| Cardiovascular diseases are the leading causes of death globally, and ischemia reperfusion injury as the major cause of acute myocardial infarction attracts much attention. Proteomic analysis is very important for exploring the inherent mechanism of ischemia reperfusion injury and seeking for effective targets for drugs.This study investigated the mechanism of ischemia reperfusion injury and protective effects of STVNa in rat in-vivo hearts by differential proteomics, and came to preliminary conclusions that ischemia and reperfusion injury caused a metabolic switch and protective effect of STVNa could attribute to the increase of fatty acid metabolism. Specifically, we established three models:(1) ischemia: ligated left anterior descending of the coronary artery(LAD) for 30 min(three rats for one model as biological replicates, n=3);(2) reperfusion: ligated LAD for 30 min and loosened LAD for 120 min(n=3);(3) STVNa postconditioning: treated with STVNa from jugular vein while loosening LAD for 120 min(n=3). To reduce the individual difference, the ischemia and normal area form the same heart was used as self-control. We used nondenaturing one-dimensional gel electrophoresis(1DE)–grid cutting–quantitative liquid chromatography-tandem mass spectrometry(LC-MS/MS) techniques:(1) soluble proteins were extracted, separated by nondenaturing micro 1DE;(2) grid gel-cutting, i.e. 1DE gel strips(1.1 mm × 42-44 mm × 1 mm) were indiscriminately and equably cut into 1.1 mm × 1.1 mm squares during the whole length(two gel strips from one PAGE gel as analytical replicates, n=2);(3) proteins in all the squares were subjected to in-gel digestion, peptides extraction and label free quantitation standard added;(4) extracted peptides were applied to LC-MS/MS analysis and label free quantitation;(5) summarized the species and quantity of all the proteins, and reconstructed the quantity distribution of the 1DE gel strips.Each proteomic sample contained 1300-1900 species of proteins, quantity range of proteins covered 5 magnitudes, and each protein had its specific quantity distribution(i.e. peptide map). Data processing procedures:(1) extracted the same proteins that both existed in analytical replicates(n=2);(2) compared the differential proteins in ischemia and normal area from the same rat heart(both existed and fold-change ≥ 1.5 or ≤ 0.67 as upregulated or downregulated proteins);(3) compared the differential proteins in different individuals(n=3) between the same model, and extracted proteins which had the same variation trend in at least two individuals. Results showed that ischemia, reperfusion and STVNa postconditioning respectively leaded to 100, 392 and 397 proteins differentially expressed, and upregulation/downregulation of proteins respectively had 23/77, 178/214 and 166/231.Preliminary analysis results showed that differentially expressed proteins mainly focused on glucose metabolism, fatty acid metabolism, mitochondrial respiratory chain and oxidative stress response. Ischemia caused some key enzymes in fatty acid oxidation and mitochondrial respiratory chain downregulated. Reperfusion caused some key enzymes in fatty acid oxidation upregulated and some key enzymes in fatty acid transport downregulated, some key enzymes in glucose metabolism upregulated, suppressed mitochondrial respiratory chain, and increased some key enzymes of energy synthesis(such as creatine kinase). Meanwhile, reperfusion could result in further injury of structural constitution and the emergence of inflammation and apoptosis factors compared to ischemia. The protective effect of STVNa on heart reflected that it could increase the expression of some key enzymes(or proteins) involved in fatty acid and glucose metabolism, respiratory chain, oxidative phosphorylation and structural constitution. All these results suggested that ischemia and reperfusion injury caused a metabolic switch, and reperfusion injury could cause further cell structure injury, inflammation and cell apoptosis. Compared to reperfusion, STVNa not only could stabilize cell structure, but also significantly improved the fatty acid metabolism, respiration and oxidative phosphorylation levels. It implied that STVNa improve the energy pathway transformation caused by ischemia and reperfusion injury.In conclusion, this study aimed at discovering the mechanism of ischemia reperfusion injury and protective effects of STVNa in rat heart.(1) Established SD rat heart ischemia, reperfusion and STVNa postconditioning models, and designed 3 × 3 animal grouping;(2) conducted nondenaturing differential proteomic analysis of ischemia and normal areas in each individual;(3) preliminary analysis of the big proteomics data, raised the lists of differentially expressed proteins and possible mechanism about ischemia and reperfusion injury, raised a preliminary conclusion that STVNa protected hearts by increasing fatty acid metabolism and stabilizing cell structure. |
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