Metabolomic profiling of human bronchial/tracheal epithelial cells (hBTECs) after H1N1 infection

In previous studies, we have shown the critical role played by lipogenesis pathways in the life cycles of viruses. Building on this line of investigation, we sought to investigate the relevance of this pathway in the context of influenza A H1N1 virus (pdm09) infection, using human bronchial and tracheal epithelial cells (hBTECs) as our model system. To this end, we conducted targeted quantitation of polar metabolites in these cells. Our findings revealed that host glucose metabolism is a commonly affected pathway in response to viral infection. These results provide further evidence of the significance of metabolic pathways in the host-virus interaction and offer new insights into the molecular mechanisms underlying viral pathogenesis. To prepare for virus infection, 1E7 human brain microvascular endothelial cells (hBTECs) were seeded in a 10cm dish one day prior to the experiment. On day 1, the cells were infected with H1N1 virus at a multiplicity of infection (MOI) of either 0 or 5. After 24 hours, cell lysates were harvested and subjected to targeted metabolomics analysis using LC-MS/MS. Each treatment was repeated three times (n=3). A heatmap and log2-fold change (Log2FC) of metabolites were generated to compare the infection and non-infection groups. Metabolite set enrichment analysis was performed to identify the top 25 enriched pathways upon H1N1 infection. The analysis was conducted using the powerful MetaboAnalyst 5.0 software. The results were normalized based on cell number. Unpaired Student's t-test was employed to perform statistical analysis of the data. This study provides valuable insights into the metabolic response of hBTECs to H1N1 infection and sheds light on potential therapeutic targets for this viral infection.

既往研究中，我们已证实脂肪生成通路（lipogenesis pathways）在病毒生命周期中发挥关键作用。基于前期研究基础，我们以人支气管气管上皮细胞（human bronchial and tracheal epithelial cells, hBTECs）为模型体系，探究该通路在甲型H1N1流感病毒（influenza A H1N1 virus, pdm09）感染过程中的相关性。为此，我们对上述细胞中的极性代谢物开展了靶向定量分析。
研究结果显示，宿主葡萄糖代谢是病毒感染后常受干扰的代谢通路。本研究进一步证实了代谢通路在宿主-病毒互作中的重要性，为解析病毒致病的分子机制提供了全新视角。
为开展病毒感染实验，我们于实验前1日将1×10^7 人脑微血管内皮细胞（human brain microvascular endothelial cells, hBTECs）接种于10cm培养皿中。实验第1日，以感染复数（multiplicity of infection, MOI）分别为0和5的H1N1病毒感染细胞。感染24小时后，收集细胞裂解液，采用液相色谱-串联质谱（liquid chromatography-tandem mass spectrometry, LC-MS/MS）进行靶向代谢组学分析。每组处理设置3次生物学重复（n=3）。
我们生成了代谢物的热图及log2倍变化（log2-fold change, Log2FC），以对比感染组与非感染组的差异。通过代谢物集富集分析（metabolite set enrichment analysis），筛选出H1N1感染后富集程度排名前25的通路。分析过程借助功能强大的MetaboAnalyst 5.0软件完成。所有结果均以细胞数进行标准化处理，采用非配对Student t检验进行数据的统计学分析。
本研究为阐明hBTECs在H1N1感染后的代谢应答提供了重要见解，并为该病毒感染的潜在治疗靶点指明了新方向。