| Objective: Along with the wide spread application of coronary intervention, coronary artery bypass, percutaneous transluminal coronary angioplasty (PTCA) and thrombolysis in clinic, the reperfusion injury has become a serious problem after coronary artery recanalization. Arrhythmia is the main clinical manifestation of ischemia-reperfusion injury. Some serious ventricular arrhythmia even threat to life. In this sense, it is of great importance to investigate the pathomechanism of arrhythmia that induced by myocardial I/R injury and the effective medicine to prevent and cure it.When preventing and curing arrhythmia induced by myocardial I/R injury, TCM is characterized by multistrata and multiangular, including cleaning up oxygen free radical, inhibiting calcium overload, affecting nitrogen monoxidum level and sympathic activity, ect. TCM has been proved to be effective in prevention and curing arrhythmia induced by myocardial I/R injury. In this study, an animal model of the myocardial I/R injury in rats was constructed by ligating and unstucking the left anterior descending coronary artery, and an intervention of complex traditional Chinese medicine named DingXin Recipe was conducted. In this study, we investigated the influence of DingXin Recipe on myocardial apoptosis and the expression of NF-κB and caspase-3 in myocardial cell. Meanwhile, proteome technique was used to detect the proteome expression spectra in animal model with arrhythmia after myocardial I/R injury and the role of DingXin Recipe, which will deeply reveal the mechanism of DingXin Recipe preventing and curing arrhythmia after myocardial I/R injury, and set a found for exploiting effective medicine for preventing and curing arrhythmia after myocardial I/R injury.Method: 30 Wistar rats were randomly divided into 3 groups: Sham operated group (SH), ischemia-reperfusion group (I/R), ischemia-reperfusion plus DingXin Recipe group (DXR). In the proteome experiment, according to the appearance of arrhythmia, DXR group was divided into two groups: the group without arrhythmia (DXRN) and the group with arrhythmia (DXRY). All the rats were fed for 2 days for accommodation. Rats in both SH group and I/R group were drenched with normal sodium, rats in DXR group were drenched with DXR physic liquor, both kinds of administration was performed twice a day, 1ml/100g.The animal model was made according to Pharmacological Empirical Methodology. First, anesthetized rats, then ligated coronary artery and gave reperfusion to induce the appearance of arrhythmia. Combined the rat with electrocardiograph and electrocardioscope to observe the occurrence of arrhythmia and made a note of arrhythmia that happened during reperfusion stage and gave a mark of ventricular arrhythmia. After the operation, took out of the heart, washed it in normal sodium, separated the left ventricle and divided it into two parts. One part was dipped into 10% neutral formaldehyde for fixation, another part was stored in refrigerator with the temperature of -80℃.The muscular tissues that fixed by formaldehyde was paraffin imbedded and made into slices. Used HE dyeing to observe the morphous changes of myocardium in each group. Detected myocardial cell apoptosis by TUNEL method. Detected the expression of NF-κB, caspase-3 by immunohistochemical method.The muscular tissues that stored in refrigerator was schizolysis with tissue lysate to extract the total protein. Quantitated myocardial protein with Bradford. Proteomic two-dimensional gel electrophoresis (2-DE) technique was used to separate total myocardial protein. IPG3- 10L, 24cm gel was used in first dimension for level ampholine electrophoresis. The second dimension was SDS- PAGE vertical electrophoresis. Silver nitrate was used to dye the gel when electrophoresis was completed. We use gel formatter to collect image. Image Master? 2D platinum version5.0 was used to analyze the image, including detecting protein spots, adjusting protein spots, decreasing background and correcting molecular weight and isoelectric point. Set the gel images in SH group as a reference, gel images in other groups was matched with them respectively. The matching was a combination of software automatic matching and artificial matching. Differential expression scope was defined as the expression amount of a kind of protein in one group was more than twice as much in another group. Searched the protein spots that has been identified from RAT HEART-2DEPAGE database in the network, and make a contradistinction and analysis of protein spots in the experiment with the above protein spots (error range: PI:±0.2, MW:±1KDa) . We also identified the similarity of some protein spots in the experiment for later use in mass spectra identification.Results: (1 )The effect of DingXin Recipe on arrhythmia induced by I/R injury in rats. In this experiment, the model of arrhythmia was successfully made, and DingXin Recipe showed a great effect on the prevention against arrhythmia induced by I/R injury. The incidence of premature ventricualr contraction in SH group was low and without other kinds of arrhythmia. In I/R group, 10 rats developed arrhythmia, mainly including premature ventricualr contraction, ventricular tachycardia and ventricular fibrillation, with a few atrial ventricular blocks. 