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| 亚甲蓝对大鼠机械通气相关肺损伤中核苷酸结合寡聚化结构域样受体蛋白3炎性小体的影响 |
| Effect of methylene blue on nucleotide-binding oligomerization domain-like receptor protein 3 inflammasome in rats with ventilator-induced lung injury |
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| DOI：10.12089/jca.2020.08.015 |
| 中文关键词: 亚甲蓝 核苷酸结合寡聚化结构域样受体蛋白3炎性小体 机械通气 机械通气相关肺损伤 |
| 英文关键词: Methylene blue Nucleotide-binding oligomerization domain-like receptor protein 3 inflammasome Mechanical ventilation Ventilator-induced lung injury |
| 基金项目:青岛市医疗卫生优秀学科带头人项目（VDTR2017Y11） |
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| 中文摘要: |
目的 评价亚甲蓝对大鼠机械通气相关损伤（VILI）中核苷酸结合寡聚化结构域样受体蛋白3（NLRP3）炎性小体的影响。
方法 清洁级成年雄性SD大鼠36只，8周龄，体重240~260 g，采用随机数字表法分为三组：自主呼吸对照组（C组）、大潮气量模型组（H组）和大潮气量+亚甲蓝组（MB组），每组12只。三组大鼠均行气管切开插管术，MB组经腹腔注射1%亚甲蓝50 mg/kg，10 min后以VT 20 ml/kg行机械通气；C组经腹腔注射等容量生理盐水后保持自主呼吸；H组经腹腔注射等容量生理盐水，10 min后以VT 20 ml/kg行机械通气。通气参数：FiO2 21%，I∶E 1∶1，RR 80次/分，PEEP 0，通气时间4 h。于基础状态、通气结束时经颈总动脉取血行血气分析，通气结束后颈总动脉放血处死大鼠，收集肺组织标本、支气管肺泡灌洗液（BALF）。光镜下观察肺组织病理学改变，记录肺损伤评分，计算肺组织湿/干重比值（W/D）。采用ELISA法测定BALF中总蛋白含量以及血清、BALF中白细胞介素（IL）-1β、IL-18浓度；鲁米诺化学发光法检测肺组织中活性氧（ROS）含量；RT-PCR法及Western blot法分别检测肺组织NLRP3、凋亡相关斑点样蛋白（ASC）、天冬氨酸半胱氨酸蛋白酶-1（caspase-1）mRNA表达量及蛋白含量。
结果 与C组比较，H组肺损伤评分、W/D明显升高（P＜0.01），BALF中总蛋白以及血清、BALF中IL-1β和IL-18浓度明显升高（P＜0.01），肺组织中ROS含量、NLRP3、ASC、caspase-1 mRNA表达量及蛋白含量明显升高（P＜0.01）。与H组比较，MB组肺损伤评分、W/D明显降低（P＜0.05），BALF中总蛋白以及血清、BALF中IL-1β和IL-18浓度明显降低（P＜0.05），肺组织中ROS含量、NLRP3、ASC、caspase-1 mRNA表达量及蛋白含量明显降低（P＜0.05）。
结论 亚甲蓝通过抑制大鼠肺组织中NLRP3炎性小体的激活，阻碍促炎因子IL-1β及IL-18的形成，减轻大鼠VILI。 |
| 英文摘要: |
Objective To evaluate the effect of methylene blue on nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) inflammasome in ventilator-induced lung injury (VILI) in rats, and explore its function in VILI.
Methods Thirty-six clean adult male Sprague-Dawley (SD) rats, weighing 240-260 g, aged 8 weeks, were randomly divided into three groups (n = 12): spontaneous breathing control group (group C), high tidal volume group (group H, VT 20 ml/kg) and high tidal volume + methylene blue group (group MB, VT 20 ml/kg). All of the rats underwent tracheotomy intubation. Rats in group C were kept spontaneous breathing. Rats in group H received mechanical ventilation with VT 20 ml/kg. Methylene blue 50 mg/kg was injected intraperitoneally 10 minutes before mechanical ventilation with VT 20 ml/kg in rats of group MB. Mechanical ventilation parameter settings were as following: FiO2 21%, I∶E 1∶1, ventilation frequency 80 times/min, PEEP 0 for 4 hours. Blood gas analysis was performed through the common carotid artery at the basic state and at the end of ventilation, the rats were sacrificed due to carotid artery bleeding, as well as bronchoalveolar lavage fluid (BALF) and lung tissue were collected. Pathological changes of lung tissue were observed under light microscope, lung injury score and wet/dry (W/D) ratio of lung tissue were measured. The contents of total protein in BALF and the levels of interleukin (IL)-1β and IL-18 in blood serum and BALF were determined by enzyme-linked immunosorbent assay (ELISA). The content of reactive oxygen species (ROS) in lung tissue was determined by chemiluminescence technique. Reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting were used to detect the mRNA and protein expression of NLRP3, apoptosis-related spot-like protein (ASC), and caspase-1 in lung tissue.
Results Compared with group C, the lung injury score and W/D ratio of group H were significantly increased (P < 0.01). The levels of IL-1β and IL-18 in serum and BALF of group H were increased (P < 0.01). The mRNA and protein expression of NLRP3, ASC, and caspase-1 of group H were up-regulated in lung tissue (P < 0.01). Compared with group H, the lung injury score and W/D ratio of group MB were significantly decreased (P < 0.05). The levels of IL-1β and IL-18 in serum and BALF of group MB were decreased (P < 0.05). The mRNA and protein expression of NLRP3, ASC, and caspase-1 of group MB were down-regulated in lung tissue (P < 0.05).
Conclusion Methylene blue alleviates VILI in rats by inhibiting the activation of NLRP3 inflammasome, as well as hindering the formation of IL-1β and IL-18 in the lung tissue of rats. |
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