NOD样受体蛋白3炎症小体在呼吸机相关性肺损伤中的作用机制研究
NOD양수체단백3염증소체재호흡궤상관성폐손상중적작용궤제연구
Role and mechanism of the NOD-like receptor 3 inflammasome in ventilator-induced lung injury in rats
  • 万方数据
    中华危重病急救医学 중화위중병급구의학
    Chinese Critical Care Medicine
    2015年 10期 821-825 ,共5页

    张维康%潘灵辉

    장유강%반령휘

    机械通气%呼吸机相关性肺损伤%NOD样受体蛋白3炎症小体%肺泡巨噬细胞 機械通氣%呼吸機相關性肺損傷%NOD樣受體蛋白3炎癥小體%肺泡巨噬細胞
    궤계통기%호흡궤상관성폐손상%NOD양수체단백3염증소체%폐포거서세포
    Mechanical ventilation%Ventilator-induced lung injury%NOD-like receptor 3 inflammasome%Alveolar macrophage
    目的：探讨NOD样受体蛋白3（NLRP3）炎症小体在呼吸机相关性肺损伤（VILI）中的作用及其机制。方法将30只清洁级雄性SD大鼠按随机数字表法分为自主呼吸对照组、正常潮气量（VT）组（VT为8 mL/kg）、大VT组（VT为40 mL/kg）3组，每组10只。所有大鼠均行气管切开插管术，自主呼吸对照组保持自主呼吸，两个VT组分别行不同VT的机械通气；4 h后颈总动脉放血处死大鼠，收集支气管肺泡灌洗液（BALF）、血清和肺组织标本。测定肺组织湿/干质量（W/D）比值，光镜下观察肺组织病理学改变，透射电镜下观察肺泡巨噬细胞超微结构改变；用酶联免疫吸附试验（ELISA）测定BALF中总蛋白含量以及血清和BALF中白细胞介素（IL-1β、IL-18）含量；用反转录-聚合酶链反应（RT-PCR）和蛋白质免疫印迹试验（Western Blot）分别检测肺泡巨噬细胞中NLRP3、凋亡相关斑点样蛋白（ASC）、天冬氨酸特异性半胱氨酸蛋白酶1（caspase-1）和核转录因子-κB（NF-κB）的mRNA及蛋白表达。结果镜下观察显示，自主呼吸对照组和正常VT组肺组织及肺泡巨噬细胞结构基本正常，大VT组则有明显的炎症性改变。与自主呼吸对照组和正常VT组比较，大VT组肺组织W/D比值明显升高（8.89±0.90比5.18±0.86、5.71±0.82，均P＜0.05），BALF中总蛋白、IL-1β、IL-18含量明显升高〔总蛋白（g/L）：2.34±0.41比1.77±0.14、1.81±0.06， IL-1β（ng/L）：133.48±10.48比81.54±3.12、83.80±5.22， IL-18（μg/L）：4.57±0.45比3.04±0.51、3.43±0.43，均P＜0.05〕，血清中IL-1β、IL-18含量也明显升高〔IL-1β（ng/L）：105.06±10.18比65.11±8.58、75.30±10.62， IL-18（μg/L）：2.27±0.09比1.18±0.34、1.43±0.15，均P＜0.05〕。大VT组肺泡巨噬细胞中NLRP3、 ASC、caspase-1和NF-κB的mRNA及蛋白表达均较自主呼吸对照组和正常VT组明显升高，大VT组NLRP3、ASC、caspase-1和NF-κB的mRNA表达分别是自主呼吸对照组的（8.53±2.21）、（5.75±1.17）、（7.47±1.23）、（10.86±2.38）倍，NLRP3、ASC、 caspase-1和NF-κB的蛋白表达分别是自主呼吸对照组的（1.50±0.14）、（1.49±0.04）、（1.53±0.15）、（1.51±0.11）倍，差异均有统计学意义（均P＜0.01）；而正常VT组各指标与自主呼吸对照组比较差异均无统计学意义。结论 NLRP3炎症小体参与了大鼠机械通气所致的肺损伤。
    목적：탐토NOD양수체단백3（NLRP3）염증소체재호흡궤상관성폐손상（VILI）중적작용급기궤제。방법장30지청길급웅성SD대서안수궤수자표법분위자주호흡대조조、정상조기량（VT）조（VT위8 mL/kg）、대VT조（VT위40 mL/kg）3조，매조10지。소유대서균행기관절개삽관술，자주호흡대조조보지자주호흡，량개VT조분별행불동VT적궤계통기；4 h후경총동맥방혈처사대서，수집지기관폐포관세액（BALF）、혈청화폐조직표본。측정폐조직습/간질량（W/D）비치，광경하관찰폐조직병이학개변，투사전경하관찰폐포거서세포초미결구개변；용매련면역흡부시험（ELISA）측정BALF중총단백함량이급혈청화BALF중백세포개소（IL-1β、IL-18）함량；용반전록-취합매련반응（RT-PCR）화단백질면역인적시험（Western Blot）분별검측폐포거서세포중NLRP3、조망상관반점양단백（ASC）、천동안산특이성반광안산단백매1（caspase-1）화핵전록인자-κB（NF-κB）적mRNA급단백표체。결과경하관찰현시，자주호흡대조조화정상VT조폐조직급폐포거서세포결구기본정상，대VT조칙유명현적염증성개변。여자주호흡대조조화정상VT조비교，대VT조폐조직W/D비치명현승고（8.89±0.90비5.18±0.86、5.71±0.82，균P＜0.05），BALF중총단백、IL-1β、IL-18함량명현승고〔총단백（g/L）：2.34±0.41비1.77±0.14、1.81±0.06， IL-1β（ng/L）：133.48±10.48비81.54±3.12、83.80±5.22， IL-18（μg/L）：4.57±0.45비3.04±0.51、3.43±0.43，균P＜0.05〕，혈청중IL-1β、IL-18함량야명현승고〔IL-1β（ng/L）：105.06±10.18비65.11±8.58、75.30±10.62， IL-18（μg/L）：2.27±0.09비1.18±0.34、1.43±0.15，균P＜0.05〕。대VT조폐포거서세포중NLRP3、 ASC、caspase-1화NF-κB적mRNA급단백표체균교자주호흡대조조화정상VT조명현승고，대VT조NLRP3、ASC、caspase-1화NF-κB적mRNA표체분별시자주호흡대조조적（8.53±2.21）、（5.75±1.17）、（7.47±1.23）、（10.86±2.38）배，NLRP3、ASC、 caspase-1화NF-κB적단백표체분별시자주호흡대조조적（1.50±0.14）、（1.49±0.04）、（1.53±0.15）、（1.51±0.11）배，차이균유통계학의의（균P＜0.01）；이정상VT조각지표여자주호흡대조조비교차이균무통계학의의。결론 NLRP3염증소체삼여료대서궤계통기소치적폐손상。
