| Objective:To establish a stable and repeatable disease and syndrome combination animal model on myocardial infarction rats, and to quest the biomarkers through the application of label free quantitative proteomics strategy.To inquiry the variation of protein expression during the syndrome and syndrome transformation of "Qi deficiency-Qi deficiency and Blood stasis-Qi deficiency", in order to provide the new proof for the biological basis, the new biomarkers, and the new therapeutic target for the syndrome of Qi deficiency and Blood stasis after acute myocardial infarction.To check against the formation and to provide the basis to the determination of therapeutic targets of Chinese medicine, which based on the proteins cluster according to the expression changes resulted from the detection, identification, and the analysis by the bioinformatics technology on the myocardium proteomics of the model animals.Methods:1. The Data mining technology based on the Shannon entropy mutual information were。used to analysis the complicated correlations of the statistical distribution of coronary heart disease syndromes associated physical and chemical indexes.2. SD rats were subjected to either proximal LAD ligation or sham operation.20 rats (10 in each group) were subjected to forced swimming test (FST) to measure the exhaustive swimming time in every time point. Echocardiography was used to evaluate the cardiac function in rats at each time point, and the respiratory rate in both peaceful state and post-stress were counted. After the images of ear concha, foot plant and facies lingualis were took, the color indexes were analyzed by Photoshop software, and the hemorheology were measured at the same time. LARS was subjected to calculate the correlation between the color and hemorheology.3. Label-free nanoscale LC-MS quantitative proteomics was applied in this study to inquiry the variation of protein expression during the syndrome and syndrome transformation of "Qi deficiency-Qi deficiency and Blood stasis-Qi deficiency" on the myocardium of myocardial infarction model animals at 4th,14th,28th,45th after operation. Fuzzy Clustering, GOslimviewer, and KEGG signaling pathway was used to do the annotation of time course expression, molecular function, cellular component, biological process, and the signaling pathway of the differential proteins.4. Western blotting was used to corroborate the proteomic results. Results:1.7 in 13 syndrome factors including qi deficiency,blood stasis,turbid phlegm,yin deficiency,cold coagulation,yang deficiency,qi stagnation which have been most researched are involved in about 134 physical and chemical indexes mentioned in the literature.The top 10 physical and chemical indicators of each syndrome factors were obtained after analysis calculation.Echocardiography is the most relevant indicator of Qi deficiency,and hemorheology and blood stasis has the highest correlation.2.Compared to sham operation group,the respiratory rate in the peaceful state was slower,but faster post stress,and the cardiac function of rats is significant decreased in model group during the period from 4th day to 60th day.The exhaustive swimming time is much shorter,and both color and hemorheology indexes showed significant differences between the two groups during the period from 7th day to 28th after operation.LARS showed that there was significant correlation between color indexes and results of hematocrit and platelet aggregation rate.All the results shows that the myocardial infarction rats induced by LAD ligation have Qi-deficiency indications,can be diagnosed as the syndrome of Qi-deficiency at 4th day；have both Qi-deficiency and Blood stasis during the period of 7th day to 28th day after operation, can be diagnosed as Qi-deficiency and Blood stasis syndrome；and only have Qi-deficiency indications,can be diagnosed as Qi-deficiency syndrome during the period of 45th day to 60th day,after operation.3.HE staining and myocardium in situ cell death detection showed that myocardial necrosis is the major pathological change in the infarct area at different time points after myocardial infarction, and infarct border zone in left ventricular showed a series of pathological changes as inflammatory cell infiltration,apoptosis,and cell hypertrophy.4.The myocardium proteome analysis showed that samples were identified in 8 groups of 526 proteins.Among which 467 proteins in the four time points were identified.45 different proteins may be asso-iated with"Qi deficiency-Qi deficiency and Blood stasis-Qi deficiency" evolution-related.The Fuzzy clustering,GO functional annotation,and KEGG cell signaling pathway annotation showed these proteins include cytoskeletal proteins, metabolic enzymes,oxidative stress related proteins and ion channel protein.They might play important roles in maintain cell structure,increase cardiac contractility,regulate intracellular energy metabolism,induce apoptosis and intracellular signal transduction.5.The dynamic changes of the expression of actin, alphaB crystallin, L-lactate dehydrogenase,and heat shock protein 8 which was tested by the Western blotting assay was concordant with the label-free quantitative proteomic results.Conclusion:1.The rats underwent acute myocardial infarction caused by the left coronary artery ligation showed different syndromes in different stages after operation.The animal model can be used in the biological basis research and Pharmacological studies of Chinese medicine on Qi deficiency syndrome or Qi deficiency and blood stasis syndrome in the myocardial ischemia caused by coronary artery disease.2. Myocardial proteins results reflected the progress of the disease and myocardial ischemia syndrome evolution. The dynamic changes of cardiac proteins related to the syndromes dynamic evolution in myocardial ischemic disease.
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