mtDNA对脓毒症肺损伤的放大作用及机制研究

Systemic inflammatory response syndrome (SIRS) is a serious disease condition upon severe trauma, shock and sepsis. Recent studies indicated that bacterial infection was not the only factor under SIRS. mtDNA release was detrimentally affect bacterial infection-induced SIRS. mtDNA/TLR9 plays a crucial role in acute respiratory distress syndrome (ARDS) in sepsis-induced lung injury. Our previous studies suggested that mtDNA enlarged inflammatory responses triggered by LPS in alveolar macrophages. However, inhibition of sepsis-induced lung injury through suppressing TLR9 expression and blocking mtDNA release has not been clearly clarified. In our current proposal, we will interrupt TLR9 expression by TLR9 siRNA and subsequently focus on investigating the role of mtDNA in LPS-provoked inflammatory responses in alveolar macrophages to further elucidate underlying mechanisms by which mtDNA release exacerbates inflammatory pathologies.we will inject mtDNA into the mice with LPS-induced lung injury by intravenous injection to imitate sepsis-induced lung injury in clinic,and we will inject improved in vivo-jetPEITM mediated TLR9 siRNA by intratracheal instillation to inhibit the enlargement induced by mtDNA release in sepsis. We will offer new avenues for the clinical treatment of lung injury in sepsis.

最新研究提示细菌感染并不是导致SIRS的唯一致病因素。严重创伤、休克及脓毒症时，mtDNA释放入血可放大细菌感染诱发的SIRS，mtDNA介导TLR9活化导致肺内炎症的放大是脓毒症肺损伤发生发展的另一关键。我们的前期研究提示mtDNA能放大LPS诱导的肺泡巨噬细胞炎症反应。然而，抑制肺泡巨噬细胞TLR9表达，阻断mtDNA对脓毒症肺损伤的放大作用的研究未见报道。本项目拟将TLR9 siRNA转染肺泡巨噬细胞，阻断mtDNA对肺泡巨噬细胞TLR9的作用，观察并分析其对mtDNA放大LPS诱导的肺泡巨噬细胞炎症反应作用的影响及机制；并将纯化的mtDNA静脉注入LPS诱导小鼠肺损伤模型中，以模拟临床上脓毒症肺损伤的过程，将改进型聚乙烯亚胺in vivo-jetPEITM介导的TLR9 siRNA经小鼠气道内滴入，阻断mtDNA对脓毒症肺损伤放大的作用，为临床治疗脓毒症肺损伤提供新途径。

脓毒症是机体对感染反应失调引起的危及生命的器官功能障碍，最易引起急性肺损伤，其死亡率高达40％，已成为脓毒症患者死亡的首位原因。近来研究发现，严重创伤、休克、脓毒症时，血浆mtDNA水平显著升高，表明mtDNA与炎症密切相关。mtDNA含有大量未甲基化CpG序列，可特异性识别tlr9从而引发炎症反应。在细胞水平，本课题选用肺泡巨噬细胞作为研究对象，将TLR9 siRNA转染肺泡巨噬细胞下调tlr9表达，再用lps及mtDNA刺激肺泡巨细胞，通过观察tlr9/MyD88/NF-KB信号通路及下游炎症因子表达的影响，分析mtDNA对lps诱导肺泡巨噬细胞炎症反应调控的可能机制。在动物水平，本课题采取腹腔注射lps构建脓毒症肺损伤模型，气管滴入tlr9 siRNA，再用lps及mtDNA对小鼠进行刺激，观察其肺组织HE染色、W/D、炎症因子等表达，探讨抑制tlr9的表达可以阻断mtDNA对脓毒症肺损伤的放大作用。我们按原计划开展了实验研究，并取得了预期的研究成果。（1）成功提取mtDNA及下调tlr9，为后续实验奠定基础；（2）体外实验中，mtDNA+lps组的tlr9、MyD88、NF-KB、炎症因子的表达及阳性细胞凋亡数比lps组显著升高，而转染tlr9 siRNA抑制mtDNA对lps炎症的放大作用；（3）体内实验中，同lps组相比，mtDNA+lps组肺组织损伤程度更为严重，W/D、炎症因子及免疫组化检测到的tlr9、MyD88、NF-KB表达显著升高，而气管滴入tlr9 siRNA可显著改善上述表现。体内外实验结果表明，mtDNA通过tlr9/MyD88/NF-KB通路放大脓毒症肺损伤。该研究的发现或许为未来临床上治疗脓毒症肺损伤提供新途径。