| Objective:Molecular biology techniques and Gas Chromatography and Mass Spectrometry (GC-MS) metabolomics is used to reveal the different biological basis of heart dysfunction with different syndromes based on the myocardial ischemia model of integrative disease and syndrome. Yixinjiedu formula which has exact clinical efficacy is given during time window of the model qi deficiency and blood stasis syndrome (QDBS), thus to reveal mechanism of the pharmacological effect of the compound. At the same time, the possible drug targets of Yixinjiedu formula is predicted by its compounds using bioinformatics techniques, and the results are validated by molecular biology techniques, to provide new ideas and methods for Chinese herbal formula target prediction and pharmacological mechanism.Methods:1. Myocardial ischemiafunctional insufficiency animal model is preparation through the left coronary artery ligation. Syndromes are judged by the collection of dynamic observation and behavioral indicators at different disease phrase.2.Enzyme-linked immunosorbent assay is used to detect the concentrations of plasma TNF-alpha, IL-1, IL-6, MMP2 and MMP9 and other indicators in 4d,7d,21d,45d,60d; Pearson correlation analysis is applied to reveal the role of TNF-alpha in the evolution of syndromes in coronary heart disease;3. GC-MS metabolomics technology is used to detect the serum metabolites of myocardial ischemia cardiac insufficiency with different syndromes (including qi deficiency and qi deficiency and blood stasis syndrome); and the principal component analysis (PCA) method is applied to investigate the characterized metabolites of myocardial ischemia with different syndromes;4. The Yixinjiedu formula is given during the myocardial ischemia heart dysfunction when qi deficiency and blood stasis syndrome happens. Through a combination of observations of symptoms and detection of microscopic physical and chemical indicators, the efficacy of the Yixinjiedu formula is evaluated comprehensively both form the disease level and the syndrome level; 5. The key node molecular which is closely related to myocardial ischemia heart dysfunction is investigated by literature research, and then molecular biology techniques such as ELISA, IHC, WB and other methods are applied to detect these proteins, thus to reveal the multi-target, multi-channel treatment characteristics of Chinese formula pharmacological mechanism in treating coronary heart disease;6. We present an idea that multi-target for herbs should be investigated by combing bioinformatics and experimental verification to finally determine drug targets. Firstly, herbal components are investigated by data mining from database. Secondly bioinformatics is applied to predict the drug target for all compounds based on the principle that similar structure has similar function in FDA and Drugbank database. Then bioinformatics including Go function analysis and KEGG pathway analysis are used to look for the pathway the proteins belong to. Finally, experimental verification is made to confirm how and what the herbs work on the body, thus to provide a credible method to investigate the complicated multi-target mechanism of herbs.Results:1. The syndromes were judged through a combination of observations of symptoms and detection of microscopic physical and chemical indicators. The results showed that model prepared by the left anterior descending coronary ligation performs as the stable qi deficiency and blood stasis from 7d to 28d, but the following 45d to 60d, this model turned to qi deficiency syndrome. Overall the syndrome evolution has occured from qi deficiency and blood stasis to qi deficiency syndrome.2. Elisa detection results showed that:7-60 days after the surgery, development of the syndrome transformed from'blood stasis and qi deficiency'to'qi deficiency'. The TNF-a was over-expression and lasted all over the pathological progress. At the stage of blood stasis and qi deficiency, the concentration of TNF-a was up-regulated with the increase of inflammatory cytokines IL-1 and IL-6, however, during the stage of qi deficiency in 45-60d, the inflammatory down-regulated to normal level. But it mainly performed as cardiac hypertrophy and heart dysfunction, including the heart function decreasing, EF value was below 50%, MMP-2\MMP-9 which closely related to cardiac hypertrophy increased by 9.91% and 20.57% respectively. We can see that different syndrome had different pathology in same disease. TNF-a first mediated the inflammation, and then the cardiac hypertrophy and heart dysfunction. We can conclude that it may be the TNF-a which mediate the development of myocardial ischemia syndromes.3. After operated for 4 d, blood metabolites have changed significantly between model and sham group. CAMP, Citric acid in energy metabolism, amino metabolites such as Glycine, a-D-glucopyranoside in glucose metabolism, lipid metabolites such as Octadecanoic acid were up-regulated. Amino metabolites such as Urea andL-Serine, d-Glucose in glucose metabolism and lipid metabolites such as Butanoic acid were down-regulated.After operated for 21d, blood metabolites have also changed significantly between model and sham group. Citric acid in energy metabolism; amino metabolites such as Urea and L-Serine; glucose metabolites such as a-D-glucopyranoside and lipid metabolites such as Hexadecanoic acid,Octadecanoic acid levels have increased. While the energy metabolites CAMP and Amino metabolites such as Glycine decreased.After operated for 45d, blood metabolites still have changed significantly between model and sham group. L-Serine and Butanoic acid went up. Energy metabolites such as Citric acid and CAMP, amino metabolites such as Urea and L-Serine, glucose metabolites such as a-D-glucopyranoside and lipid metabolites such as Hexadecanoic acid and Octadecanoic acid were down-regulated.4. Efficacy evaluation in disease level:in hemodynamic aspect, Yixinjiedu and the positive drug could well improve arterial SBP and DBP, and can effectively improve