目的:研究自噬在失血性休克小鼠急性肺损伤(ALI)中的生物学作用及相关机制。
方法:根据随机数字表法,将野生型雄性C57BL/6小鼠分为对照组,ALI集团,雷帕霉素组和3-甲基腺嘌呤(3-MA)组,每组8只小鼠。将具有C57BL/6背景的轻链3(LC3)基因敲除小鼠分为LC3敲除组和LC3敲除+ALI组,每组8只小鼠。对照组,ALI集团,LC3基因敲除组,LC3敲除+ALI组腹腔注射生理盐水2mL/kg,雷帕霉素组腹腔注射自噬激活剂雷帕霉素3mg/kg,3-MA组腹腔注射自噬抑制剂3-MA15mg/kg,所有这些都连续给予3天。上次给药后2小时,建立失血性休克诱导的ALI模型。建模后24小时,计算肺指数。采用苏木精-伊红(HE)染色观察肺组织病理变化,并进行肺损伤评分。免疫印迹法检测肺组织中自噬基因LC3-Ⅱ/LC3-Ⅰ和Beclin-1的表达。肿瘤坏死因子-α(TNF-α)的含量,按照试剂盒的步骤检测肺组织中的白细胞介素-6(IL-6)和丙二醛(MDA)。
结果:与对照组相比,肺组织结构被破坏,渗出增加,肺指数,肺损伤评分,LC3-II/LC3-I的表达式,Beclin-1和TNF-α的含量,ALI组肺组织中IL-6和MDA显著增高。与ALI组相比,雷帕霉素组肺组织的结构损伤和渗出减少,肺指数,肺损伤评分和TNF-α含量,肺组织中IL-6和MDA降低,而肺组织中LC3-II/LC3-I和Beclin-1的表达增加[肺指数:(7.56±0.39)%vs.(9.12±0.59)%,肺损伤评分:3.04±0.58vs.9.32±2.14,TNF-α(ng/mg):1.85±0.32vs.3.51±0.62,IL-6(ng/mg):1.61±0.32vs.2.52±0.44,MDA(nmol/mg):1.03±0.16vs.1.88±0.24,LC3-II/LC3-I:1.21±0.12vs.0.39±0.05,Beclin-1/β-肌动蛋白:1.10±0.12vs.0.58±0.06,均P<0.05],而3-MA组肺组织结构损伤加重,渗出物进一步增多,肺指数,肺损伤评分和TNF-α含量,肺组织中IL-6和MDA升高,肺组织中LC3-II/LC3-I和Beclin-1的表达降低[肺指数:(10.44±0.62)%vs.(9.12±0.59)%,肺损伤评分:11.59±2.28vs.9.32±2.14,TNF-α(ng/mg):4.77±0.71vs.3.51±0.62,IL-6(ng/mg):3.44±0.52vs.2.52±0.44,MDA(nmol/mg):2.71±0.42vs.1.88±0.24,LC3-II/LC3-I:0.25±0.04vs.0.39±0.05,Beclin-1/β-肌动蛋白:0.21±0.03vs.0.58±0.06,均P<0.05。肺指数,肺损伤评分和TNF-α含量,LC3敲除ALI小鼠肺组织中IL-6和MDA高于野生型ALI小鼠[肺指数:(10.44±0.75)%vs.(9.12±0.59)%,肺损伤评分:12.41±2.86vs.9.32±2.14,TNF-α(ng/mg):4.85±0.72vs.3.51±0.62,IL-6(ng/mg):3.28±0.51vs.2.52±0.44,MDA(nmol/mg):2.75±0.41vs.1.88±0.24,均P<0.05]。
结论:自噬对失血性休克小鼠ALI具有保护作用,相关的分子机制是抑制炎症反应和氧化应激反应。
OBJECTIVE: To study the biological role and related mechanism of autophagy in acute lung injury (ALI) of hemorrhagic shock mice.
