Kwon Oh Kwang, Lee Wonhwa, Kim Sun Ju, Lee You-Mie, Lee Ju Yeon, Kim Jin Young, Bae Jong-Sup, Lee Sangkyu
College of Pharmacy, Research Institute of Pharmaceutical Sciences, Kyungpook National University, Daegu, Republic of Korea.
Department of Biochemistry and Cell Biology, CMRI, BK21 Plus KNU Biomedical Convergence Program, School of Medicine, Kyungpook National University, Daegu, Republic of Korea.
Electrophoresis. 2015 Dec;36(23):2851-8. doi: 10.1002/elps.201500198. Epub 2015 Sep 13.
Sepsis and septic shock, which are conditions triggered by infection, occur with high incidence in emergency departments and are among the most common causes of death in hospitalized patients worldwide. Therefore, the identification of sepsis biomarkers for the rapid diagnosis is a major goal for researchers in the field of critical care. Endothelial cells play a pivotal role in orchestrating the inflammatory response triggered by sepsis. In this study, we used proteomics to investigate the secretome of EA.hy926 endothelial cells following lipopolysaccharide (LPS) stimulation with 1 μg/mL LPS for 12 or 24 h. SILAC in cell cultures and an online 2D-LC-MS/MS system were used to analyze the secretome dynamics in response to LPS. We found that 22 of the 77 secreted proteins identified in both the presence and absence of LPS and that 19 of the secreted proteins were quantified more strongly after LPS treatment for 24 h than after treatment for 12 h. By Gene Ontology and KEGG pathway analyses, we found that proteins related to the regulation of the actin cytoskeleton showed the highest secretion response to LPS stimulation. Out of the 19 candidate proteins, we focused on moesin, which is involved in the function of endothelial cells, and confirmed its amount in cellular lysates and media taken from primary human umbilical vein endothelial cells treated with LPS. To our knowledge, this study provides the first in-depth analysis of the LPS-induced secretome in human endothelial cells, and we propose 19 new biomarker candidates for sepsis, including moesin.
脓毒症和脓毒性休克是由感染引发的病症，在急诊科发病率很高，是全球住院患者最常见的死亡原因之一。因此，识别用于快速诊断的脓毒症生物标志物是重症监护领域研究人员的主要目标。内皮细胞在协调脓毒症引发的炎症反应中起关键作用。在本研究中，我们使用蛋白质组学来研究用1μg/mL脂多糖（LPS）刺激12或24小时后EA.hy926内皮细胞的分泌蛋白组。细胞培养中的稳定同位素标记氨基酸法（SILAC）和在线二维液相色谱-串联质谱（2D-LC-MS/MS）系统用于分析对LPS反应的分泌蛋白组动态变化。我们发现，在有和没有LPS的情况下鉴定出的77种分泌蛋白中有22种，并且在LPS处理24小时后，19种分泌蛋白的定量比处理12小时后更强。通过基因本体论和KEGG通路分析，我们发现与肌动蛋白细胞骨架调节相关的蛋白质对LPS刺激表现出最高的分泌反应。在这19种候选蛋白中，我们重点关注参与内皮细胞功能的埃兹蛋白（moesin），并确认了其在用LPS处理的原代人脐静脉内皮细胞的细胞裂解物和培养基中的含量。据我们所知，本研究首次对人内皮细胞中LPS诱导的分泌蛋白组进行了深入分析，并且我们提出了19种新的脓毒症生物标志物候选物，包括埃兹蛋白。
