摘要
目的探讨线粒体自噬在呼吸机相关性肺损伤(VILI)中的作用及其机制。方法将36只SPF级SD大鼠按随机数字表法分为自主呼吸组(CON组)、正常潮气量(VT)组(NVT组,VT为8mL/kg)、大VT组(HVT组,VT为40mL/kg)3组,每组12只。所有大鼠均气管切开后行气管插管,CON组保持自主呼吸,NVT组和HVT组分别给予相应VT的机械通气。通气4h后收集血清、支气管肺泡灌洗液(BALF)和肺组织标本,测定肺组织湿/干重比值(W/D),分别于光镜和透射电镜下观察肺组织病理学改变和Ⅱ型肺泡上皮细胞(AECⅡ)超微结构改变;采用酶联免疫吸附试验(ELISA)测定血清和BALF中白细胞介素(IL-1β、IL-6)及肿瘤坏死因子-α(TNF-α)水平;采用BCA法测定BALF中总蛋白水平,并进行细胞计数;采用实时荧光定量反转录一聚合酶链反应(RT—qPCR)和蛋白质免疫印迹试验(Western Blot)分别检测肺组织中微管相关蛋白1轻链3B(LC3B)、线粒体DNA编码的细胞色素C氧化酶Ⅳ(COX—IV)和核转录因子-κBp65(NF—κBp65)的mRNA及蛋白表达。结果CON组和NVT组肺组织及AECⅡ超微结构基本正常,HVT组则有明显的炎症性改变和病理学损伤。与CON组和NVT组比较,HVT组肺组织W/D比值明显升高(8.53±1.05比5.12±0.65、5.57±0.55,均P〈0.05),BALF中总蛋白、渗出细胞、IL-1β、IL-6和TNF-α含量明显升高[总蛋白(g/L):2.35±0.45比1.46±0.34、1.76±0.51,细胞计数(×10^5/mL):2.05±0.48比0.40±0.08、0.60±0.23,IL-1β(ng/L):119.82±6.56比76.15±3.32、79.39±4.44,IL-6(μL):4.10±0.52比1.97±0.40、2.27±0.36,TNF—α(mg/L):1.49±0.28比0.43±0.23、0.61±0.24,均P〈0.05],血清IL-1β、IL-6和TNF—α含量明显升高[IL-1β(ng/L):127.53±7.10比79.40±2.80、82.95±2.25,IL-6(μg/L):6.28±0.82比2.96±0.35、3.36±0.72,TNF—α(mg/L):1.59±0.42比0.53±0.22、0.78±0.25,均P〈0.05]。HVT组肺组织LC3B、COX—Ⅳ和NF—KBp65的mRNA及蛋白表达均较CON组和NVT组明显上调,HVT组LC3B-Ⅱ、COX—Ⅳ和NF—KBp65的mRNA表达分别是CON组的(3.52±0.90)、(3.76±1.16)和(9.54±2.06)倍,蛋白表达分别是CON组的(1.76±0.24)、(1.65±0.20)和(1.91±0.12)倍,差异均有统计学意义(均P〈0.05)。NVT组与CON组各项指标比较差异均无统计学意义。结论线粒体自噬参与了大鼠VILI,可能与逃逸的线粒体DNA激活炎症反应有关。
Objective To explore the role and mechanism of mitophagy in ventilator-induced lung injury (VILI) in rats. Methods Thirty-six adult Sprague-Dawley (SD) rats were randomly divided into three groups (each n = 12): spontaneous breathing group (CON group), normal tidal volume (VT) group (NVT group, VT was 8 mL/kg) and high VT group (HVT group, VT was 40 mL/kg). All rats received endotracheal tube by tracheostomy. The rats in CON group were maintained spontaneous breathing, while those in NVT and HVT groups received mechanical ventilation with corresponding VT. After 4 hours of ventilation, the serum, bronchoalveolar lavage fluid (BALF) and lung tissues were harvested. The lung wet/dry weight (W/D) ratio was assessed, and the histopathology changes were observed by light microscopy, and the ultra structure changes in type Ⅱ alveolar epithelial cell (AEC Ⅱ ) were observed by electron microscopy. The levels of interleukin (IL-1β and IL-6) and tumor necrosis factor- α (TNF- α ) in serum and BALF were determined by enzyme linked immunosorbent assay (ELISA). The total protein in BALF was measured by bicinchoninic acid methods, and the infiltrated cells in BALF were counted. The mRNA expressions and protein levels of microtube associated light chain 3B (LC3B), mitochondrial DNA coded cytochrome C oxidase Ⅳ (COX- Ⅳ) and nuclear factor-κB p65 (NF-κB p65) in lung tissues were determined by real-time fluorescent quantitation reverse transcriptionpolymerase chain reaction (RT-qPCR) and Western Blot. Results The histopathology of lung tissue and the ultra structure of AEC Ⅱ were normal in CON group and NVT group, and the obvious inflammatory changes and pathological injury were found in HVT group. Compared with CON and NVT groups, the W/D ratio in HVT group was significantly increased (8.53 ± 1.05 vs. 5.12 ± 0.65, 5.57 ± 0.55, both P 〈 0.05), and total protein, infiltrated cells, and IL-1β, IL-6 and TNF- α in BALF were significantly increased [total protein (g/L): 2.35 ± 0.45 vs. 1.46 ± 0.34, 1.76 ± 0.51; infiltrated cells ( × 10^5/mL): 2.05 ± 0.48 vs. 0.40 ± 0.08, 0.60 ± 0.23; IL-1β (ng/L): 119.82 ± 6.56 vs. 76.15 ± 3.32, 79.39 ± 4.44; IL-6 (μg/L): 4.10±0.52 vs. 1.97±0.40, 2.27±0.36; TNF-α (mg/L): 1.49±0.28 vs. 0.43±0.23, 0.61±0.24; all P 〈 0.05], IL-1β, IL-6 and TNF- α levels in serum were also elevated [IL-1β (ng/L): 127.53 ± 7.10 vs. 79.40 ± 2.80, 82.95 ± 2.25; IL-6 (ng/L): 6.28 ± 0.82 vs. 2.96 ± 0.35, 3.36 ± 0.72; TNF- α (mg/L): 1.59 ± 0.42 vs. 0.53 ± 0.22, 0.78± 0.25; all P 〈 0.05]. The mRNA expressions and protein levels of LC3B, COX-IV and NF-nB p65 in lung tissue of HVT group were significantly higher than those of CON group and NVT group, the mRNA expressions of LC3B- Ⅱ, COX- Ⅳ and NF-κB p65 were (3.52 ± 0.90), (3.76 ± 1.16) and (9.54 ± 2.06) folds of those in CON group, and the protein expressions were (1.76±0.24), (1.65±0.20) and (1.91 ± 0.12) folds of those in CON group, with significantly statistical differences (all P 〈 0.05). There were no significant differences in the parameters mentioned above between NVT group and CON group. Conclusion Mitophagy may be associated with VILI resulting from escaped mitoehondrial DNA for activation of inflammation.
出处
《中华危重病急救医学》
CAS
CSCD
北大核心
2017年第1期6-10,共5页
Chinese Critical Care Medicine
基金
国家自然科学基金(81460016)