Yu Zhaoxia, Rayile Aisa, Zhang Xiangyang, Li Ying, Zhao Qiang
Intensive Care Unit, The First Affiliated Hospital of Xinjiang Medical University, Urumqi, Xinjiang 830054, P.R. China.
Department of Cardiac Center, The First Affiliated Hospital of Xinjiang Medical University, Urumqi, Xinjiang 830054, P.R. China.
Int J Mol Med. 2017 May;39(5):1269-1276. doi: 10.3892/ijmm.2017.2920. Epub 2017 Mar 15.
The present study aimed to evaluate the effects of ulinastatin on the permeability and apoptosis of lipopolysaccharide (LPS)-induced cardiac microvascular endothelial cells (CMVECs), and investigate its molecular mechanisms in sepsis. The sepsis rat model was induced by cecal ligation and puncture (CLP), and rat CMVECs were isolated and treated with LPS or/and ulinastatin. Then, cell permeability was evaluated by transendothelial electrical resistance, reactive oxygen species (ROS) levels were assessed by 2,7-dichlorofluorescein diacetate, cell apoptosis was detected using Annexin V-FITC apoptosis detection kit, and the expression levels of Bcl-2, Bax, caspase-3, metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) and EZH2 were detected by RT-qPCR and/or western blotting. In addition, the relationship of MALAT1 and EZH2 was evaluated by RNA immunoprecipitation (RIP) assay and chromatin immunoprecipitation (ChIP) assay. Compared with LPS-induced CMVECs, treatment with ulinastatin significantly reduced CMVEC permeability and the percentage of apoptotic cells, as well as an increased level of Bcl-2 and inhibited the levels of ROS, caspase-3 and Bax (all p<0.05). In addition, long non-coding RNA (lncRNA) MALAT1 was confirmed to interact with EZH2 in CMVECs. Overexpression of lncRNA MALAT1 and EZH2 was found in the hearts of the sepsis rat and LPS-induced CMVECs, while ulinastatin inhibited the upregulation of lncRNA MALAT1 and EZH2 in the LPS-induced CMVECs (all p<0.05). Ulinastatin protected against LPS-induced CMVEC cell hyperpermeability and apoptosis via downregulation of lncRNA MALAT1 and EZH2 in sepsis.
本研究旨在评估乌司他丁对脂多糖（LPS）诱导的心脏微血管内皮细胞（CMVECs）通透性和凋亡的影响，并探讨其在脓毒症中的分子机制。采用盲肠结扎穿孔术（CLP）诱导脓毒症大鼠模型，分离大鼠CMVECs并用LPS或/和乌司他丁处理。然后，通过跨内皮电阻评估细胞通透性，用二氯荧光素二乙酸酯评估活性氧（ROS）水平，使用膜联蛋白V-FITC凋亡检测试剂盒检测细胞凋亡，并用RT-qPCR和/或蛋白质免疫印迹法检测Bcl-2、Bax、半胱天冬酶-3、转移相关肺腺癌转录本1（MALAT1）和EZH2的表达水平。此外，通过RNA免疫沉淀（RIP）试验和染色质免疫沉淀（ChIP）试验评估MALAT1和EZH2的关系。与LPS诱导的CMVECs相比，乌司他丁处理显著降低了CMVECs的通透性和凋亡细胞百分比，同时增加了Bcl-2水平并抑制了ROS、半胱天冬酶-3和Bax的水平（均p<0.05）。此外，证实长链非编码RNA（lncRNA）MALAT1在CMVECs中与EZH2相互作用。在脓毒症大鼠心脏和LPS诱导的CMVECs中发现lncRNA MALAT1和EZH2表达上调，而乌司他丁抑制LPS诱导的CMVECs中lncRNA MALAT1和EZH2的上调（均p<0.05）。在脓毒症中，乌司他丁通过下调lncRNA MALAT1和EZH2来保护LPS诱导的CMVECs细胞免受高通透性和凋亡。