2 rats in the group died of ventricular fibrillation. The score of ventricular arrhythmia in I/R group was significantly higher then that in SH group. In DXR group, 7 rats developed arrhythmia, mainly premature ventricualr contraction and ventricular tachycardia. The score of ventricular arrhythmia was lower compared with I/R group. (2) The effect of DingXin Recipe on the structure of myocardium in rats with arrhythmia induced by I/R injury. The observation of HE dyeing from light microscope: myocardium structure in SH group was characterized with well-arranged myocardial cells, well-distributed HE dyeing and integrated cell membrane. While in I/R group, the myocardial cells were chaotic, HE dyeing was uneven , cells in some parts of the heart were cloudy swelling, the cardiac muscle fibers was unclear or disappeared. In DXR group, some myocardial cells developed degeneration and necrosis, but it was not severe. HE dyeing was relatively even, cell hydropic degeneration was lightened, necrosis scope was small. (3) The effect of DingXin Recipe on myocardial cell apoptosis in rats with arrhythmia induced by I/R injury. Apoptosis index in VR group was significantly higher than that in SH group (P<0.01) .Apoptosis index in DXR group was lower than that in I/R group (P<0.01) . (4) The effect of DingXin Recipe on the expression of caspase-3 and NF-κB in rats that developed arrhythmia induced by I/R injury. In SH group, expression of caspase-3 and NF-κB was at a low level. While in I/R group, the expression level of the two proteins up-regulated significantly (P<0.01) . In DXR group, the expression level of caspase-3 and NF-κB was lower then that in I/R group (P<0.01). (5) Proteome expression spectra of myocardium of rats with arrhythmia induced by I/R injury and the role of DingXin Recipe. After two dimensional gel electrophoresis and silver staining, 751 + 36 protein spots were obtained in each group, most of the protein spots were centralized between PH4-7 and 20-80KD. Make VR as group as the reference, protein spots in the other two group were matched with VR group, the matching rate was 81%±4.3%. Image analysis reveled that some proteins SHowed qualitative or quantitative changes after VR injury or plus DXR. Compared SH group with I/R group, there were 33 protein spots changed, including 19 protein spots up-regulated,6 protein spots down-regulated, 6 protein spots only expressed in SH group,2 protein spots protein spots only expressed in I/R group. Compared DXRN group with I/R group, there were 36 protein spots changed, with 4 protein spots up-regulated, 23 protein spots down-regulated, 7 protein spots only expressed in SH group, 2 protein spots only expressed in DXRN. Compared DXRY group with I/R group, there were 11 protein spots changed, with 3 protein spots up-regulated, 7 protein spots down-regulated, 1 protein spots only expressed in I/R group. Compared and analyzed protein spots in the experiment with the protein spots that has been identified in RAT HEART -2DEAGE database searched from network( error range: PI:±0.2, MW:±lKDa). 5 protein spots were identified. They were SSP1883, SSP2079, SSP2211, SSP545, SSP1766, representing Fructose-bisphosphate aldolase, ATP synthase alpha chain, Creatine kinase M chain, Myosin heavy chain1 and Alpha-actin 2 respectively. These protein spots may play an important role in the pathological process of arrhythmia induced by I/R injury or in the prevention of DingXin Recipe against arrhythmia induced by I/R injury.Conclusion: (1) Arrhythmia can easily be induced by I/R injury in rats, and it can effectively be prevented by DxingXin Recipe. (2) DingXin Recipe showed great protection against myocardium histological structure destruction in rats with arrhythmia induced by I/R injury. (3) The number of apoptosis myocardium cells increased in rats with arrhythmia induced by VR injury, and DingXin Recipe could reduce the incidence of apoptosis. (4) The expression of caspase-3 and NF-κB increased in myocardium in rats with arrhythmia induced by VR injury, and DingXin Recipe could decrease the expression. (5) The myocardial proteome expression spectrain rats with arrhythmia induced by I/R injury were distinctively different from that in normal rats, and DingXin Recipe could intervene the quality and the quantity of some proteins in some extent.
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