Chen Shengsong, Zhang Yi, Zhan Qingyuan
Department of Pulmonary and Critical Care Medicine, China-Japan Friendship Hospital, Center of Respiratory Medicine, National Center for Respiratory Medicine, National Clinical Research Center for Respiratory Diseases, Institute of Respiratory Medicine, Chinese Academy of Medical Sciences, WHO Collaborating Centre for Tobacco Cessation and Respiratory Diseases Prevention, Beijing 100029, China.
Graduate School of Peking Union Medical College, Chinese Academy of Medical Sciences, Beijing 100730, China. Corresponding author: Zhan Qingyuan, Email:
Zhonghua Wei Zhong Bing Ji Jiu Yi Xue. 2022 Jan;34(1):41-47. doi: 10.3760/cma.j.cn121430-20210823-01260.
To investigate differential expression gene (DEG) in mice with ventilator-induced lung injury (VILI) by bioinformatics analysis, and to verify the key genes by reproducing the VILI mouse model.
(1) Experiment 1 (bioinformatics analysis): the microarray dataset of GSE9368 and GSE11662 regarding VILI mice and those in the spontaneous breathing control group were downloaded from the gene expression omnibus (GEO) database. DEG obtained by R and Venn map was further used to obtain common DEG. DAVID online database was used to obtain gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. Finally, the protein-protein interaction (PPI) analysis of common DEG was carried out by using Search Tool for the Retrieval of Interacting Genes Database (STRING) and the key genes were screened out by using CytoScape software, molecular complex detection (MCODE) analysis plug-in and CytoHubba plug-in with maximum cluster centrality (MCC), maximum neighbor connectivity (MNC) and degree. (2) Experiment 2 (related protein verification): VILI mouse model was reproduced by high tidal volume (20 mL/kg) ventilator. Spontaneous breathing control group was set up. Hematoxylin-eosin (HE) staining was performed to assess lung injury and the key genes screened in experiment 1 were verified by immunohistochemical staining.
(1) Experiment 1 results: a total of 114 DEG were screened from GSE9368 dataset, including 99 up-regulated genes and 15 down-regulated genes. A total of 258 DEG were screened from GSE11662 dataset, including 188 up-regulated genes and 70 down-regulated genes. Furthermore, 66 common DEG were obtained, including 61 up-regulated genes and 5 down-regulated genes. GO analysis showed that the common DEG were mainly involved in inflammatory response, immune response, leukocyte and neutrophil chemotaxis. KEGG analysis showed that the common DEG were involved cell adhesion, cytokine receptor interaction and tumor necrosis factor (TNF) signaling pathway. STRING and CytoScape analysis were used to construct gene PPI network diagram and important sub modules. And the CytoHubba plug-in with MCC, MNC and degree algorithms was used to perform topology analysis and then taken an intersection to obtain eight genes including suppressor of cytokine signaling 3 (SOCS3), interleukin-1β (IL-1β), matrix metalloproteinase-9 (MMP-9), integrin Itgam, CXC chemokine ligand 2 (CXCL2), CXC chemokine receptor 2 (CXCR2), Sell and CC chemokine receptor 1 (CCR1). (2) Experiment 2 results: a mouse model of high tidal volume VILI was reproduced. Compared with the spontaneous breathing control group, the lung tissue was injured slightly at 0 hour after the end of ventilation, and the lung tissue structure was significantly damaged at 6 hours after the end of ventilation, showing bleeding in alveolar cavity, significant increase and collapse of alveolar wall thickness, and infiltration of inflammatory cells. The top three genes from intersection and topological analysis including IL-1β, SOCS3 and MMP-9 were verified by immunohistochemical staining. The results showed that the expressions of IL-1β, SOCS3 and MMP-9 were gradually increased with time of ventilation, the differences were found at 6 hours as compared with those in the spontaneous breathing control group [IL-1β (integral A value): 8.40±2.67 vs. 5.10±0.94, SOCS3 (integral A value): 9.74±1.80 vs. 5.95±1.31, MMP-9 (integral A value): 11.45±6.20 vs. 5.36±1.28, all P < 0.05].
Bioinformatics analysis based on GSE9368 and GSE11662 data sets found that VILI is mainly related to inflammatory injury, cytokines and immune cell infiltration; IL-1β, SOCS3 and MMP-9 might be biomarkers of VILI.
通过生物信息学分析研究呼吸机诱导性肺损伤（VILI）小鼠中的差异表达基因（DEG），并通过复制VILI小鼠模型验证关键基因。
（1）实验1（生物信息学分析）：从基因表达综合数据库（GEO）下载关于VILI小鼠及自主呼吸对照组小鼠的GSE9368和GSE11662芯片数据集。通过R和韦恩图获得的DEG进一步用于获取共同的DEG。利用DAVID在线数据库进行基因本体（GO）和京都基因与基因组百科全书（KEGG）通路分析。最后，使用检索相互作用基因数据库（STRING）对共同DEG进行蛋白质-蛋白质相互作用（PPI）分析，并使用CytoScape软件、分子复合物检测（MCODE）分析插件和具有最大聚类中心性（MCC）、最大邻居连通性（MNC）和度的CytoHubba插件筛选关键基因。（2）实验2（相关蛋白验证）：通过高潮气量（20 mL/kg）呼吸机复制VILI小鼠模型。设立自主呼吸对照组。进行苏木精-伊红（HE）染色评估肺损伤，并通过免疫组织化学染色验证实验1中筛选出的关键基因。
（1）实验1结果：从GSE9368数据集中共筛选出114个DEG，包括99个上调基因和15个下调基因。从GSE11662数据集中共筛选出258个DEG，包括188个上调基因和70个下调基因。此外，获得66个共同DEG，包括61个上调基因和5个下调基因。GO分析表明，共同DEG主要参与炎症反应、免疫反应、白细胞和中性粒细胞趋化。KEGG分析表明，共同DEG参与细胞黏附、细胞因子受体相互作用和肿瘤坏死因子（TNF）信号通路。使用STRING和CytoScape分析构建基因PPI网络图和重要子模块。并使用具有MCC、MNC和度算法的CytoHubba插件进行拓扑分析，然后取交集获得8个基因，包括细胞因子信号转导抑制因子3（SOCS3）、白细胞介素-1β（IL-1β）、基质金属蛋白酶-9（MMP-9）、整合素Itgam、CXC趋化因子配体2（CXCL2）、CXC趋化因子受体2（CXCR2）、Sell和CC趋化因子受体1（CCR1）。（2）实验2结果：复制了高潮气量VILI小鼠模型。与自主呼吸对照组相比，通气结束后0小时肺组织轻度损伤，通气结束后6小时肺组织结构明显受损，表现为肺泡腔内出血、肺泡壁厚度显著增加和塌陷以及炎症细胞浸润。通过免疫组织化学染色验证了交集和拓扑分析中排名前三位的基因，即IL-1β、SOCS3和MMP-9。结果表明，IL-1β、SOCS3和MMP-9的表达随通气时间逐渐增加，与自主呼吸对照组相比，6小时时差异显著[IL-1β（积分A值）：8.40±2.67对5.10±0.94，SOCS3（积分A值）：9.74±1.80对5.95±1.31，MMP-9（积分A值）：11.45±6.20对5.36±1.28，均P<０.05]。
基于GSE9368和GSE11662数据集的生物信息学分析发现，VILI主要与炎症损伤、细胞因子和免疫细胞浸润有关；IL-1β、SOCS3和MMP-9可能是VILI的生物标志物。
