Xu Xiaotao, Zhu Qingwei, Zhang Rong, Wang Yan, Niu Fangfang, Wang Wenying, Sun Dawei, Wang Aizhong
Department of Anesthesiology, Shanghai Jiao Tong University Affiliated Sixth People's Hospital, Shanghai, China.
Department of Neurosurgery, Shanghai Jiao Tong University Affiliated Sixth People's Hospital, Shanghai, China.
Cell Physiol Biochem. 2017;44(5):1949-1964. doi: 10.1159/000485885. Epub 2017 Dec 8.
BACKGROUND/AIMS: This study was conducted to investigate the relationship between differentially expressed proteins (DEPs) and the pathogenesis of oleic acid (OA)-induced acute lung injury (ALI) in mice.
Eight-week-old male C57BL/6 mice were injected with OA through the tail vein and sacrificed 6 hours after OA administration to identify protein expression levels in lung tissue using isobaric tags for relative and absolute quantification (iTRAQ) technology. Then, DEPs such as antithrombin III (AT III), 12-lipoxygenase (12-LO), dedicator of cytokinesis 2 (DOCK2), polycystin-2 and plasminogen were identified by western blotting. Subsequently, we focused on investigating the effect of AT III on endothelial integrity using siRNA interference technology. The levels of IL-6, IL-1β, TNF-α and TGF-β expression were detected using an enzyme-linked immunosorbent assay (ELISA). Alterations in the tight junction component ZO-1 and the phosphorylation of myosin light chain (pMLC) were determined by western blotting. The stress fiber F-actin were also detected by immunofluorescence staining. In addition, endothelial permeability was determined via a transwell permeability assay.
A total of 5152 proteins were found to be expressed in lung tissues from the OA-treated and saline-treated mice. Among these proteins, 849 were differentially expressed between the two groups, including 545 upregulated and 304 downregulated proteins. After AT III knockdown, the levels of inflammatory factors and endothelial permeability were elevated, the expression of ZO-1 was decreased, and the expression of F-actin and pMLC was increased. All these results illustrated that AT III knockdown exaggerated the disruption of endothelial integrity mediated by OA.
These findings using iTRAQ technology demonstrate, for the first time, differences in the lung tissue expression levels of proteins between OA-treated mice and saline-treated mice. This study reveals that 12-LO, DOCK2 and especially AT III may be candidate biomarkers for OA-induced acute lung injury.
背景/目的：本研究旨在探讨差异表达蛋白（DEPs）与油酸（OA）诱导的小鼠急性肺损伤（ALI）发病机制之间的关系。
将8周龄雄性C57BL/6小鼠通过尾静脉注射OA，并在给予OA 6小时后处死，使用相对和绝对定量等压标签（iTRAQ）技术鉴定肺组织中的蛋白质表达水平。然后，通过蛋白质印迹法鉴定抗凝血酶III（AT III）、12-脂氧合酶（12-LO）、胞质分裂 dedicator 2（DOCK2）、多囊蛋白-2和纤溶酶原等差异表达蛋白。随后，我们使用小干扰RNA（siRNA）技术重点研究AT III对内皮完整性的影响。使用酶联免疫吸附测定（ELISA）检测白细胞介素-6（IL-6）、白细胞介素-1β（IL-1β）、肿瘤坏死因子-α（TNF-α）和转化生长因子-β（TGF-β）的表达水平。通过蛋白质印迹法测定紧密连接成分闭合蛋白-1（ZO-1）的变化和肌球蛋白轻链（pMLC）的磷酸化。应激纤维F-肌动蛋白也通过免疫荧光染色进行检测。此外，通过Transwell通透性测定法测定内皮通透性。
在OA处理组和生理盐水处理组小鼠的肺组织中共发现5152种蛋白质表达。在这些蛋白质中，两组之间有849种差异表达，包括545种上调蛋白和304种下调蛋白。AT III基因敲低后，炎症因子水平和内皮通透性升高，ZO-1表达降低，F-肌动蛋白和pMLC表达增加。所有这些结果表明，AT III基因敲低加剧了OA介导的内皮完整性破坏。
这些使用iTRAQ技术的研究结果首次证明了OA处理组小鼠和生理盐水处理组小鼠肺组织中蛋白质表达水平的差异。本研究表明，12-LO、DOCK2，尤其是AT III可能是OA诱导的急性肺损伤的候选生物标志物。
