Zhu Jiang, Chen Gaoli
Department of Respiratory and Critical Care Medicine, Sichuan Academy of Medical Sciences & Sichuan Provincial People's Hospital, University Hospital of Electronic Science & Technology of China, Chengdu, China.
Department of Blood Transfusion, Teaching Hospital of Chengdu University of TCM, Chengdu, China.
Ann Transl Med. 2020 Dec;8(24):1651. doi: 10.21037/atm-20-7703.
As a key transcription factor, forkhead box protein 3 (FOXP3) plays an important role in the development and function of natural cluster of differentiation 4 [CD4 (+)] regulatory T cells (Treg cells). However, the function of FOXP3 in Lipopolysaccharide (LPS)-induced acute lung injury (ALI) through regulating miR-146b-5p is unclear. This research aimed to disclose the regulatory effect of the FOXP3-mediated miR-146b-5p/Roundabout 1 (Robo1)/NF-κB system on LPS-induced ALI in mice.
The mice were subjected to 5 mg/kg of LPS via intratracheal instillation to induce ALI and generate the ALI model. Mice was divided into five group, including control group, ALI group, ALI + FOXP3 group, the ALI + miR antagomir group and ALI + miR antagomir+ FOXP3 group. Lung tissue injury were detected by hematoxylin and eosin (HE) staining. Lung wet/dry weight ratio, total cells in bronchoalveolar lavage fluid (BALF), total protein in BALF and the polymorphonuclear leukocyte (PMN) in BALF were detected. The levels of tumor necrosis factor-α (TNF-α), Interleukin 6 (IL-6) and IL-1β were detected by enzyme-linked immunosorbent assay (ELISA) kit. The dual-luciferase reporter assay were used to detect the target relationship between FOXP3 and Robo1. Mice was divided into five group, including control group, ALI group, ALI + FOXP3 group, ALI + Robo1 group and ALI + FOXP3+ Robo1 group. The protein levels of FOXP3, Robo1 and p-p65 were detected by western bolt. The mRNA levels of miR-146b-5p and Robo1 were detected by quantitative reverse transcription polymerase chain reaction (qRT-PCR).
Although protein expression levels of FOXP3 were significantly down-regulated in the ALI model, the increased FOXP3 levels promoted an increase in miR-146b-5p. Compared with the control group, the ALI model group exhibited severe histopathologic injury, such as thickening of the alveolar wall, pulmonary congestion, and decreased alveolar numbers. By mediating the overexpression of miR-146b-5p, FOXP3 also increased alveolar clearance and inhibited inflammatory responses in the ALI model. Importantly, Robo1 is a potential target of miR-146b-5p.
FOXP3 could inhibit NF-κB activation, reduce lung pathological damage, and inhibit inflammatory responses by mediating the miR-146b-5p/Robo1/NF-κB system in the ALI model. These results may provide a new potential target for the treatment of ALI disease.
作为一种关键转录因子，叉头框蛋白3（FOXP3）在自然分化簇4[CD4(+)]调节性T细胞（Treg细胞）的发育和功能中发挥重要作用。然而，FOXP3通过调节miR-146b-5p在脂多糖（LPS）诱导的急性肺损伤（ALI）中的功能尚不清楚。本研究旨在揭示FOXP3介导的miR-146b-5p/轮状蛋白1（Robo1）/核因子κB（NF-κB）系统对LPS诱导的小鼠ALI的调节作用。
通过气管内滴注5mg/kg LPS诱导小鼠ALI并建立ALI模型。将小鼠分为五组，包括对照组、ALI组、ALI+FOXP3组、ALI+miR拮抗剂组和ALI+miR拮抗剂+FOXP3组。采用苏木精-伊红（HE）染色检测肺组织损伤。检测肺湿/干重比、支气管肺泡灌洗液（BALF）中的总细胞数、BALF中的总蛋白以及BALF中的多形核白细胞（PMN）。采用酶联免疫吸附测定（ELISA）试剂盒检测肿瘤坏死因子-α（TNF-α）、白细胞介素6（IL-6）和IL-1β的水平。采用双荧光素酶报告基因检测法检测FOXP3与Robo1之间的靶向关系。将小鼠分为五组，包括对照组、ALI组、ALI+FOXP3组、ALI+Robo1组和ALI+FOXP3+Robo1组。采用蛋白质免疫印迹法检测FOXP3、Robo1和p-p65的蛋白水平。采用定量逆转录聚合酶链反应（qRT-PCR）检测miR-146b-5p和Robo1的mRNA水平。
虽然ALI模型中FOXP3的蛋白表达水平显著下调，但FOXP3水平的升高促进了miR-146b-5p的增加。与对照组相比，ALI模型组表现出严重的组织病理学损伤，如肺泡壁增厚、肺充血和肺泡数量减少。通过介导miR-146b-5p的过表达，FOXP3还增加了ALI模型中的肺泡清除率并抑制了炎症反应。重要的是，Robo1是miR-146b-5p的潜在靶点。
在ALI模型中，FOXP3可通过介导miR-146b-5p/Robo1/NF-κB系统抑制NF-κB激活，减轻肺病理损伤并抑制炎症反应。这些结果可能为ALI疾病的治疗提供一个新的潜在靶点。
