HIF-1α介导炎性因子转录激活在重症H7N9禽流感致肺纤维化中作用机制研究

Inflammatory cascade plays an important role in the H7N9 virus infection which induced pulmonary fibrosis. The viral infection could induce and stabilize the transcription factor HIF-1α in certain target cells, which up-regulates inflammatory reactions. Our previous studies implied that HIF-1α could be a potential regulatory point in the process of pulmonary fibrosis. Thus we made a hypothesis of the pathogenic mechanism in severe H7N9 infection, which is "H7N9-HIF-1α induced inflammatory factor transcription-pulmonary fibrosis". The exact mechanism through which HIF-1α regulates the transcription of inflammatory factors is not well understood. Based on clinical cases as well as H7N9 infected animal models and cells, this study is going to further investigate different stages of H7N9 induced pulmonary fibrosis, analyzing the changes in dynamic ventilation, blood gas analysis, radiological and pathological features, as well as events on the molecular level. The technologies employed in our study would include but not limited to chromatin immunoprecipitation (CHIP),RT-PCR, etc. The emphasis of our study would be focused on the recruitment of transcription factor HIF-1α in inflammatory factor gene promoters and the level of histone acetylation or methylation. Furthermore, the results would be verified in clinical samples in an attempt to figure out the effect of HIF-1α-regulated transcription of inflammatory factors on clinical outcomes of severe H7N9 infection induced pulmonary fibrosis. The aim of this study was to clarify the dynamic molecular mechanisms and their multi-dimensional regulation in the process of pulmonary fibrosis induced by H7N9 infection.

瀑布样炎症反应是重症H7N9病毒感染致肺纤维化高死亡率的重要原因。病毒感染可诱导转录因子HIF-1α在特定靶细胞的稳定性，上调炎症反应。课题组前期研究提示HIF-1α是调控肺纤维化发生的潜在关键节点。因此我们推测H7N9致肺纤维化中存在"H7N9-HIF-1α介导炎性因子转录-肺纤维化"反馈环路，HIF-1α调控炎性因子转录激活的具体机制尚需阐明。本研究从临床病例着手，在细胞及已建立的H7N9模式动物中，运用染色体免疫共沉淀和反转录PCR相结合等手段，分析H7N9感染肺纤维化不同阶段临床、影像、病理学特征及分子事件，深入研究重症H7N9致肺纤维化中转录因子HIF-1α在炎性因子基因启动子区域招募，组蛋白乙酰化及甲基化水平变化；同时在临床样本中验证，从整体层面对HIF-1α介导炎性因子转录激活在重症H7N9致肺纤维化中作用进行解析，揭示重症H7N9禽流感肺纤维化的动态分子机制及多维调控规律。

瀑布样炎症反应是重症流感病毒感染致死的重要原因。如何阻止过度的炎症反应是降低重症流感患者死亡率的关键。本项目主要探讨HIF-1α在流感病毒所致炎症风暴中的作用及机制，为重症流感感染寻找新的治疗靶点提供理论和实验依据。本项目通过分析临床标本发现H1N1流感病毒感染患者血清中HIF-1α较健康对照明显上升。我们成功构建了H1N1流感病毒感染的体外细胞模型和动物模型，研究发现HIF-1α在H1N1感染的肺泡上皮细胞（A549）及巨噬细胞（THP-1）中表达明显上升且存在向细胞核转移的现象，H1N1感染小鼠的血清和肺组织中HIF-1α的表达也明显上升。使用HIF-1α入核抑制剂2ME2处理细胞和小鼠，明显降低了细胞上清中以及小鼠肺组织匀浆中TNF-α、IL-6的表达，改善了小鼠肺部病理损伤情况，提示HIF-1α在H1N1感染所致炎症风暴中发挥重要作用。同时本项目还深入研究了H1N1流感病毒激活宿主HIF-1α信号通路的机制。研究发现H1N1感染不改变HIF-1α mRNA的转录，也不抑制HIF-1α的脯氨酸羟化及泛素化步骤，而是抑制了宿主细胞的蛋白酶体的功能，从而抑制了HIF-1α的降解，使HIF-1α蛋白发生累积。因此，我们认为除了炎症反应所致的组织细胞缺氧可累积HIF-1α之外，流感病毒自身也可直接抑制宿主细胞的蛋白酶体从而加剧HIF-1α的累积。同时，在H1N1感染后，HIF-1α抑制蛋白FIH-1的表达明显降低，可能进一步增强HIF-1α的促进炎症因子转录的作用。因此本研究为流感病毒所致炎症风暴的治疗提供了一定的理论基础和可能的靶点。

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