lncRNA-EPS通过转录调控巨噬细胞炎症反应参与呼吸机诱导肺损伤的机制研究

Biotrauma caused by inflammatory response plays a key role in ventilator induced lung injury (VILI), causing secondary hit to pulmonary cells, which is closely related to a poor prognosis. It may be a novel strategy to alleviate lung injury that inhibiting excessive pro-inflammation of macrophages by transcriptional and post–transcriptional regulation. It has been well proved that long noncoding RNAs (lncRNAs) regulate gene expression in diverse biological contexts in epigenetic level. However, it is still poorly known what the role of lncRNAS in inflammation or tissue injury in mechanical ventilation. Previously, we found a number of differentially expressed lncRNAs by RNA-seq. lncRNA-EPS was significantly downregulated via macrophages phagocytosis of the dead cells. lncRNA-EPS might affect activation of NLRP3 inflamasome as well. Consequently, summing lastest reported mechanism of lncRNA-EPS, we hypothesize: expression of novel lncRNA-EPS is downregulated in infiltrated macrophages through TRL4/MD2 pathway in VILI; decreased lncRNA-EPS, which transcriptionally inhibits asc gene expression, leads to NLRP3 inflammasome activation; the changes of inflammatory status aggravate alveolar epithelial cells damage; exogenous lncRNA-EPS is expected to become a therapeutic target for VILI. In this study, we plan to prove our hypothesis in vivo using the wild type and lncRNA-EPS overexpressed mice with VILI model. Moreover, macrophages with alveolar epithelial cells will be co-cultured in vitro. Lentivirus transfection, RNA interference, RIP, ChIP and other experimental methods will be applied to reveal lncRNA-EPS-mediated inflammation in acute phage of VILI. Within this study, we anticipate to investigate the underlying mechanism of lncRNA-EPS as a key "brake molecular" in tissue impair following VILI, and explore a novel therapeutic target to improve outcome based on theoretical and experimental evidence.

炎症反应造成的生物伤在呼吸机诱导肺损伤(VILI)中发挥关键作用，对肺组织造成二次打击，影响预后。如能在转录及转录后水平抑制炎症激活可减轻VILI。lncRNA参与损伤后表观调控的研究日益深化，但在VILI炎症调控中作用尚不清楚。我们通过转录组测序发现VILI早期巨噬细胞吞噬死亡细胞后lncRNA-EPS表达下调，可能参与炎症小体激活。结合最新研究进展，我们提出假说：VILI形成后巨噬细胞清除死亡细胞时诱导lncRNA-EPS下调，对asc基因转录抑制作用削弱，导致NLRP3炎症小体激活加重损伤，外源性lncRNA-EPS有望成为治疗靶点。拟采用小鼠VILI模型，利用慢病毒转染、ChIP等手段，在分子、细胞及整体水平阐明正压通气中lncRNA-EPS作为刹车分子，在转录水平调控VILI相关炎症反应。研究结果有助于明确VILI炎症激活和组织损伤新机制，为进一步优化通气策略提供理论和实验依据。

炎症反应造成的生物性损伤在呼吸机诱导肺损伤（VILI）中发挥关键作用，对肺组织造成二次打击，影响预后。如能在转录及转录后水平抑制炎症激活可减轻VILI。lncRNA参与损伤后表观调控的研究日益深化，但在VILI炎症调控中作用尚不清楚。我们的研究工作发现，大潮气量机械通气成功构建VILI模型，转录组学分析显示炎症反应、氧化应激、免疫炎症细胞浸润及相应调控信号通路参与VILI发生发展；然后，VILI小鼠肺组织及肺泡巨噬细胞NLRP3炎症小体激活，肺泡巨噬细胞向M1极化，予以TAK242或SN50干预TLR4/NF-κB信号通路后可抑制NLRP3炎症小体活化，逆转巨噬细胞而向M2极化，从而减轻VILI；最后，基于TLR4/NF-κB信号通路调控，lncEPS在VILI小鼠模型肺泡巨噬细胞中表达受抑制，过表达lncEPS可抑制NLRP3炎症小体激活，减轻VILI炎症反应。lncEPS可能是防治VILI生物性损伤的潜在靶点。我们以肺泡巨噬细胞炎症反应与细胞焦亡为切入点，构建小鼠VILI模型及分离纯化肺泡巨噬细胞，利用转录组测序、原位杂交、腺相关病毒转染等方法，在分子、细胞及动物水平阐明VILI肺泡巨噬细胞NLRP3炎症小体活化调控与功能变化，揭示了lncEPS作为关键“炎症刹车分子”在转录水平调控VILI炎症反应，lncEPS在VILI防治提供的潜在价值。