巨噬细胞lncRNA-Cox2调控的炎症反应在脓毒症心肌损伤中的作用和机制

Myocardial injury is one of the main lethal factors in patients with sepsis. It has been reported that long non-coding RNAs (lncRNAs) are involved in the synthetic regulation of inflammatory factors in macrophages. Our previous studies found that lncRNA-Cox2 expression and cellular infiltration of macrophages were significantly increased in the myocardial tissue of the septic mouse model, indicating that inflammatory responses mediated by macrophage-produced lncRNA-Cox2 are probably related with sepsis-induced myocardial injury. In the present study, we will further (1) investigate the correlation between the expression of lncRNA-Cox2 and macrophages infiltration in the myocardium of the septic mouse model by cecal ligation and perforation, (2) confirm the effects induced by over-expression or knock-down of lncRNA-Cox2 in macrophages on the myocardial injury of the septic mouse model using bone marrow reconstruction, (3) clarify the molecular mechanisms of macrophage-produced lncRNA-Cox2 and its targeted inflammatory factor genes involved in sepsis-induced myocardial injury. The findings would be helpful to elucidate the role and related mechanism of macrophage-produced lncRNA-Cox2 in sepsis-induced myocardial injury and develop new therapeutic strategies for the treatment of septic myocardial injury.

心肌损伤是脓毒症患者重要致死因素之一。已有报道表明，长链非编码RNA（lncRNA）可参与调控巨噬细胞介导的炎症反应。我们前期研究发现，小鼠脓毒症模型心肌组织中lncRNA-Cox2表达显著增加、并有大量巨噬细胞浸润，提示有可能巨噬细胞通过lncRNA-Cox2调控的炎症反应参与脓毒症心肌损伤。本研究以lncRNA-Cox2为切入点，以小鼠脓毒症模型为研究对象，进一步（1）在盲肠结扎法构建的小鼠脓毒症模型中，研究巨噬细胞lncRNA-Cox2的表达水平与脓毒症心肌损伤的相关性；（2）应用小鼠骨髓重建技术，研究特异性干预巨噬细胞lncRNA-Cox2表达对脓毒症心肌损伤的影响；（3）应用体外脂多糖处理的细胞模型，明确脓毒症诱导巨噬细胞lncRNA-Cox2表达从而调控炎症因子合成的机制。本项目研究结果将为阐明脓毒症心肌损伤的新机制、并为发现脓毒症心肌损伤新的防治策略提供依据。

心肌损伤是脓毒症患者重要致死因素之一。长链非编码RNA（lncRNA）可参与调控吞噬细胞介导的炎症反应。我们前期研究发现，脓毒症心肌组织lncRNA-Cox2表达显著增加、并有巨噬细胞浸润，提示巨噬细胞可能通过lncRNA-Cox2调控的炎症反应参与脓毒症心肌损伤。本研究以lncRNA-Cox2为切入点，（1）在小鼠脓毒症模型中，发现巨噬细胞lncRNA-Cox2在脓毒症小鼠心肌组织中表达增加，与心肌巨噬细胞浸润、心肌病理学、组织炎症因子（TNF-α、IL-1β和IL-6）、心肌损伤标记物（CTnI和NT-proBNP）和心功能指标具有一定的相关性。分别采用慢病毒载体系统上调lncRNA-Cox2表达，siRNA下调lncRNA-Cox2表达水平，尾静脉注射小鼠体内，构建脓毒症模型。发现，慢病毒过表达载体处理组小鼠心肌组织lncRNA-Cox2、巨噬细胞浸润、炎症因子（TNF-α、IL-1β和IL-6）水平、心肌组织病理学病变增加，siRNA处理组小鼠上述指标发生不同程度的改善。（2）在脂多糖刺激的细胞模型中，发现心肌细胞经脂多糖刺激后hnRNP A/B、A2/B1表达增加，shRNA特异性干预hnRNP A/B、A2/B1后可见hnRNP A/B、A2/B1表达相应降低。表明，巨噬细胞lncRNA-Cox2通过hnRNP A/B、A2/B1上调TNF-α、IL-1β、IL-6等炎症因子水平，促进脓毒症心肌细胞损伤。在本实验的基础上，我们还发现脓毒症心肌焦亡相关蛋白NLRP3、caspase-1及细胞因子IL-10、IL-1β表达增加，TUNEL和Hoechst-PI染色可见心肌细胞凋亡。进一步，我们分离培养骨髓间充质干细胞（MSCs）并提取外泌体，经尾静脉注射大鼠体内。发现，注射外泌体的脓毒症大鼠心肌焦亡相关蛋白NLRP3、caspase-1及细胞因子IL-10、IL-1β表达下降。体外细胞水平，脂多糖刺激心肌细胞JAK2、STAT3信号通路活化，焦亡相关蛋白IL-18、IL-1β、caspase-1蛋白表达明显增强，经外泌体处理的心肌细胞上述指标下降。表明，骨髓间充质干细胞来源的外泌体通过JAK2/STAT3信号通路减少脓毒症心肌焦亡，对脓毒症心肌具有保护作用。上述研究结果为今后临床干预脓毒症心肌损伤提供新的思路和方法，具有深远的经济效应和社会意义。