arterial MAP (P <0.05);in ventricular hemodynamic aspect, the Yixinjiedu formula and fosinopril sodium can increase heart failure in rats LVSP and LV+dP/dt max in LV-dP/dt max, thus improving the blood hemodynamic conditions of rats with heart failure.The blood chemistry results showed that compared with the sham group, the plasma myocardial enzyme of rats in model group such as alanine aminotransferase (ALT), aspartate aminotransferase (AST), serum creatine kinase (CK), creatine kinase isoenzyme (CK-MB), lactate dehydrogenase (LDH), troponin I (cTnl) increased significantly. After given the Yixinjiedu formula, it can effectively down-regulate these myocardial enzymes, corresponding improvement was also found in regulating the heart enzymes when compared with positive drug control group.The Yixinjiedu formula and fosinopril sodium could reduce the plasma AngⅡconcentration, and reduce plasma BNP levels in heart failure rats (P<0.05). At the same time, the results of lmmunoturbidimetry showed that the serum hs-CRP decreased significantly (P<0.05), indicating Yixinjiedu formula has anti-inflammatory effect.Efficacy evaluation of the syndrome level:the Yixinjiedu formula Treatment reduced the rat plantar skin and scrotal skin of the R value, G value, B value (P<0.05). The RGB values to the plantar skin and scrotal skin effectively reflected the rat skin blood stasis, indicating that the Yixinjiedu formula had a significant improvements in the rat plantar skin, dark purple, reducing the related characterization of the rat skin, dark purple. Respiratory rate results showed that when compared with the sham-operated rats, the respiratory rate of model rats in the quiet state was significantly accelerated, the Yixinjiedu formula can effectively slow down the rats with heart failure breathing frequency (P<0.05). When compared with the model group, positive drug group has no significant difference in improving respiratory rate of heart failure rats.The ultrasound results showed that the Yixinjieud formula group could be effective in reducing heart failure rat expansion LVEDd and the LVEDs (decreased by 22.10% and 28.57%, P<0.05), especially in improving the end-systolic inner diameter. And in further improving the EF (P<0.05); Fosinopril sodium can only improve EF value in heart failure rats.Its effects on LVEDd and LVEDs were not obvious, and had no statistical difference.5. Indicators of blood detection results show that in regulating the expression of inflammatory cytokines aspect, after given Yixinjiedu formula for 28 days, the levels of inflammatory cytokines TNF-alpha, IL-6 in the serum significantly decreased. It also can effectively reduce the circulating MDA levels and increasing SOD levels, thus to inhibit oxidative stress.Myocardial tissue index test results showed that Yixinjiedu formula could reduce the MMP9, MMP2 expression in myocardial tissue. Simultaneous inhibition of the myocardial tissue RAAS activity and significant resisted the oxidative stress injury. It could significantly inhibit NADPH oxidase including NOX2 and NOX4 expression; inhibit the pathway protein of STAT3 and of NFκB expression;6. Drug targets are predicted base on the composition of the herbs. The results show that the compound's pharmacological mechanism mainly focus on the nervous endocrine link, amino acid metabolism, energy metabolism and other process, RAAS is selected to validate. The animal model and drug intervention are taken to deeply validate. Yixinjiedu formula has not only the effective role in the reduction in the myocardium and the circulation of the renin of AngⅡlevel of ATI R, but also proved that the multi-targets regulation mechanism of Chinese medicine.Conclusion1. Within 60 days after surgery, the myocardial ischemia heart dysfunction artery model induced by left coronary artery ligation has a performance as "qi deficiency→qi deficiency and blood stasis→qi deficiency syndrome evolution; and lasting high TNF-a expression mediated myocardial ischemia syndromes in evolution through different pathological processes of inflammation and matrix remodeling.2. In the process of the syndrome evolution "qi deficiency→qi deficiency and blood stasis→qi deficiency syndrome", the plasma metabolites have characteristic changes; in the myocardial ischemia ventricular dysfunction disease states, sugar, fat, amino acid metabolism disorders, eventually leading to the TAC pathway abnormalities, in which the specific molecular abnormalities of citric acid, a-D-glucopyranoside, lactic acid, Octadecanoic acid expression may become the target of Myocardial ischemia in clinical diagnosis and treatment Data mining method to find the specific model can be used to the clinical Qi deficiency and blood stasis diagnosis and evaluation of traditional Chinese medicines.3. The experimental results show that Yixinjiedu formula can improve cardiac function, hemodynamic indicators and reduce various types of cardiac enzymes and myocardial protection. Besides it also can improve the characterization level of indicators, such as rats breathing exercises, claws, skin and dark purple.4. Establishment of myocardial ischemia with qi deficiency and blood stasis regulation mechanism marked by the core of NADPH oxidase activity, through the intervention of Yixinjiedu formula to prove that the network regulation of the compound depends on the core roles of the NADPH oxidase enzyme, which RAAS, the MMPs network equilibrium and the oxidative stress injury are the key effect points.5. By a combination of bioinformatics and experimental validation, complex the target prediction system is applied to establish the molecular mechanism of Chinese herbal formula. Yixinjiedu formula is selected because its ensuring efficacy, to clarify the multi-targets, multi-pathway mechanism of the prescription. Through this study, we have expected to establish a reliable and effective way for traditional Chinese medicine complex, multi-target mechanism pharmacological efficacy research methods. |
PDF Full Text Request