Zhao Ying, Zhang Hui, Zhang Shu-Li, Wei Juan, Zheng Liang-Liang, Du Jian-Kui, Zhu Xiao-Yan, Jiang Lai, Liu Yu-Jian
School of Kinesiology, Shanghai Frontiers Science Research Base of Exercise and Metabolic Health, The Key Laboratory of Exercise and Health Sciences of Ministry of Education, Shanghai University of Sport, Shanghai, China.
Department of Anesthesiology and Surgical Intensive Care Unit, Xinhua Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China.
Ann Transl Med. 2022 Jun;10(11):630. doi: 10.21037/atm-21-5380.
Autophagy is activated during the pathogenesis of endothelial dysfunction and sepsis-associated acute lung injury (ALI). This study aimed to investigate whether autophagy affected endothelial barrier dysfunction and lung injury in a murine model of lipopolysaccharide (LPS)-induced ALI, and then further clarify whether forkhead box O1 (), an autophagy-related transcriptional factor, contributed to autophagy activation and ALI induced by LPS.
Male C57BL/6 mice were treated with LPS (30 mg/kg), and then were allocated to a control group and an LPS group with or without FOXO1 inhibitor (AS1842856) treatment, respectively. Primary cultured mouse lung vascular endothelial cells (MLVECs) were treated with LPS, autophagy inhibitor 3-methyladenine (3-MA), AS1842856, and small interfering RNA (siRNA) targeting autophagy-related gene 5 () or . Endothelial autophagic flux was assessed by transfection of MLVECs with red fluorescent protein (RFP)-green fluorescent protein (GFP) tandem fluorescent-tagged LC3 (RFP-GFP-LC3) adenovirus. Endothelial permeability was analyzed by the diffusion of fluorescein isothiocyanate-carboxymethyl (FITC)-dextran through the endothelial monolayer. Evans blue albumin tracer was used to measure the pulmonary transvascular permeability, and hematoxylin and eosin (H&E) staining was used to observe pathological changes in the lung tissues. Immunofluorescence staining was also used to detect the expression of zonula occludens-1 (ZO-1) and FOXO1.
This study found autophagy induction in lung tissues of endotoxemic mice and LPS-treated MLVECs, as evidenced by elevated expression of light chain 3 II (LC3-II) and Unc-51-like kinase (ULK1) and autophagic flux. LPS treatment decreased vascular endothelial (VE)-cadherin and ZO-1 expression and increased endothelial permeability in MLVECs, which were significantly alleviated by autophagy inhibitor 3-MA and siRNA. It was found that both phosphorylated FOXO1 and FOXO1 were upregulated in the lung tissues of endotoxemic mice and LPS-treated MLVECs. Both FOXO1 inhibitor AS1842856 and siRNA suppressed LPS-induced autophagy and endothelial cell injury in MLVECs. Moreover, FOXO1 inhibition profoundly alleviated autophagy, lung endothelial hyperpermeability, and ALI in endotoxemic mice.
This work demonstrated that upregulation is an important contributor to LPS-induced autophagy in pulmonary VE cells. The detrimental effects of FOXO1 in endotoxemia-associated endothelial dysfunction and ALI are partly due to its potent pro-autophagic property. Inhibition of FOXO1 may be a potential therapeutic option for the treatment of ALI.
自噬在内皮功能障碍和脓毒症相关急性肺损伤（ALI）的发病过程中被激活。本研究旨在探讨在脂多糖（LPS）诱导的ALI小鼠模型中自噬是否影响内皮屏障功能障碍和肺损伤，进而进一步阐明自噬相关转录因子叉头框O1（FOXO1）是否促成LPS诱导的自噬激活和ALI。
雄性C57BL/6小鼠接受LPS（30 mg/kg）处理，然后分别分配至对照组和LPS组，LPS组又分为接受或不接受FOXO1抑制剂（AS1842856）处理。原代培养的小鼠肺血管内皮细胞（MLVECs）用LPS、自噬抑制剂3-甲基腺嘌呤（3-MA）、AS1842856以及靶向自噬相关基因5（ATG5）或ATG7的小干扰RNA（siRNA）处理。通过用红色荧光蛋白（RFP）-绿色荧光蛋白（GFP）串联荧光标记的微管相关蛋白1轻链3（RFP-GFP-LC3）腺病毒转染MLVECs来评估内皮自噬通量。通过异硫氰酸荧光素-羧甲基（FITC）-葡聚糖透过内皮单层的扩散来分析内皮通透性。用伊文思蓝白蛋白示踪剂测量肺血管通透性，并用苏木精和伊红（H&E）染色观察肺组织的病理变化。免疫荧光染色也用于检测紧密连接蛋白1（ZO-1）和FOXO1的表达。
本研究发现内毒素血症小鼠肺组织和LPS处理的MLVECs中有自噬诱导，这通过轻链3 II（LC3-II）和Unc-51样激酶（ULK1）表达升高及自噬通量得以证明。LPS处理降低了MLVECs中血管内皮（VE）-钙黏蛋白和ZO-1的表达并增加了内皮通透性，自噬抑制剂3-MA和ATG5 siRNA可显著减轻这些变化。发现内毒素血症小鼠肺组织和LPS处理的MLVECs中磷酸化FOXO1和FOXO1均上调。FOXO1抑制剂AS1842856和ATG7 siRNA均抑制LPS诱导的MLVECs自噬和内皮细胞损伤。此外，抑制FOXO1可显著减轻内毒素血症小鼠的自噬、肺内皮高通透性和ALI。
这项研究表明FOXO1上调是LPS诱导肺VE细胞自噬的重要促成因素。FOXO1在内毒素血症相关内皮功能障碍和ALI中的有害作用部分归因于其强大的促自噬特性。抑制FOXO1可能是治疗ALI的一种潜在治疗选择。
