Research On The Animal Model Of Blood Stasis (Myocardial Ischemia) And Its Related Proteome

Since syndrome is the core theory of Chinese medicine, the revealing of its scientific connotation is considered as the key point to the modernization of Chinese medicine. However it depends on the breakthrough on the biology basis research of syndromes. As other biological researches, animal models are necessary. Considerd to the lack of recognized standards, there is a huge controversy in practical ways to establish or evaluate syndrome animal models. so, an objective and admitted standard is necessary. We will try to determine its syndromes'attributes based on dynamic observation and strict evaluation on a disease animal model, which may be a practical way to establish an integrated disease and sydrome animal model with more reproducibility and better accordance to the theories of Chinese medicine.Meanwhile, research on the biological basis of syndromes encountered a bottleneck due to the aims to find single indicator for the diagnosis of syndromes in the past years. However syndrome is an integritical concept and its scientific content may need an overall review of the organism. There is a beam of hope now attributing to development of systematic biology research techniques and methods. Plasma is one of the most informative bodily fluids, as it provides a link between human organs, tissues and cells. Because of its important role in vital movement, there is great information of the overall physiological and pathological conditions in plasma. Therefore, the plasma proteomic research may provide some evidence for the protein patterns of syndrome.Blood stasis is the most common syndrome type of coronary heart disease, and there were many related researches before. So the research on the animal model of blood stasis syndrome can be used as the starting point. The time window of blood stasis in a myocardial ischemia model was determined depend on the dynamic observation, and then the characteristics of its plasma proteome was explored.Aims:To establish a stable blood stasis (myocardial ischemia) animal model, and find the characteristics of its plasma proteome.Methods:1 Establishment and assessment of a blood stasis model with myocardial ischemiaChinese experimental mini swines (25±4 kg) were instrumented with a size-matched Ameroid constrictor on the anterior descending branch under general anesthesia in sterile conditions. And a sham group was also included. Based on early results of dynamic observations, evaluations were performed four weeks after operation. Clinical performances of animals were collected and electrocardiogram, echocardiography, coronary angiography were detected. Meanwhile, blood was obtained from former cava vena to detect blood rheological features and the concentration of prostacyclin, thromboxane A2, endothelin-1 and calcitonin gene-related peptide. Then the diseases of model animals were diagnosed and their syndromes were differentiated.Then, blood stasis animals with myocardial ischemia were randomly divided into therapeutic group and control group (n = 6). Nine gram Danqi tablet were administrated once a day to a therapeutic animal for 4 weeks. At the end of experiment, to determine the diagnosis of control animals and the effect of Danqi tablet, all animals were evaluated in the same way as which performed 4 weeks after operation. After sacrificed, myocardial samples destined to histological evaluation were fixed in 4% phosphate buffered paraformaldehyde, routinely dehydrated and embedded in paraffin. Then serial sections were stained with HE stain and Masson's trichrome stain to evaluate the general morphology.2 Research on plasma proteome of swines of blood stasis with myocardial ischemiaBlood was obtained from the animals with blood stasis syndrome in myocardial ischemia and matched sham operated animals 4 weeks after operation. Plasma samples were centrifuged and then stored at -80℃.All plasma samples were quantitated by Bradford assay, and then separated by two-dimensional electrophoresis (2-DE) to identify differentially expressed proteins. Precast IPG strips (pH3-11 nonlinear, 24 cm) were used in the first dimension, and SDS-polyacrylamide gel electrophoresis (PAGE) was performed using 1 mm thick, 13% SDS-PAGE gels. The Coomassie Brilliant Blue R350-stained 2-DE were scanned with digital scanner and analyzed with ImageMaster 2D Platinum 6.0 software. A cluster of protein spots differentially expressed were selected and identified with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS).After 4 weeks'therapy of Danqi tablet, plasma samples were obtained from animals in therapeutic, control and sham operated group, and then analyzed by 2-DE as described above to evaluate the differentially expressed proteins got from previous experiment.Results:1 Establishment and assessment of a blood stasis model with myocardial ischemia1.1 Evaluations performed 4 weeks after operation1.1.1 Clinical performances:Model animals appeared mental stress, irritation, fear, violent behaviors, strong self-defense, pilose disorderly and lack of luster, slightly dark red or