沉默lncRNA-5657对脓毒症大鼠肺损伤的保护作用及机制研究

Inhibition of over-activation of phagocytes in lungs is the main way to protect from septic lung injury. The current studies showed that lncRNA was not only involved in the pathogen-related immune reaction, but also modulated the monocyte-phagocyte inflammatory response through the interaction with protein factors. However, there is no report on the roles of lncRNA in the phagocytes and inflammatory response in the lungs of rats. Our previous study showed that the lncRNA-5657 expression in the alveolar phagocytes of LPS challenged rats was increased measured by HTseq and RT-qPCR, and the LPS induced TNF-α release was decreased by silencing the lncRNA-5677. Thus we hypothesize that the lncRNA-5657 may enhance the inflammatory reaction in the alveolar phagocytes through the interaction with a special protein. This study will explore the protective effects and mechanism on lung injury using vivo-jetPEI mediated lncRNA-5657 siRNA in the rats of the acute lung injury, and detect the special protein interacting with lncRNA-5657 by in vitro RNA-pulldown and MS analysis. This study will provide a new way to treat acute lung injury through the mechanism of lncRNA-5657.

抑制肺泡巨噬细胞（AM）过度激活是防止脓毒症肺损伤根本措施，研究表明lncRNA参与了单核-巨噬细胞炎症反应的调控，与蛋白相互作用是其发挥生物学功能的重要途径，但lncRNA在大鼠AM炎症反应中的作用未见报道。我们前期采用高通量测序及RT-qPCR验证发现，lncRNA-5657在LPS诱导的大鼠AM炎症反应中表达上调，沉默lncRNA-5657能减少TNFα等炎症因子释放。因此，我们假设lncRNA-5657通过与大鼠AM内某一特异蛋白相互作用，进而促进炎症反应。本项目拟在前期研究基础上，将in vivo-jetPEI介导的lncRNA-5657siRNA经气道滴入大鼠脓毒症肺损伤模型中，探讨其对肺损伤的保护作用；并在大鼠AM内通过RNA-pulldown实验联合质谱分析，鉴定与lncRNA-5657相互作用的蛋白，探讨lncRNA-5657的促炎机制，为治疗脓毒症肺损伤提供新途径。

研究表明lncRNAs与多种人类重大疾病的发生发展密切相关，然而有关lncRNA在脓毒症肺损伤中的作用尚不明确。近来研究表明lncRNA能够通过调节炎症相关介质的转录从而调控炎症反应，然而有关lncRNA对肺泡巨噬细胞炎症反应的调控作用及机制仍未明确。本项目通过RNA高通量测序鉴定出LPS刺激肺泡巨噬细胞后早期上调最明显的lncRNA-5657，并通过过表达和沉默lncRNA-5657，探讨其对脓毒症时肺内炎症反应的作用及机制，结果发现：（1）LPS刺激肺泡巨噬细胞后lncRNA 表达谱显示lncRNA-5657表达上调最早且最显著；（2）在肺泡巨噬细胞中过表达或沉默lncRNA-5657后，炎症因子和spns2的表达相应的增加或降低，双荧光素酶报告基因结果表明lncRNA-5657作用于靶基因spns2的启动子区域；沉默spns2使得LPS诱导的肺泡巨噬细胞炎症反应减弱，并且能阻断lncRNA-5657在肺泡巨噬细胞内的促炎作用；（3）脓毒症肺损伤病人BALF及CLP脓毒症肺损伤大鼠肺内lncRNA-5657表达增加，沉默lncRNA-5657后CLP脓毒症肺损伤大鼠肺内炎症反应减轻。综上所述：lncRNA-5657与脓毒症肺损伤密切相关，体内外实验均证实沉默lncRNA-5657能够减轻脓毒症肺损伤，其机制是通过抑制spns2的表达从而减轻肺内炎症反应。