[Discovery and preliminary verification of direct targets of active ingredients of Shuangshen Ningxin Capsules in preventing and treating myocardial ischemia-reperfusion injury].

摘要

Based on the magnetic bead "fishing" technology, this study explored the direct targets of the active ingredients of Shuangshen Ningxin Capsules in preventing and treating myocardial ischemia-reperfusion injury. Three active ingredients of Shuangshen Ningxin Capsules, namely dehydrocorydaline, salvianolic acid B, and ginsenoside Rg_1, were selected based on previous work. Firstly, three complexes of magnetic bead-single active ingredient of Shuangshen Ningxin Capsules were synthesized. Then, they were incubated with the protein solutions of SD rat heart tissue, mouse myocardial microvascular endothelial cells, and rat H9c2 cardiomyocytes separately to form magnetic bead-active ingredient-target protein complexes. The target proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE), and then in-gel digestion was performed. The proteins were identified by high-resolution mass spectrometry. Potential target proteins were screened by bioinformatics analysis and molecular docking. A rat model of myocardial ischemia-reperfusion injury was established, and the biological function was verified preliminarily through Western blot experiments. The results showed that three complexes of magnetic bead-TCM monomer were successfully prepared, and the potential direct targets of the three active ingredients of Shuangshen Ningxin Capsules were obtained through the "fishing" method. Through mass spectrometry identification and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway enrichment analysis, it was found that most of the target proteins were involved in metabolic pathways related to glucose, fatty acids, amino acids, and energy. After Venn analysis and molecular docking, proteins such as electron transfer flavoprotein subunit alpha(ETFA), pyruvate kinase M1/M2(PKM), acyl-CoA dehydrogenase medium chain(ACADM), ATP synthase F1 subunit alpha(ATP5F1A), isocitrate dehydrogenase(NADP~+) 2(IDH2), and aspartyl-tRNA synthetase(DARS) were finally screened out. Through the rat myocardial ischemia-reperfusion injury model, it was found that Shuangshen Ningxin Capsules significantly up-regulated the expression of ATP energy production-related proteins such as ATP5F1A, ETFA, 3-oxoacid CoA-transferase 1(OXCT1), and succinate-CoA ligase GDP-forming subunit beta(SUCLG2). This suggested that restoring mitochondrial energy supply was an important aspect for Shuangshen Ningxin Capsules to exert myocardial protective effects. In conclusion, ATP5F1A, ETFA, OXCT1, SUCLG2, etc. may be the direct targets of the active ingredients of Shuangshen Ningxin Capsules in preventing and treating myocardial ischemia-reperfusion injury.