METHODS: According to random number table method, wild-type male C57BL/6 mice were divided into control group, ALI group, rapamycin group and 3-methyladenine (3-MA) group, with 8 mice in each group. Light chain 3 (LC3) gene knockout mice with C57BL/6 background were divided into LC3 knockout group and LC3 knockout+ALI group, with 8 mice in each group. Control group, ALI group, LC3 knockout group, LC3 knockout+ALI group were intraperitoneally injected with 2 mL/kg normal saline, rapamycin group was intraperitoneally injected with 3 mg/kg autophagy activator rapamycin, 3-MA group was intraperitoneally injected with 15 mg/kg autophagy inhibitor 3-MA, all of which were given for 3 consecutive days. 2 hours after the last administration, the hemorrhagic shock induced ALI model was established. 24 hours after modeling, the lung index was calculated. Hematoxylin-eosin (HE) staining was used to observe the pathological changes of lung tissue and lung injury score was performed. The expressions of autophagy genes LC3- II/LC3- I and Beclin-1 in lung tissue were detected by Western blotting. The contents of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and malondialdehyde (MDA) in lung tissue were detected according to the steps of the kit.
RESULTS: Compared with the control group, the lung tissue structure was destroyed and exudation increased, lung index, lung injury score, the expressions of LC3- II/LC3- I, Beclin-1, and the contents of TNF-α, IL-6 and MDA in lung tissue significantly increased in the ALI group. Compared with the ALI group, the structural damage and exudation of lung tissue were reduced in the rapamycin group, lung index, lung injury score and the contents of TNF-α, IL-6 and MDA in lung tissue decreased, while the expressions of LC3- II/LC3- I and Beclin-1 in lung tissue increased [lung index: (7.56±0.39)% vs. (9.12±0.59)%, lung injury score: 3.04±0.58 vs. 9.32±2.14, TNF-α (ng/mg): 1.85±0.32 vs. 3.51±0.62, IL-6 (ng/mg): 1.61±0.32 vs. 2.52±0.44, MDA (nmol/mg): 1.03±0.16 vs. 1.88±0.24, LC3- II/LC3- I: 1.21±0.12 vs. 0.39±0.05, Beclin-1/β-actin: 1.10±0.12 vs. 0.58±0.06, all P < 0.05], while lung tissue structure damage was aggravated and exudation was further increased in the 3-MA group, lung index, lung injury score and the contents of TNF-α, IL-6 and MDA in lung tissue increased, the expressions of LC3- II/LC3- I and Beclin-1 in lung tissue decreased [lung index: (10.44±0.62)% vs. (9.12±0.59)%, lung injury score: 11.59±2.28 vs. 9.32±2.14, TNF-α (ng/mg): 4.77±0.71 vs. 3.51±0.62, IL-6 (ng/mg): 3.44±0.52 vs. 2.52±0.44, MDA (nmol/mg): 2.71±0.42 vs. 1.88±0.24, LC3- II/LC3- I: 0.25±0.04 vs. 0.39±0.05, Beclin-1/β-actin: 0.21±0.03 vs. 0.58±0.06, all P < 0.05]. Lung index, lung injury score and the contents of TNF-α, IL-6 and MDA in lung tissue of LC3 knockout ALI mice were higher than those of wild-type ALI mice [lung index: (10.44±0.75)% vs. (9.12±0.59)%, lung injury score: 12.41±2.86 vs. 9.32±2.14, TNF-α (ng/mg): 4.85±0.72 vs. 3.51±0.62, IL-6 (ng/mg): 3.28±0.51 vs. 2.52±0.44, MDA (nmol/mg): 2.75±0.41 vs. 1.88±0.24, all P < 0.05].
CONCLUSIONS: Autophagy plays a protective role in ALI of hemorrhagic shock mice, and the related molecular mechanism is the inhibition of inflammatory response and oxidative stress response.