Abstract 532: Protein Analysis of Tongxinluo-modulated Cytokines in Cardiac Microvascular Endothelial Cells via Tandem Mass Tag Quantitative Proteomics After Ischemia/Reperfusion Injury

Abstract

Background: Proteomics is a potential tool to study the large-scale expression, function and interaction of the complement of proteins in an organism. In this study, we used the TMT-labeled proteomics to detect the various cytokines in an in vitro model of cardiac microvascular endothelial cells (CMECs) ischemia/reperfusion injury with Tongxinluo(TXL) treatment. Our aims are to investigate whether TXL could modulate the secretion function of CMECs, and to synthetically analysis the underlying mechanism of the regulation. Methods: Human CMECs were exposed to different concentrations of TXL, and incubated to scavenge free oxygen for 2 h of hypoxia and were moved to normal conditions for 2 h of reoxygenation. Cell apoptosis was assessed by flow cytometric analysis. CMECs were divided into three groups for TMT-labeled proteomics analysis: CMECs in normal condition (Group N), CMECs in hypoxia and serum deprivation condition (Group HR), CMECs treatment with TXL in hypoxia and serum condition(Group HR+TXL) . We utilized bioinformatics analysis to identify differential proteins. Results: TXL concentration-dependently decreased apoptosis of CMECs. The optimal concentration of TXL to have the maximum protection for CMECs was 800 μg/mL. Both hypoxia/reoxygenation and TXL treatment significantly changed the cytokines level of CMECs. 32 differential proteins between group N and group HR were detected. TXL treatment up-regulated 6 cytokines and down-regulated 6 cytokines in ischemia/reperfusion injury. These proteins mainly had vital functions such as cell proliferation, stress response, regulation of multicelluler organismal metabolic process. We evaluated several proteins played important roles in ischemia/reperfusion injury including Human Heme Oxygenase 1 (HMOX1), angiopoietin 2 (ANGPT2), sequestosome 1 (SQSTM1), and connective tissue growth factor (CTGF). Conclusion: The study presented differential proteins responsible for ischemia/reperfusion injury through TMT-labeled proteomic analysis. We assessed some vital proteins including their characters and roles. These findings may provide new mechanisms of TXL treatment in acute myocardial diseases.