    ObjectiveTo investigate the role and its mechanism of the NOD-like receptor 3 (NLRP3) inflammasome in alveolar macrophages in ventilator-induced lung injury (VILI) in rats.Methods Thirty adult male Sprague-Dawley (SD) rats were randomly divided into three groups, with 10 rats in each group: spontaneous breathing control group, normal tidal volume (VT) group (VT 8 mL/kg) and high VT group (VT 40 mL/kg). All of the rats underwent tracheotomy. Then rats in spontaneous breathing control group were kept to have spontaneous breathing, while rats in normal VT group and high VT group received mechanical ventilation. After 4 hours, the rats were sacrificed by carotid artery bleeding, and the bronchoalveolar lavage fluid (BALF), blood serum and lung tissue were collected. Lung wet/dry ratios (W/D) were measured. Light microscopy and electron microscopy were performed to observe the pathological changes in lung tissue, and the ultrastructural changes in alveolar macrophages. Enzyme linked immunosorbent assay (ELISA) was performed to measure the total protein content in the BALF and the interleukins (IL-1β and IL-18) in the serum and BALF. The mRNA expressions and protein levels of NLRP3, apoptosis-associated speck-like protein containing CARD (ASC), caspase-1, and nuclear factor-κB (NF-κB) in alveolar macrophages were&nbsp;assayed by real-time fluorescent quantization reverse transcription-polymerase chain reaction (RT-PCR) and Western Blot.Results The structure of lung tissue and alveolar macrophages of rats in spontaneous breathing control group and normal VT group appeared normal, while obvious inflammatory changes were found in high VT group. Compared with spontaneous breathing control group and normal VT group, the ratio of W/D (8.89±0.90 vs. 5.18±0.86, 5.71±0.82, bothP< 0.05), contents of total protein, IL-1β, IL-18 in BALF were significantly increased [total protein (g/L):2.34±0.41 vs. 1.77±0.14, 1.81±0.06, IL-1β (ng/L): 133.48±10.48 vs. 81.54±3.12, 83.80±5.22, IL-18 (μg/L):4.57±0.45 vs. 3.04±0.51, 3.43±0.43, allP< 0.05], and IL-1β and IL-18 in serum were also increased [IL-1β(ng/L): 105.06±10.18 vs. 65.11±8.58, 75.30±10.62, IL-18 (μg/L): 2.27±0.09 vs. 1.18±0.34, 1.43±0.15, all P< 0.05]. The mRNA and protein expressions of NLRP3, ASC, caspase-1 and NF-κB in alveolar macrophages of high VT group were significantly increased compared with that of spontaneous breathing control group and normal VT group. The mRNA expressions of NLRP3, ASC, caspase-1 and NF-κB in high VT group were (8.53±2.21), (5.75±1.17), (7.47±1.23) and (10.86±2.38) folds of those in spontaneous breathing control group, and the protein expressions of NLRP3, ASC, caspase-1 and NF-κB were (1.50±0.14), (1.49±0.04), (1.53±0.15) and (1.51±0.11) folds of those in spontaneous breathing control group (allP< 0.01). There were no significant differences in all the indexes between normal VT group and spontaneous breathing control group.ConclusionNLRP3 inflammasome in alveolar macrophages may be involved in the mechanism of occurrence of VILI.
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