Liu Bo, Wu Yanqi, Wang Yahui, Cheng Yumei, Yao Ling, Liu Yuqin, Qian Hong, Yang Huilin, Shen Feng
a Department of Critical Care Medicine , The Affiliated Hospital of Guizhou Medical University , Guiyang , China.
b Department of Critical Care Medicine , The Second Affiliated Hospital of Guizhou Medical University , Kaili China.
Exp Lung Res. 2018 May-Jun;44(4-5):241-251. doi: 10.1080/01902148.2018.1505975. Epub 2018 Nov 19.
Purpose/aim: Activated coagulation and reduced fibrinolysis in alveolar compartment are an important characteristics in acute respiratory distress syndrome (ARDS). Alveolar epithelial cell type II (AECII) participates in regulating the intra-alveolar abnormalities of coagulation and fibrinolysis mainly through adjusting the productions of tissue factor (TF), plasminogen activator inhibitor (PAI)-1 and activated protein C (APC) in ARDS. NF-κB signal pathway may be involved in coagulation regulation in sepsis-induced ALI. The purpose of this study was to testify the hypothesis that NF-κB p65 (p65) knock-down would improve the abnormalities of coagulation and fibrinolysis mediated by lipopolysaccharide (LPS) stimulation in AECII.
p65 gene knock-down in AECII was achieved by small interfering RNA (siRNA) transfection. Rat AECII (RLE-6TN) with or without p65 gene knock-down were stimulated by LPS for 24 hours. And then cytolysate was used for TF, PAI-1 expression examination, and supernatant was collected for TF, PAI-1 and PC concentrations determination. Activation of NF-κB canonical pathway was simultaneously checked by western-blotting, RT-PCR and immunofluorescence respectively.
TF, PAI-1 expressions in normal cells obviously increased under LPS stimulation with NF-κB canonical pathway activation represented by high levels of p65, p-p65, p-IκB with increased nuclear translocation of p-p65. Cells with NF-κB p65 knock-down, however, showed significant decreases in TF, PAI-1, p65, p-p65, p-IκB expressions following LPS stimulation with significant reduction in p-p65 nuclear translocation as compared to normal and siRNA control cells. The high concentrations of TF, PAI-1 and low level of APC in supernatant induced by LPS in normal cells were significantly reversed through p65 knock-down.
The experimental findings demonstrate that NF-kB signaling pathway is involved in regulating the expressions of coagulation and fibrinolysis factors in LPS-stimulated AECII, which suggest that NF-kB signaling pathway may be a new target to correct intra-alveolar coagulation and fibrinolytic abnormalities in ARDS.
目的/目标：肺泡腔中凝血激活和纤溶降低是急性呼吸窘迫综合征（ARDS）的重要特征。II型肺泡上皮细胞（AECII）主要通过调节ARDS中组织因子（TF）、纤溶酶原激活物抑制剂（PAI）-1和活化蛋白C（APC）的产生来参与调节肺泡内凝血和纤溶异常。NF-κB信号通路可能参与脓毒症诱导的急性肺损伤（ALI）中的凝血调节。本研究的目的是验证以下假设：敲低NF-κB p65（p65）可改善脂多糖（LPS）刺激介导的AECII中凝血和纤溶异常。
通过小干扰RNA（siRNA）转染实现AECII中p65基因的敲低。用LPS刺激有或无p65基因敲低的大鼠AECII（RLE-6TN）24小时。然后将细胞裂解物用于TF、PAI-1表达检测，并收集上清液用于测定TF、PAI-1和PC浓度。分别通过蛋白质免疫印迹法、逆转录-聚合酶链反应（RT-PCR）和免疫荧光同时检测NF-κB经典途径的激活。
在LPS刺激下，正常细胞中TF、PAI-1表达明显增加，以高水平的p65、磷酸化p65（p-p65）、磷酸化IκB（p-IκB）以及p-p65核转位增加为代表的NF-κB经典途径被激活。然而，与正常细胞和siRNA对照细胞相比，敲低NF-κB p65的细胞在LPS刺激后TF、PAI-1、p65、p-p65、p-IκB表达显著降低，p-p65核转位明显减少。通过敲低p65，LPS诱导的正常细胞上清液中高浓度的TF、PAI-1和低水平的APC得到显著逆转。
实验结果表明，NF-κB信号通路参与调节LPS刺激的AECII中凝血和纤溶因子的表达，这表明NF-κB信号通路可能是纠正ARDS肺泡内凝血和纤溶异常的新靶点。
