Yan Huiyuan, Wu Junsong, Yan Huilian
Department of Internal Medicine, Children's Hospital of Fudan University, Shanghai 201102, P.R. China.
Medical College, Qinghai University, Xining, Qinghai 810016, P.R. China.
Exp Ther Med. 2023 May 15;26(1):319. doi: 10.3892/etm.2023.12018. eCollection 2023 Jul.
Acute lung injury (ALI) is associated with increased lung inflammation and lung permeability. The present study aimed to determine the role of inactive rhomboid-like protein 2 (iRHOM2) in ALI in lipopolysaccharide (LPS)-induced pulmonary microvascular endothelial cell model. Human pulmonary microvascular endothelial cells (HPMVECs) were transfected with small interfering RNA targeting iRHOM2 and C-X3-C motif chemokine ligand 1 (CX3CL1) overexpression plasmids and treated with LPS. Cell viability was detected using a Cell Counting Kit-8 assay, while levels of TNFα, IL-1β, IL-6 and p65 were measured by reverse transcription-quantitative PCR and western blotting. Apoptosis levels were measured using a TUNEL assay. Endothelial barrier permeability was detected, followed by analysis of zonula occludens-1, vascular endothelial-cadherin and occludin by immunofluorescence staining or western blotting. The interaction of iRHOM2 and CX3CL1 was analyzed using an immune-coprecipitation assay. Through bioinformatics analysis, it was found that CX3CL1 was upregulated in the LPS group compared with the control. Kyoto Encyclopedia of Genes and Genomes pathway analysis demonstrated that the TNF signaling pathway affected by iRHOM2 and cytokine-cytokine receptor interaction, including CX3CL1, served a key role in ALI. HPMVECs treated with LPS exhibited a decrease in cell viability and an increase in inflammation, apoptosis and endothelial barrier permeability, while these effects were reversed by iRHOM2 silencing. However, CX3CL1 overexpression inhibited the effects of iRHOM2 silencing on LPS-treated HPMVECs. The present study demonstrated a novel role of iRHOM2 as a regulator that affects inflammation, apoptosis and endothelial barrier permeability; this was associated with CX3CL1.
急性肺损伤（ALI）与肺部炎症增加和肺通透性增加有关。本研究旨在确定无活性类菱形蛋白2（iRHOM2）在脂多糖（LPS）诱导的肺微血管内皮细胞模型中ALI中的作用。用靶向iRHOM2的小干扰RNA和C-X3-C基序趋化因子配体1（CX3CL1）过表达质粒转染人肺微血管内皮细胞（HPMVECs），并用LPS处理。使用细胞计数试剂盒-8检测细胞活力，同时通过逆转录定量PCR和蛋白质印迹法测量肿瘤坏死因子α（TNFα）、白细胞介素-1β（IL-1β）、白细胞介素-6（IL-6）和p65的水平。使用TUNEL检测法测量凋亡水平。检测内皮屏障通透性，然后通过免疫荧光染色或蛋白质印迹法分析紧密连接蛋白-1、血管内皮钙黏蛋白和闭合蛋白。使用免疫共沉淀检测法分析iRHOM2与CX3CL1的相互作用。通过生物信息学分析发现，与对照组相比，LPS组中CX3CL1上调。京都基因与基因组百科全书通路分析表明，受iRHOM2影响的TNF信号通路以及包括CX3CL1在内的细胞因子-细胞因子受体相互作用在ALI中起关键作用。用LPS处理的HPMVECs表现出细胞活力下降以及炎症、凋亡和内皮屏障通透性增加，而这些作用通过iRHOM2沉默得以逆转。然而，CX3CL1过表达抑制了iRHOM2沉默对LPS处理的HPMVECs的影响。本研究证明了iRHOM2作为一种调节因子的新作用，其影响炎症、凋亡和内皮屏障通透性；这与CX3CL1有关。
