Supplementary Material for: Quantitative Proteomics Analysis of Ischemia/Reperfusion Injury-Modulated Proteins in Cardiac Microvascular Endothelial Cells and the Protective Role of Tongxinluo

<i>Background:</i> The protection of endothelial cells (ECs) against reperfusion injury has received little attention. In this study, we used Tandem Mass Tag (TMT) labeling proteomics to investigate the modulated proteins in an <i>in vitro</i> model of cardiac microvascular endothelial cells (CMECs) subjected to ischemia/reperfusion (I/R) injury and their alteration by traditional Chinese medicine Tongxinluo (TXL). <i>Methods:</i> Human CMECs were subjected to 2 h of hypoxia followed by 2 h of reoxygenation with different concentrations of TXL Protein expression profiles of CMECs were determined using tandem mass spectrometry. We evaluated several proteins with altered expression in I/R injury and summarized some reported proteins related to I/R injury. <i>Results:</i> TXL dose-dependently decreased CMEC apoptosis, and the optimal concentration was 800 µg/mL. I/R significantly altered proteins in CMECs, and 30 different proteins were detected between a normal group and a hypoxia and serum deprivation group. In I/R injury, TXL treatment up-regulated 6 types of proteins including acyl-coenzyme A synthetase ACSM2B mitochondrial (ACSM2B), cyclin-dependent kinase inhibitor 1B (CDKN1B), heme oxygenase 1 (HMOX1), transcription factor SOX-17 (SOX17), sequestosome-1 isoform 1 (SQSTM1), and TBC1 domain family member 10B (TBC1D10B). Also, TXL down-regulated 5 proteins including angiopoietin-2 isoform c precursor (ANGPT2), cytochrome c oxidase assembly factor 5 (COA5), connective tissue growth factor precursor (CTGF), cathepsin L1 isoform 2 (CTSL), and eukaryotic elongation factor 2 kinase (LOC101930123). These types of proteins mainly had vital functions, including cell proliferation, stress response, and regulation of metabolic process. <i>Conclusions:</i> The study presented differential proteins upon I/R injury through a proteomic analysis. TXL modulated the expression of proteins in CMECs and has a protective role in response to I/R.

**背景**：内皮细胞（Endothelial Cells, ECs）对抗再灌注损伤的保护作用尚未得到足够关注。本研究采用串联质量标签（Tandem Mass Tag, TMT）标记蛋白质组学技术，在缺血再灌注（Ischemia/Reperfusion, I/R）损伤的心脏微血管内皮细胞（Cardiac Microvascular Endothelial Cells, CMECs）体外模型中，探究受调控的蛋白及其受中药通心络（Tongxinluo, TXL）调控的变化情况。
**方法**：将人源心脏微血管内皮细胞置于缺氧环境2小时，随后在不同浓度通心络干预下复氧2小时。采用串联质谱技术检测心脏微血管内皮细胞的蛋白质表达谱。本研究对缺血再灌注损伤中表达异常的多种蛋白进行了分析，并总结了部分已报道的与缺血再灌注损伤相关的蛋白。
**结果**：通心络可剂量依赖性地抑制心脏微血管内皮细胞凋亡，其最佳作用浓度为800 μg/mL。缺血再灌注可显著改变心脏微血管内皮细胞的蛋白表达，正常组与缺氧血清剥夺组间共检测到30种差异表达蛋白。在缺血再灌注损伤模型中，通心络干预可上调6种蛋白，包括线粒体酰基辅酶A合成酶ACSM2B（ACSM2B）、细胞周期蛋白依赖性激酶抑制剂1B（CDKN1B）、血红素氧合酶1（HMOX1）、转录因子SOX-17（SOX17）、隔离体1亚型1（SQSTM1）以及TBC结构域家族成员10B（TBC1D10B）。同时，通心络可下调5种蛋白，包括血管生成素-2亚型c前体（ANGPT2）、细胞色素c氧化酶组装因子5（COA5）、结缔组织生长因子前体（CTGF）、组织蛋白酶L1亚型2（CTSL）以及真核延伸因子2激酶（LOC101930123）。上述差异蛋白主要参与细胞增殖、应激反应及代谢过程调控等关键生物学功能。
**结论**：本研究通过蛋白质组学分析鉴定了缺血再灌注损伤后的差异表达蛋白。通心络可调控心脏微血管内皮细胞的蛋白表达，在对抗缺血再灌注损伤中发挥保护作用。