| Objective:Along with the wide application of thrombolytic therapy, percutaneous transluminal coronary angioplasty and intracoronary stening to myocardial infarction(MI),myocardial ischemia-reperfusion after acute MI(AMI) becomes more and more popular.According to the experiments and clinic research on AMI,early myocardial ischemia-reperfusion can not only minify infract size and degrade the death rate,but also induce myocardial ischemia-reperfusion injury that causes arrhythmia,myocardial textural change,myocardial stunning and so on. Arrhythrnia is the main clinical manifestation of myocardial ischemia-reperfusion injury,and serious ventricular arrhythmia is the important cause of cardiogenic sudden death,so,it is very significant to study the mechanism of the arrhythmia induced by myocardial ischemia-reperfusion injury and to find the effective medicine to prevent and cure it.Drug treatment is now still the main therapy for arrhythmia though operation and electronic machine have been greatly developed in western medicine.Long-term taking antiarrhythmic drug always bring some adverse reaction,one kind of which the arrhythmia induced by antiarrhythmic drug,have been highly thought of by pharmacist and physician.Inversely,Traditional Chinese Medicine against arrhythmia is characterized by multistrata,multiangular,multitarget and has no significant adverse reaction.So,in this research,by models of the arrhythmia induced by myocardial ischemia-reperfusion injury in rats,we studied the influence of DingXin Recipe and Oxymatrine on arrhythmia,the level of intercellular adhesion molecule-1(ICAM-1) and proteome expression spectra through proteome technique. Through the above-mentioned studies,we expect to reveal the molecular mechanism of arrhythmia induced by myocardial ischemia-reperfusion injury,and the mechanism of DingXin Recipe(DXR) and Oxymatrine(OMT) on arrhythmia,and provide evidence for preventing and curing arrhythmia in clinic induced by myocardial ischemia-reperfusion injury.Methods:32 adult male Wistar rats were randomized into 4 equal groups:Sham operated group(SH),Ischemia-reperfusion group(I/R),DingXin Recipe group(DXR) and Oxymatrine group(OMT).All the rats were fed for 2 days for accommodation. Rats of SH,I/R and OMT groups were drenched with normal sodium,and rats in DXR group were drenched with DXR physic liquor.After successive intragastric administration for 7 days,the rats of I/R,DXR and OMT groups accepted operation, and the rats of SH group also accepted the operation except no ligation and reperfusion.We made models of arrhythmia according to Pharmacological Empirical Methodology.Rats were combined 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 above-mentioned operation,took blood from abdominal aorta and then prepared serum.Eventually,took out of the heart,washed it in cool normal sodium and separated the left ventricle,then stored it in refrigerator with the temperature of -80℃.Arrhythmia induced by myocardial ischemia-reperfusion injury mainly was ventricular arrhythmias(VA),including premature ventricualr contraction(VE), ventricular tachycardia(VT) and ventricular fibrillation(VF),so we gave a mark of ventricular arrhythmia based on the method of Curtis.We detected ICAM-1 of Serum from abdominal aorta by ABC-ELISA,and observed the effect of DXR and OMT on expression of serum ICAM-1.Total protein was extracted from partial myocardium with lysate,and was quantitated by the method of Bradford.After quantitation,we separated total protein by two-dimensional gel electrophoresis(2DE) technique.IPG3-10L,24cm gel was used in first dimension for level ampholine electrophoresis,and the second dimension was SDS-PAGE vertical electrophoresis.Then Coomassie Brilliant Blue was used to dye the gel when electrophoresis was completed,and images were collected by Umax PowerLook 1100.We analyzed images by PDQuest 8.0 of Bio-Rad company,and software automaticly gave information of differential protein spots.We cut partial differential spots and sent them to Genome Research Centre of the University of Hong Kong.Peptide Mass Fingerprints(PMF) obtained by matrix assisted laser desorption/ionization time-of-flight mass spectrometry/mass spectrometry (MALDI-TOF MS/MS) were searched in NCBInr by Mascot to identify differential protein spots.Total