis obstruction, phlegm obstruction, qi stagnation groups(P < 0.01 or P < 0.05).(3)Multivariate analysis of myocardial tissue metabonomic data showed that the four groups of different etiological factors were distributed respective areas and could be distinguished from each other clearly. Moreover, all of the four model groups could be obviously distinguished from the corresponding control group. Analysis of differential metabolites showed that myocardial energy metabolism related metabolites(e.g. glucose, fructose, maltose, etc.) were up-regulated in syndrome of stasis obstructing the heart vessels group, syndrome of cold congealing the heart vessels group and syndrome of qi stagnation the heart vessels group, while down-regulated in syndrome of phlegm obstructing the heart vessels group. Metabolites of fatty acid were up-regulated in syndrome of stasis obstructing the heart vessels group, while down-regulated in syndrome of phlegm obstructing the heart vessels group, syndrome of cold congealing the heart vessels group and syndrome of qi stagnation the heart vessels group. Pathway analysis showed that there was no shared differential pathways in four model groups of different etiological factors induced blood stasis syndrome in CHD. The differential metabolites mainly enriched in carbohydrate metabolism, lipid metabolism, amino acid metabolism pathways.(4)Analysis of FBA showed that energy metabolic substrate(Gluc) and key enzymes in glycolysis(Hk, Pfk, Pk) in syndrome of stasis obstructing the heart vessels group and syndrome of cold congealing the heart vessels group were significantly higher than those in C1, C3 control group(P<0.01), while in syndrome of phlegm obstructing the heart vessels group and syndrome of qi stagnation the heart vessels group were significantly lower than C2, C4 control group(P<0.05 or P<0.01). The energy metabolic substrates(FA, O2)and key enzymes in fatty acid oxidation(FAO, FACo AT, FAcl), TCA cycle(Cs, Icdh, Od), oxidative phosphorylation(NADHd, Sdh2) and ATP generation in four model groups of different etiological factors induced blood stasis syndrome in CHD were significantly lower than the corresponding healthy control group(P<0.01 or P<0.05).Analysis of SPA showed the sensitivity of Gluc in the syndrome of stasis obstructing the heart vessels group and syndrome of cold congealing the heart vessels group were significantly higher than those in C1, C3 control group(P<0.01 or P<0.05), while with no significant difference in phlegm obstructing the heart vessels group and syndrome of qi stagnation the heart vessels group(P>0.05).The sensitivity of FA in our model groups of different etiological factors induced blood stasis syndrome in CHD were significantly higher than the corresponding healthy control group(P<0.01), while the sensitivity of FA had the opposite variation trend(P<0.01).Conclusion(1) The pathogenesis of blood stasis syndrome is complex, moreover, the basic pathogenesis is "heart vessel blockage stasis", which usually induced by stasis obstruction, phlegm obstruction, congealing cold and qi stagnation. The pathogenesis of blood stasis syndrome in CHD is closely related to the myocardial energy metabolism disturbance.(2) Both acute and chronic blood stasis syndrome in CHD could cause changes in myocardial energy metabolism. Further more, changes of myocardial cell energy metabolism tended to increase the use of glycolysis of glucose in acute stasis syndrome in CHD. While cardiomyocytes aerobic metabolism and anaerobic metabolism were depressed under the state of chronic blood stasis syndrome in CHD.(3) Different etiological factors induced blood stasis syndrome in CHD could cause myocardial energy metabolism changes. As a whole, the variation trend was up-regulation of glucose metabolism pathway and down-regulation of fatty acid metabolism pathway. In addition, myocardial energy metabolism in syndrome of stasis obstructing the heart vessels group, syndrome of cold congealing the heart vessels group and syndrome of qi stagnation the heart vessels group tended to increase the uptake of glucose metabolic substrates, while in syndrome of phlegm obstructing the heart vessels group tended to heighten the glycolytic pathway.(4) On the whole, the established myocardial energy metabolism network model of blood stasis syndrome in CHD based on the Flux Explorer platform could accurately predict the changes of myocardial energy metabolic substrates and key enzymes in energy metabolism pathways as well as the change of ATP generation in acute and chronic blood stasis syndrome in CHD and different etiological factors induced blood stasis syndrome in CHD. The content of O2 had a key role on the objective function to generate ATP in the network model. |
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