脓毒症巨噬细胞焦亡调控新机制：circRNA_101401/ miRNA-590-3p/PP2A信号通路研究

Sepsis is characterized by high mortality and dysregulated inflammatory response is its essential feature. Macrophage pyroptosis is an important trigger in inflammatory cascade of sepsis and blocking this process has important implications. In previous study we have found that circRNA_101401 and PP2A was significantly elevated in patients with sepsis, and there is a close correlation between PP2A, TAK1 and pyroptosis genes. This result shows that circRNA_101401 can be used as a new target for the pyroptosis regulation, but the exact mechanism is still unclear. MiRNA-590-3p is predicted to be the intermediate link between circRNA_101401 and PP2A by bioinformatics. PP2A can bind to and negatively regulate TAK1, which is a key molecule in the caspase-8 mediated macrophage pyroptosis. We hypothesize that circRNA_101401 can adsorb miRNA-590-3p, which inhibits the action of target gene PP2A and regulate macrophage pyroptosis through PP2A/TAK1 signaling pathway. In this study, we plan to further demonstrate that circRNA_101401 plays an important role in macrophage pyroptosis regulation in the septic model in vitro and vivo and clarify the molecular mechanism of circRNA_101401/miRNA-590-3p/PP2A signaling pathway. This study would provide novel target for sepsis therapy.

脓毒症病死率居高不下，失控炎症反应是其本质特征。巨噬细胞焦亡是失控炎症反应的重要环节，阻断该过程意义重大。Caspase-8是最新发现介导焦亡的新途径，上游机制不明。我们预实验发现与焦亡相关的circRNA_101401与PP2A在脓毒症患者中显著增加，提示circRNA_101401可作为调控焦亡重要靶点。生信分析提示miRNA-590-3p与circRNA_101401和PP2A均存在结合位点，而PP2A可能与caspase-8介导的焦亡途径关键分子TAK1相结合、负向调控TAK1。推测circRNA_101401可吸附miR-590-3p，解除其对PP2A的抑制，激活PP2A/TAK1通路调控焦亡。本课题拟分子层面阐明circRNA_101401/miR-590-3p/PP2A通路调控焦亡的机制，动物层面证实下调circRNA_101401抑制巨噬细胞焦亡，为脓毒症治疗提供新靶点。

脓毒症（sepsis）是临床常见的危重症，失控的炎症反应是其本质特征。既往研究证实，巨噬细胞焦亡是导致脓毒症失控炎症反应的重要环节，探寻巨噬细胞焦亡的细胞分子机制是临床急需解决的重要课题。课题组既往对不同严重程度的患者全血进行转录组水平测序发现circRNPS1在不同严重程度的脓毒症患者外周血中差异表达，且与脓毒症严重程度显著相关，生信分析提示circRNPS1与调控巨噬细胞焦亡的下游分子TAK1具有良好的相关性。本课题通过构建慢病毒载体并感染THP-1细胞调控circRNPS1的表达，在此基础上采用LPS+Nig诱导巨噬细胞焦亡，并通过WB检测GSDMD-N表达水平、ELISA检测IL-1β、IL-18水平及LDH释放试验，结果发现高表达circRNPS1可加重巨噬细胞的焦亡程度，使得炎症因子释放增加，而低表达circRNPS1减少巨噬细胞焦亡，从而证实了circRNPS1对巨噬细胞焦亡的调控作用。为进一步明确circRNPS1调控的机制，针对circRNPS1进行了成环鉴定实验、核质分离实验和原位杂交实验，结果提示，circRNPS1是一具有稳定环状结构的环状RNA，其主要分布于细胞质，ceRNA机制可能是circRNPS1发挥生物学功能的重要机制。为此，通过circBase、target scan对下游miRNA及mRNA进行预测，并通过qPCR与WB进行验证，发现circRNPS1通过ceRNA机制调控miR-590-3p，进而影响靶基因TAK1的表达，从而调控巨噬细胞焦亡。上调miRNA-590-3p或阻断TAK1，可减少脓毒症小鼠巨噬细胞焦亡比例，改善脓毒症相关肺损伤，减少炎症因子释放，可能是未来脓毒症治疗的重要策略。考虑到circRNPS1人鼠不同源，而miR-590-3p人鼠同源性较好，因此构建高表达miR-590-3p的agomir尾静脉注射，调控全身miR-590-3p表达，发现高表达减少巨噬细胞焦亡、减轻肺损伤，改善小鼠预后。为进一步明确全血circRNPS1水平与脓毒症患者病情严重程度及预后的相关性，留取52例脓毒症患者及14例健康志愿者的外周血，发现脓毒症患者外周血中circRNPS1水平显著高于健康志愿者，且circRNPS1水平与SOFA评分正相关，circRNPS1对脓毒症严重程度及预后具有良好的预测价值。