| Objective: Here, we investigate the myocardial protein expressionfollowing ischemia-reperfusion (I/R) injury in rats using proteomictwo-dimensional gel electrophoresis (2-DE) technique combined withsearching the 2-DE database on the web, and obverse the effects ofcarvedilol (CVD), which functions as a cardio-protective agent, on themyocardial protein expression. According to the experimental results, wefurther analyze the potential pathologic mechanisms of the myocardial I/Rinjury and the pharmacologic mechanisms of CVD for cardio-protection. Methods: An animal model of the myocardial I/R injury in rats wasconstructed. The I/R injury rats were subjected to 30min of ischemia byligation of left anterior descending branch of the coronary artery(LAD)and 2h of reperfusion by release of the ligation. CVD was givenintragastrically (5mg/kg·d) for 7 consecutive days before I/R. Rats weredivided into three groups: the sham-operated group (Sham group), I/Rinjury group (I/R group), and CVD treatment group (CVD group). Serumcreatine kinase-MB (CK-MB) and malondiadehyde (MDA) weredetermined. To improve the resolving power, based on the knowledge ofthe characteristics of cardiomyocyte, subfraction rich in cytosolic proteinsextracted from the whole cardiac tissue homagenates was used as aresearch sample. After its protein concentration was quantified and nodifferential extraction among groups was confirmed, the subfraction wastwo-dimensionally separated by isoelectric focusing (IEF) and SDS-PAGEvertical electrophoresis. After being silver stained, 2-DE images (3 ratseach group, produced 3 images) were acquired following that gels werescanned. By the help of PDQuest software, scanned images underwent spotdetection, spot matching, and then quantitative or qualitative analysis ofdifferential expression of protein spots between groups. The reproducibilityof 2-DE images were evaluated. Spot identification was performed throughsearching and comparing of the ready-identified spots' data in rat'sventricle 2-DE database on the web. Results: (1) Serum MDA was remarkably increased after I/R injury(P<0.05), but its level was significantly decreased following pre-treatmentwith CVD (P<0.01), while serum CK-MB level was not changed amonggroups (P>0.05). (2) On average, 453±66 valid protein spots appeared inthe gels, and 278 spots were matched to every gel of the match-set with 365spots matched in all gels of Sham group, 326 spots in I/R group, 392 spotsin CVD group respectively. The average matching rate was 76%. Averagecorrelation coefficient among groups and samples was 0.81 and 0.74respectively. The reproducibility analysis of spot quantity showed acoefficient of variation to be 41.7%~56.2%. The reproducibility analysis ofspot position revealed a average deviation of 1.30mm in IEF direction and1.21mm in SDS-PAGE direction. (3) The quantitative analysis of proteinspots displayed that 8 protein spots were differentially expressed after I/Rinjury with all of them down-regulated, while 9 protein spots weredifferentially expressed after pre-treatment of CVD with 5 protein spotsup-regulated and 4 protein spots down-regulated. The qualitative analysisof protein spots showed that 1 protein spot was specially expressed inSham group but no protein spot in I/R group when Sham group wascompared with I/R group, while 3 unique protein spots presented in I/Rgroup and 1 protein spot in CVD group when I/R group was compared withCVD group. (4) Two differentially expressed protein spots were identifiedto be superoxide dismutase (SOD) and transthyretin (TTR) precursorrespectively through the 2-DE database searching. SOD was significantlydown-regulated after I/R injury, while TTR precursor was significantlyup-regulated after pre-treatment with CVD (P<0.05). Conclusions: (1) Serum CK-MB level is not changed after I/R injurybecause of the short duration of I/R injury. The oxidative stress in I/Rinjury may play an important role in the heart damage, while pre-treatmentwith CVD can function as an anti-oxidative stress resulting in preventingthe injury of hear...
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