Gu Xiaoling, Wu Guannan, Yao Yanwen, Zeng Junli, Shi Donghong, Lv Tangfeng, Luo Liang, Song Yong
Department of Respiratory Medicine, Jinling Hospital, Nanjing University School of Medicine, Nanjing, Jiangsu Province 210002, People's Republic of China.
Department of Medical Imaging, Jinling Hospital, Nanjing University School of Medicine, Nanjing, Jiangsu Province 210002, People's Republic of China.
Free Radic Biol Med. 2015 Jun;83:149-58. doi: 10.1016/j.freeradbiomed.2015.02.034. Epub 2015 Mar 13.
An increasing number of studies have focused on the phenomenon that mitochondrial DNA (mtDNA) activates innate immunity responses. However, the specific role of mtDNA in inflammatory lung disease remains elusive. This study was designed to examine the proinflammatory effects of mtDNA in lungs and to investigate the putative mechanisms. C57BL/6 mice were challenged intratracheally with mtDNA with or without pretreatment with chloroquine. Changes in pulmonary histopathology, cytokine concentrations, and phosphorylation levels of p38 MAPK were assayed at four time points. In in vitro experiments, THP-1 macrophages were pretreated or not pretreated with chloroquine, TLR9 siRNA, p38 MAPK siRNA, or SB203580 and then incubated with mtDNA. The levels of cytokines and p-p38 MAPK were detected by ELISA and Western blot, respectively. The intratracheal administration of mtDNA induced infiltration of inflammatory cells, production of proinflammatory cytokines (including IL-1β, IL-6, and TNF-α), and activation of p38 MAPK. The chloroquine pretreatment resulted in an abatement of mtDNA-induced local lung inflammation. In vitro experiments showed that the exposure of THP-1 macrophages to mtDNA also led to a significant upregulation of IL-1β, IL-6, and TNF-α and the activation of p38 MAPK. And these responses were inhibited either by chloroquine and TLR9 siRNA or by SB203580 and p38 MAPK siRNA pretreatment. The intratracheal administration of mtDNA induced a local inflammatory response in the mouse lung that depended on the interactions of mtDNA with TLR9 and may be correlated with infiltrating macrophages that could be activated by mtDNA exposure via the TLR9-p38 MAPK signal transduction pathway.
越来越多的研究聚焦于线粒体DNA（mtDNA）激活先天免疫反应这一现象。然而，mtDNA在炎症性肺病中的具体作用仍不清楚。本研究旨在检测mtDNA在肺中的促炎作用并探究其潜在机制。将C57BL/6小鼠经气管内给予mtDNA，部分小鼠在给予mtDNA前用氯喹预处理。在四个时间点检测肺组织病理学变化、细胞因子浓度以及p38丝裂原活化蛋白激酶（p38 MAPK）的磷酸化水平。在体外实验中，用氯喹、Toll样受体9（TLR9）小干扰RNA（siRNA）、p38 MAPK siRNA或SB203580对人单核细胞白血病细胞系（THP-1）巨噬细胞进行预处理或不进行预处理，然后与mtDNA共同孵育。分别通过酶联免疫吸附测定（ELISA）和蛋白质印迹法检测细胞因子水平和磷酸化p38 MAPK（p-p38 MAPK）水平。气管内给予mtDNA可诱导炎症细胞浸润、促炎细胞因子（包括白细胞介素-1β（IL-1β）、白细胞介素-6（IL-6）和肿瘤坏死因子-α（TNF-α））的产生以及p38 MAPK的激活。氯喹预处理可减轻mtDNA诱导的局部肺部炎症。体外实验表明，THP-1巨噬细胞暴露于mtDNA也会导致IL-1β、IL-6和TNF-α显著上调以及p38 MAPK的激活。并且这些反应可被氯喹和TLR9 siRNA或SB203580和p38 MAPK siRNA预处理所抑制。气管内给予mtDNA可在小鼠肺中诱导局部炎症反应，该反应依赖于mtDNA与TLR9的相互作用，并且可能与浸润的巨噬细胞相关，这些巨噬细胞可通过TLR9-p38 MAPK信号转导途径被mtDNA暴露激活。