purple tongue, etc, while sham operated animals'performances gradually returned to normal, with increased appetite, neat and shiny pilose, light red or light white tongue.1.1.2 ECG:More than 0.1mV ST segment depression and inversion T waves were observed in several leads of surface electrocardiogram of model animals. But no significant arrhythmia was found in experimental period.1.1.3 EchocardiographyThe structure and function of left ventricular were evaluated by echocardiography. Compared with sham operated animals, there was great increase of end-systolic volume (P < 0.05), end-diastolic volume (P < 0.05), end-systolic diameter (P < 0.01) and end-diastolic diameter (P < 0.01) in model animals. Anterior wall thickness of both papillary muscle and apex level was decreased at end-diastolic and end-systolic in model animals (P < 0.01). Meanwhile, the apex of the left ventricular anterior systolic wall thickening decreased (P < 0.01). In addition, septal thickness at the end-systolic decreased (P < 0.05). All the results above showed a segmental dysfunction of the left ventricular. There were no significant changes in stroke volume, ejection faction, fraction of shortening and E/A, which suggested that cardiac function was still in compensatory period.1.1.4 Coronary angiographyExvivo angiography performed more than 90% occlusion or even completely block of the left anterior descending artery by the Ameroid constrictor in model animals. TIMI flow gradeⅠorⅡwere observed in most model animals. There was good filling the in left anterior descending artery with TIMI flow gradeⅢin Sham-operated animals.1.1.5 HemorrheologyCompared with sham-operated animals, the blood viscosity of model animals increased significantly at a shear rate of high (P < 0.05), mid (P < 0.01), low (P < 0.01). No other changes were observed.1.1.6 Other changesCompared with sham-operated animals, the concentration of 6-K-PGF_1α in plasma decreased significantly(P < 0.05), while other factors observed did not have significant change.1.2 Evaluations performed 8 weeks after operationCompared with sham-operated group, the most changes of model group detected at 4 week still existed at 8 weeks except the clinic performances. Model animals seemed recovering a little, such as appetite, urine and stool. But the other characters were also persistent such as disorderly and lackluster clothing hair, slightly dark red or purple tongue, low dose of 6-K-PGF1α, changes of ECG, echocardiography and hemorrheology, etc.1.3 Effect of Danqi tabletDue to treatment of Danqi tablet, red blood cell deformation was improved(P < 0.05), elevated volume of packed red blood cells was decreased(P < 0.05), and the decreased 6-K-PGF1αwas recovered(P < 0.05), when compared with model animals. Compared with sham oprated animals, the septal thickness of model animals at the end-systolic decreased (P < 0.05) while that of thearaputic animals didn't.The main lesion of model animal heart observed in HE staining were disarranged, swell, degeneration, necrosis myocardial cells, and infiltrated inflammatory cells. These lesions were improved in slices of therapeutic animals. In the Masson trichrome-stained slices of ischemia region of model animals'heart, increased collagen in intercellular, infiltrated inflammatory cells, injured myocardial cells with vacuolar degeneration, disarranged structure, or displaced by collagen were observed. Meanwhile in the slices of therapeutic animals, the number and size of the collagen hosted region were less.2 Differential express of plasma proteome in model animals10 up-regulated protein spots were detected in the 2-DE separation of pooled samples, while 9 up-regulated in single plasma sample test. Among these differential expressed proteins, 1 was existed in both tests. MS results suggest that the unregulated proteins includingα1-acid glycoprotein,α1-antitrypsin, fetuin A, apolipoprotein A-IV, apolipoprotein A-I, Complement C4, complement cytolysis inhibitor. Other changes were still unknown.Part of the differential expressed proteins detected in 4weeks'animal models were still differential ones with different relative concentration in 8 weeks'models. However, significant improvement was observed only in part of these proteins of Danqi treated animals.Conclusions:1 The Chinese experimental miniature swine with Ameroid constrictor around the left descending artery were performed as blood stasis syndrome with myocardial ischemia between 4 and 8 weeks after operation. Danqi tablet improved some indexes observed, which provided other evidences of the diagnosis.2 The detected differential expressed plasma proteins of animals with blood stasis syndrome were associated with inflammatory reactions. Thus, we supposed that inflammation may be one of the important pathologic processes in blood stasis syndrome. However, their exact relationship was still unknown.The proteomic feature of syndrome may be some differential expressed proteins and their different assemblies.Danqi tablet only showd effect on part of differential expressed proteins, which suggested that some of these proteins were not effective targets of Danqi tablet.