自噬在失血性休克小鼠急性肺损伤中的生物学作用及相关机制

Biological role and related mechanism of autophagy in acute lung injury of hemorrhagic shock mice

目的探讨自噬在失血性休克小鼠急性肺损伤(ALI)中的生物学作用及机制。方法按随机数字表法将野生型雄性C57BL/6小鼠分为对照组、ALI组、雷帕霉素组、3-甲基腺嘌呤(3-MA)组,每组8只;将自噬微管相关蛋白轻链3(LC3)基因敲除的C57BL/6小鼠分为LC3敲除组和LC3敲除+ALI组,每组8只。对照组、ALI组、LC3敲除组、LC3敲除+ALI组腹腔注射2 mL/kg生理盐水,雷帕霉素组腹腔注射3 mg/kg自噬激活剂雷帕霉素,3-MA组腹腔注射15 mg/kg自噬抑制剂3-MA,均连续给药3 d,末次给药后2 h制备失血性休克致ALI模型。制模后24 h处死小鼠,计算肺指数;苏木素-伊红(HE)染色观察肺组织病理改变并计算肺损伤评分;蛋白质免疫印迹试验(Western blotting)检测肺组织自噬基因LC3-Ⅱ/LC3-Ⅰ、Beclin-1的表达;按试剂盒步骤检测肺组织肿瘤坏死因子-α(TNF-α)、白细胞介素-6(IL-6)、丙二醛(MDA)含量。结果与对照组比较,ALI组肺组织结构破坏、渗出增多,肺指数、肺损伤评分、肺组织中LC3-Ⅱ/LC3-Ⅰ、Beclin-1表达及TNF-α、IL-6、MDA含量均明显升高。与ALI组比较,雷帕霉素组肺组织结构破坏减轻、渗出减少,肺指数、肺损伤评分及肺组织中TNF-α、IL-6、MDA含量降低,肺组织中LC3-Ⅱ/LC3-Ⅰ、Beclin-1表达升高〔肺指数:(7.56±0.39)%比(9.12±0.59)%,肺损伤评分(分):3.04±0.58比9.32±2.14,TNF-α(ng/mg):1.85±0.32比3.51±0.62,IL-6(ng/mg):1.61±0.32比2.52±0.44,MDA(nmol/mg):1.03±0.16比1.88±0.24,LC3-Ⅱ/LC3-Ⅰ:1.21±0.12比0.39±0.05,Beclin-1/β-actin:1.10±0.12比0.58±0.06,均P<0.05〕,而3-MA组肺组织结构破坏加重、渗出进一步增多,肺指数、肺损伤评分及肺组织中TNF-α、IL-6、MDA含量升高,肺组织中LC3-Ⅱ/LC3-Ⅰ、Beclin-1表达降低〔肺指数:(10.44±0.62)%比(9.12±0.59)%,肺损伤评分(分):11.59±2.28比9.32±2.14,TNF-α(ng/mg):4.77±0.71比3.51±0.62,IL-6(ng/mg):3.44±0.52比2.52±0.44,MDA(nmol/mg):2.71±0.42比1.88±0.24,LC3-Ⅱ/LC3-Ⅰ:0.25±0.04比0.39±0.05,Beclin-1/β-actin:0.21±0.03比0.58±0.06,均P<0.05〕。LC3敲除ALI小鼠肺指数、肺损伤评分及肺组织中TNF-α、IL-6、MDA含量均高于野生型ALI小鼠〔肺指数:(10.44±0.75)%比(9.12±0.59)%,肺损伤评分(分):12.41±2.86比9.32±2.14,TNF-α(ng/mg):4.85±0.72比3.51±0.62,IL-6(ng/mg):3.28±0.51比2.52±0.44,MDA(nmol/mg):2.75±0.41比1.88±0.24,均P<0.05〕。结论自噬在失血性休克小鼠ALI中起保护作用,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-Ⅰand 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-Ⅰand 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-Ⅰand 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].Conclusion 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.