Merk Dennis, Ptok Johannes, Jakobs Philipp, von Ameln Florian, Greulich Jan, Kluge Pia, Semperowitsch Kathrin, Eckermann Olaf, Schaal Heiner, Ale-Agha Niloofar, Altschmied Joachim, Haendeler Judith
Environmentally-Induced Cardiovascular Degeneration, Clinical Chemistry and Laboratory Diagnostics, Medical Faculty, University Hospital and Heinrich-Heine University Düsseldorf, 40225 Düsseldorf, Germany.
Institute for Virology, Medical Faculty, University Hospital and Heinrich-Heine University Düsseldorf, 40225 Düsseldorf, Germany.
Antioxidants (Basel). 2021 Sep 7;10(9):1427. doi: 10.3390/antiox10091427.
Sepsis is an exaggerated immune response upon infection with lipopolysaccharide (LPS) as the main causative agent. LPS-induced activation and apoptosis of endothelial cells (EC) can lead to organ dysfunction and finally organ failure. We previously demonstrated that the first twenty amino acids of the Apurinic/Apyrimidinic Endodeoxyribonuclease 1 (APEX1) are sufficient to inhibit EC apoptosis. To identify genes whose regulation by LPS is affected by this N-terminal APEX1 peptide, EC were transduced with an expression vector for the APEX1 peptide or an empty control vector and treated with LPS. Following RNA deep sequencing, genes upregulated in LPS-treated EC expressing the APEX1 peptide were identified bioinformatically. Selected candidates were validated by semi-quantitative real time PCR, a promising one was Selenoprotein T (SELENOT). For functional analyses, an expression vector for SELENOT was generated. To study the effect of SELENOT expression on LPS-induced EC activation and apoptosis, the SELENOT vector was transfected in EC. Immunostaining showed that SELENOT was expressed and localized in the ER. EC transfected with the SELENOT plasmid showed no activation and reduced apoptosis induced by LPS. SELENOT as well as APEX1(1-20) can protect EC against activation and apoptosis and could provide new therapeutic approaches in the treatment of sepsis.
脓毒症是一种以脂多糖（LPS）为主要病原体感染后的过度免疫反应。LPS诱导的内皮细胞（EC）活化和凋亡可导致器官功能障碍，最终导致器官衰竭。我们之前证明，脱嘌呤/脱嘧啶核酸内切酶1（APEX1）的前二十个氨基酸足以抑制EC凋亡。为了鉴定其受LPS调控且受该APEX1 N端肽影响的基因，用APEX1肽的表达载体或空对照载体转导EC，并给予LPS处理。经过RNA深度测序后，通过生物信息学方法鉴定了在表达APEX1肽的LPS处理的EC中上调的基因。通过半定量实时PCR验证了选定的候选基因，其中一个很有前景的是硒蛋白T（SELENOT）。为了进行功能分析，构建了SELENOT的表达载体。为了研究SELENOT表达对LPS诱导的EC活化和凋亡的影响，将SELENOT载体转染到EC中。免疫染色显示SELENOT表达并定位于内质网。用SELENOT质粒转染的EC未显示出LPS诱导的活化，且凋亡减少。SELENOT以及APEX1（1-20）可以保护EC免受活化和凋亡的影响，并可能为脓毒症的治疗提供新的治疗方法。
