| BACKGROUNDVentricular remodeling (VR) is the state cardiac injury or overload the heart-shaped after Acute myocardial infarction (AMI), Appear the change of complex molecular signals and re-expression of embryonic gene and protein, resulting in maladaptation of myocardial hypertrophy, myocardial apoptosis and myocardial extracellular matrix fibrosis, Clinically manifested as ventricular hypertrophy, increased ventricular volume and changed ventricular structure.With the the development of reperfusion technology in recent years, the mortality rate of patients with MI has decreased significantly, which means a large number of patients will enter a recovery phase after myocardial infarction, which makes the prevention and treatment of VR after MI has become the primary problem of treatment. The further development of VR after MI can lead to heart failure, sudden death, is the most important risk factor of long-term cardiac death. Therefore, prevention of VR is the key to reduce the incidence of heart failure after myocardial infarction.Proteomics is a high-tech platform to study the proteins related to a variety of life events,and their dynamic changes in activities of law. It refers to a full set of proteins expression of a genome, a cell or tissue. Currently, the technical system proteome research include protein separation and spectra of preparation based on two-dimensional gel electrophoresis (2D-PAGE), Computer-aided two-dimensional gel electrophoresis spectrum analysis, Identification of protein spots of biological mass spectrometry and database search techniques, Proteome database construction and functional analysis. The technology system has been applied to many areas of life science research range from proteome mapping, Identification of the protein composition to the research of cell differentiation and development and other important life events, and medical research to find the target molecules and analysis. And began to use on the gene mutation and some important pathological processes of disease. Through proteomics research, comparative study of protein expression systems map in different states, quantitative analysis of the protein changes. It can dynamic monitoring the complex biological functions of system, diagnose and treat disease early at the molecular level; find the specific molecular markers of disease; Investigate physiological and pathological processes and mechanisms of some major diseases. Protein is the implementation of a number of body functions in life activity, Each live form is specific protein groups appear at different time and space, and function of different combinations of results. Proteome response the dynamic evolution of protein expression in the disease process from the overall level, which has great similarity of traditional Chinese medicine.ObjectiveThe subject to explore the target of Buyanghuanwu Decoction anti VR by Analyzing the differences protein expression profile between model group and Buyanghuanwu Decoction, revealed the mechanism of Buyanghuanwu Decoction against VR, to provide a basis for further study of mechanism of Buyanghuanwu Decoction against VR. Methods1. Establishment of the animal modelAdoption SPF level of male Wistar rats, Mask inhalation anesthesia of isoflurane, tracheal intubation, connecting animal respirator. Cut the skin layer by layer, open the chest, exposing the heart. Ligation the left coronary artery department under The left coronary artery origin 1～2mm (sham-operated rats only open the chest without coronary artery ligation). Standard limb lead II ST-segment elevation, after ligation area myocardium white, exhausted the air and blood out of cavity, closed chest, the skin sutured. After surgery,check electrocardiogram every 10 minutes, Continuous ST-segment elevation 3 times is a successful model of acute myocardial infarction. To penicillin 40000 U, intramuscularly,1/d, total 3d.2. Preparation of Buyanghuanwu DecoctionThe proportion of traditional Chinese medicine according to the original book, Herbs casserole container, adding the equivalent volume of 5 times the amount of herbs soaked in water 2 h, Boil 30 min, filtered liquid; Then add 5 times the amount of water to boil 20 min, filtered, merge the two filtrate, The liquid condensed to the concentration of 1 g/ml, save backup at 4℃.3. Grouping and TreatmentModeling before and after modeling, 1w,2w,4w,12w using Doppler ultrasound morphology index (LVDd, LVDs, LVPWd, LVPWs) and systolic function parameters (EF, FS) of rat heart. The difference between left ventricular end diastolic diameter (△LVDd)> 5mm as a criteria to determine whether left ventricular remodeling, Eligible rats were randomly divided into 3 groups:Model group, Buyanghuanwu Decoction group, captopril group; with sham operation group, a total of 4 groups,12 in each group, Altogether of 48. Buyanghuanwu Decoction(13.6g/kg·d)group, and DDB (10ml/kg·d) Group were given daily via gavage for a period of 12 weeks. The control group and model group were given an equal volume of saline.4. Dynamic observation of Structural changes in rat heart by DopplerAdoption Doppler observe the structural changes of rat heart, the frequency of feeler is 12 MHZ; use classic left ventricle long axis view, apex cordis fore chambers heart cross section, five chambers heart cross section, at the same time recording ECG results. R wave represent end-diastole, T wave represent end-systole, carefully observed the shape of the heart and the motion rate of ventricular wall. Index of morphology concludes:left ventricular end diastolic dimension (LVDd) and end systolic diameter (LVDs), left ventricle end-diastole posterior thickness (LVPWd). Index of contractile function concludes:ejection fraction (EF), shortening fraction (FS).5. HE staining for histological observationRats were sacrificed, the rapid separation of the heart, neutral formalin fixed tissue, Paraffin were sliced with a Leica-RM2025 Wax Slicer, HE staining. methods: Dewaxing tissue sections, Immersed in hematoxylin dye 5-10min (depending on the situation of dye), distilled water rinse,1% hydrochloric acid alcohol solution separations 15s, distilled water rinse,1% of the ammonia back to blue 1min, distilled water rinse, Immersed in eosin dye 3～5min, distilled water rinse,70%,80%,95%, 95%,100%,100% gradient ethanol dehydration, lmin each, xylene 2 times, each 3min, Neutral rubber seal slide, observed under light microscope.6. Immunohistochemical detection of myocardial collagen typeⅠ,ⅢSlice off wax, PBS rinse 3min×3, eliminate endogenous peroxidase with 3 %H2O2, PBS rinse 3min×3, Repair antigen 5min, down to room temperature naturally, PBS rinse 3min×3. add the first antibody,37℃overnight, PBS rinse 3min×3. Adding the second antibody-HRP polymer 37℃for 20min, PBS rinse 3min×3, DAB (new configuration, room temperature)color 15min, hematoxylin-stained nuclei, Coverslip generally, observed. Separate negative staining on the photo, the first antibody replaced by PBS.7. To screen differentially expressed proteins using proteomics technologiesProteins in the liver tissue of the rat were extracted with histolysis step by step, and the attained proteins were separated by two-dimensional electrophoresis (2-DE),with colloidal staining. Using immobilized PH gradient isoelectric focusing as the first, SDS-PAGE as the second, prepared by two-dimensional electrophoresis spectrum.8. Identification of Proteins by Mass SpectrometryThe differential depressions of proteins were analyzed by using mass spectrometry, after peptide mass fingerprint obtained, search the corresponding protein in SwiSS-PROT/TrEMBL database using Mascot software.9. The statistics method.Statistieal software SPSS13.0 was used to analyze the experimental data.The experimental data were indieated by mean±standard deviation(x±s).One-way ANOVA was used to analyze the data.LSD method was used in multiple comparisons between groups when variance was homogeneous.Dunnett’sT3 was used when variance is not homogeneous. Kruskal Wallis was used to analysis the difference of the stage of liver fibrosis. Difference is significant when P<0.05.Results:1. The escape case of experimental animal24h after ligation, one rat of a model group dead because of cardiac rupture, the other one dead because of cardiac failure,10 experiment rats rest in model group. There are two rats dead in respiratory failure and cardiac failure at 72h after ligation, 10 experiment rats rest in captopril group. There is no death in Buyanghuanwu Decoction group. Totally there are four rats escaped,44 rats enter our experiment. 2. The effect of Buyanghuanwu Decoction on cardiac structure and function of rats with VR after MIUsing Doppler detection of cardiac structure and function of rat before modeling Id and after modeling, 1w,2w,4w,12w. The results showed that Buyanghuanwu Decoction could improve LVDd、LVDs, LVPWd、LVPWs、EF、FS of MI in rats,it has function to anti-VR.3. Histological observationThe results can be seen that myocardial wall of the sham group is thin and lumen is smooth finishing, myocardial cells lined, no significant changes in the nucleus, chromatin clear, and the cell cross-sectional area is small; Compared with the sham group the myocardial wall of rat in model group is thick, lumen is irregular, myocardial cells loosely arranged, ill-defined edges, fuzzy chromatin and cell cross-sectional area is relatively large; Myocardial tissue of Buyanghuanwu Decoction group and captopril group improved significantly compared with model group.4. The effect of Buyanghuanwu Decoction on theⅠ,Ⅲcollagen expression of myocardial tissueSham group, collagen typeⅠ,Ⅲcollagen expression was weak, while in model group was significantly higher than the sham group. Compared with model group, Buyanghuanwu Decoction group and captopril groupⅠ,Ⅲcollagen expression was significantly lower than the model; Buyanghuanwu Decoction group and captopril group was not significantly different.5. Screening of different proteins and mass spectrometrySelected out seven protein spots whose expression significant difference, identified seven kinds of proteins. Three kinds of protein (Myosin light chain 4, Myosin regulatory light chain 2, Atrial natriuretic factor) in the model group were higher than sham group and Buyanghuanwu Decoction group. Four kinds of protein (Heat shock protein 20, Peroxiredoxin-6, Regucalcin, NADH dehydrogenase) in the model group were lower than the sham group and Buyanghuanwu Decoction group, the highest expression is the Buyanghuanwu Decoction group.Conclusions:1. the rat of the left coronary artery ligation-induced acute myocardial infarction, during 12 weeks of regular feeding can occur cardiac hypertrophy, heart dysfunction, myocardial collagen remodeling and so on, which can be used as research models of ventricular remodeling.2. Buyanghuanwu Decoction could improve cardiac function, reduce myocardial collagen remodeling and maintaining myocardial morphology, have equivalence with captopril.3. the expression of 7 proteins were significantly different in model group,the sham group and Buyanghuanwu Decoction group. the expression of 3 different proteins are increased, the expression of 4 different proteins are reduced, the expression changes in these proteins may be associated with protective effects of Buyanghuanwu Decoction.The specific mechanisms of Buyanghuanwu Decoction against VR after MI still need further study. |
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