protein was extracted from partial myoeardium with the kit of Western and IP cell lysate for Western blot.We separated total protein by SDS-polyacrylamide gel electrophoresis(SDS-PAGE),transferred total protein to PVDF film after SDS-PAGE, blocked,and incubated antibodies.Then we collected images by KODAK Image Station 2000MM,and analyzed images by Image Tool 3.0 to observe the expression level of fatty acid binding protein(FABP). Results 1.The effect of DXR and OMT on arrhythmia induced by myocardial ischemia-reperfusion injury in rats.By statistics analysis,the score of arrhythmia in different groups had significant difference in total(χ~2=20.614,P=0.000＜0.01).The incidence of premature ventficualr contraction in SH group was low and without other kinds of arrhythmia.In I/R group,arrhythmia occurred in 8 rats,and started to appear when coronary artery was perfused again for 1 minute,mainly including premature ventricualr contraction,ventricular tachycardia and ventricular fibrillation, with a few atrial ventricular blocks,and 1 rat in this group died of ventricular fibrillation.The score of ventricular arrhythmia in I/R group was significantly higher than that in SH group(P=0.000＜0.01).In DXR group,arrhythmia also occurred in 8 rats,mainly premature ventricualr contraction and ventricular tachycardia,and the score of ventricular arrhythmia was lower compared with I/R group(P=0.013＜0.015) In OMT group,arrhythmias were also mainly premature ventricualr contraction and ventricular tachycardia,and there was only premature ventricualr contraction in 2 rats the score of ventricular arrhythmia was also lower compared with I/R group(P= 0.005＜0.01).2.The effect of DXR and OMT on expression of serum ICAM-1.the content of serum ICAM-1 had significant difference by statistics(F=4.233,P=0.014＜0.05).we found that compared with SH group,the content of serum ICAM-1 in I/R group rose obviously,and difference had statistics significance(P=0.007＜0.05);due to the role of DXR and OMT,the content of serum ICAM-1 decreased,and difference had statistics significance(P=0.003,0.049＜0.05),but compared DXR with OMT, difference had no statistics significance(P=0.241＞0.05).3.Changes of protein expression in arrhythmia induced by myocardial ischemia-reperfusion injury and the effect of DXR and OMT.After reperfusion, compared with I/R group,SH group has 71 differential spots,DXR group has 73 differential spots,and in the 71 differential spots of I/R group and SH group,32 differential spots recovered due to the role of DXR;OMT group has 84 differential spots compared with I/R group,and 30 differential spots recovered due to the role of OMT.After advanced comparison,8 differential spots had similar changes due to the role of DXR and OMT.4.Identify partial differential proteins.We identified acquired 5 differential proteins,and obtain five PMFs.These PMFs were respectively identified successfully as 4 proteins after searching database by Mascot:albumin,prohibitin,FABP, mitochondrial aldehyde dehydrogenase(mtALDH).5.Verify the expression of FABP.For verifying the expression of FABP,we detected FABP of myocardium by western blot,and found that the expression of FABP in four groups had significant difference(F=12.007,P=0.002＜0.05);compared with SH group,the expression of FABP in I/R group rose significantly(P= 0.000＜0.05),but due to DXR and OMT,the expression of FABP obviously decreased(P=0.007,0.002＜0.05),which were similar to the results of 2DE.Conclusions 1.Arrhythmia can easily be induced by I/R injury in rats,and it can effectively be prevented by DXR and OMT.2.The expression of serum ICAM-1 was obviously degraded by DXR and OMT, and the occurrence of arrhythmia induced by myocardial ischemia-reperfusion injury was lessened.3.Arrhythmia induced by myocardial ischemia-reperfusion injury,DXR and OMT had effect on maps of protein expression of myocardium.4.After arrhythmia induced by myocardial ischemia-reperfusion injury,proteins related with myocardium energy metabolism and the clearance of free radical had changed obviously.5.Verification of protein expression confirmed that the technology platform of 2DE and mass spectrometry we established was relatively stable,and our study was credible by means of technology platform. |
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