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ORIGINAL ARTICLE
Year : 2018  |  Volume : 131  |  Issue : 10  |  Page : 1174-1184

Mitochondrial Autophagy and NLRP3 Inflammasome in Pulmonary Tissues from Severe Combined Immunodeficient Mice after Cardiac Arrest and Cardiopulmonary Resuscitation


1 Department of Emergency, Renmin Hospital of Wuhan University, Wuhan, Hubei 430060, China
2 Department of Medicine, Central Arkansas Veterans Healthcare System, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA
3 Department of Emergency, Peking Union Medical College Hospital, Beijing 100032, China

Correspondence Address:
Dr. Jing-Jun Lyu
Department of Emergency, Renmin Hospital of Wuhan University, Wuhan, Hubei 430060
China
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0366-6999.231519

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Background: The incidence of cancer, diabetes, and autoimmune diseases has been increasing. Furthermore, there are more and more patients with solid organ transplants. The survival rate of these immunocompromised individuals is extremely low when they are severely hit-on. In this study, we established cardiac arrest cardiopulmonary resuscitation (CPR) model in severe combined immunodeficient (SCID) mice, analyzed the expression and activation of mitochondrial autophagy and NLRP3 inflammasome/caspase-1, and explored mitochondrial repair and inflammatory injury in immunodeficiency individual during systemic ischemia-reperfusion injury. Methods: A potassium chloride-induced cardiac arrest model was established in C57BL/6 and nonobese diabetic/SCID (NOD/SCID) mice. One hundred male C57BL/6 mice and 100 male NOD/SCID mice were randomly divided into five groups (control, 2 h post-CPR, 12 h post-CPR, 24 h post-CPR, and 48 h post-CPR). A temporal dynamic view of alveolar epithelial cells, macrophages, and neutrophils from bronchoalveolar lavage fluid (BALF) was obtained using Giemsa staining. Spatial characterization of phenotypic analysis of macrophages in the lung interstitial tissue was analyzed by flow cytometry. The morphological changes of mitochondria 48 h after CPR were studied by transmission electron microscopy and quantified according to the Flameng grading system. Western blotting analysis was used to detect the expression and activation of the markers of mitochondrial autophagy, NLRP3 inflammasome, and caspase-1. Results: (1) In NOD/SCID mice, macrophages were disintegrated in BALF, and many alveolar epithelial cells were shed at 48 h after resuscitation. Compared with C57BL/6 mice, the ratio of macrophages/total cells peaked at 12 h and was significantly higher in NOD/SCID mice (31.17 ± 4.13 vs. 49.69 ± 2.43, t = 14.46, P = 0.001). After 24 h, the results showed a downward trend. Furthermore, a large number of macrophages were disintegrated in the BALF. (2) Mitochondrial autophagy was present in both C57BL/6 and NOD/SCID mice after CPR, but it began late in the NOD/SCID mice. Compared with C57BL/6 mice, phos-ULK1 (Ser327) expression was significantly lower at 2 h and 12 h after CPR (2 h after CPR: 1.88 ± 0.36 vs. 1.12 ± 0.11, t = −1.36, P < 0.01 and 12 h after CPR: 1.52 ± 0.16 vs. 1.05 ± 0.12, t = −0.33, P < 0.01), whereas phos-ULK1 (Ser757) expression was significantly higher at 2 h and 12 h after CPR in NOD/SCID mice (2 h after CPR: 1.28 ± 0.12 vs. 1.69 ± 0.14, t = 1.7, P < 0.01 and 12 h after CPR: 1.33 ± 0.10 vs. 1.94 ± 0.13, t = 2.75, P < 0.01). (3) Furthermore, NLRP3 inflammasome/caspase-1 activation in the pulmonary tissues occurred early and for only a short time in C57BL/6 mice, but this phenomenon was sustained in NOD/SCID mice. The expression of the NLRP3 inflammasome increased modestly in the C57 mice, but the increase was higher in the NOD/SCID mice than in the C57BL/6 mice, especially at 12, 24, 48 h after CPR (48 h after CPR: 1.46 ± 0.13 vs. 2.97 ± 0.19, t = 5.34, P = 0.001). The expression of caspase-1-20 generally followed the same pattern as the NLRP3 inflammasome. Conclusions: There is a regulatory relationship between the NLRP3 inflammasome and mitochondrial autophagy after CPR in the healthy mice. This regulatory relationship was disturbed in the NOD/SCID mice because the signals for mitochondrial autophagy occurred late, and NLRP3 inflammasome- and caspase-1-dependent cell injury was sustained.

 

 Abstract in Chinese

严重联合免疫缺陷小鼠心脏骤停心肺复苏后肺组织线粒体自噬与NLRP3炎症小体

摘要

背景:肿瘤、糖尿病、自身免疫系统疾病发病率逐年升高,实体器官移植患者越来越多,这些免疫缺陷个体遭受严重打击时存活率极低。本研究应用严重联合免疫缺陷小鼠建立心搏骤停心肺复苏模型,分析线粒体自噬和NLRP3炎症小体/caspase-1的表达和活化,探讨在系统性缺血再灌注损伤过程中,免疫缺陷个体的线粒体修复和炎症损伤。

方法:应用C57BL/6 小鼠和严重联合免疫缺陷(NOD/SCID)小鼠建立心搏骤停心肺复苏模型。100只雄性C57BL/6小鼠和100只雄性NOD/SCID小鼠随机分组,采用Giemsa染色对各组小鼠心肺复苏前、复苏后2h、12h、24h、36h、48h支气管肺泡灌洗液(BALF)中的肺泡上皮细胞、巨噬细胞和中性粒细胞进行分类计数;采用流式细胞术分析各组小鼠心肺复苏后肺间质组织中巨噬细胞表型;使用透射电镜观察复苏后不同时间点线粒体形态,采用Flameng 分级方法对线粒体损伤进行半定量分析;采用Western blot法检测线粒体自噬流(autophagic flux),以及NLRP3炎症小体/caspase-1表达和激活。

结果:NOD/SCID小鼠肺泡灌洗液中巨噬细胞崩解焦亡,大量肺泡上皮细胞脱落。C57BL/6和NOD /SCID小鼠在心肺复苏后都出现线粒体自噬,但NOD/ SCID小鼠启动得较晚。C57BL/6小鼠肺组织中NLRP3炎症小体/caspase-1表达和激活发生在复苏后早期,并且只持续短暂时间,NOD/SCID小鼠肺组织中NLRP3炎症小体/caspase-1激活持续存在。

结论:在心搏骤停心肺复苏诱导缺血再灌注损伤过程中,线粒体自噬和NLRP3 炎症小体之间存在着负调节关系。免疫功能正常的C57BL/6 小鼠在心肺复苏早期及时启动线粒体自噬,抑制NLRP3 炎症小体持续高表达。NOD / SCID 小鼠在心肺复苏早期没有正确启动线粒体自噬,致使线粒体ROS→NLRP3 炎症小体→Caspase1→线粒体ROS 这一信号环路持续扩增,持续扩大炎症反应,导致Caspase1 依赖性细胞焦亡。



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