Abstract
This study aimed to establish an animal model of decompression-induced lung injury (DILI) secondary to repetitive diving in mice and explore the role of macrophages in DILI and the protective effects of high-concentration hydrogen (HCH) on DILI. Mice were divided into three groups: control group, DILI group, and HCH group. Mice were exposed to hyperbaric air at 600 kPa for 60 min once daily for consecutive 3 d and then experienced decompression. In HCH group, mice were administered with HCH (66.7% hydrogen and 33.3% oxygen) for 60 min after each hyperbaric exposure. Pulmonary function tests were done 6 h after decompression; the blood was harvested for cell counting; the lung tissues were harvested for the detection of inflammatory cytokines, hematoxylin and eosin (HE) staining, and immunohistochemistry; western blotting and polymerase chain reaction (PCR) were done for the detection of markers for M1 and M2 macrophages. Our results showed that bubbles formed after decompression and repeated hyperbaric exposures significantly reduced the total lung volume and functional residual volume. Moreover, repetitive diving dramatically increased proinflammatory factors and increased the markers of both M1 and M2 macrophages. HCH inhalation improved lung function to a certain extent, and significantly reduced the pro-inflammatory factors. These effects were related to the reduction of M1 macrophages as well as the increase in M2 macrophages. This study indicates that repetitive diving damages lung function and activates lung macrophages, resulting in lung inflammation. HCH inhalation after each diving may be a promising strategy for the prevention of DILI.
摘 要
目 的
探索反复潜水引起肺损伤的炎症机制以及吸入高浓度氢气 (HCH) 对这种损伤的治疗作用.
创新点
本研究首次在小鼠体内建立并评估减压诱导肺损伤 (DILI) 模型; 首次探索巨噬细胞在 DILI 中的作用; 首次探索呼吸 HCH 对于 DILI 的治疗作用.
方 法
将雄性 C57 小鼠随机分为对照组、 DILI 组和 HCH 组. DILI 组于 600 kPa 压力下暴露 60 min, 连续 3 d.HCH 组在减压处理后吸入 HCH (66.7% H 2 +33.3% O2) 干预. 减压操作 6 h 后检测小鼠肺功能和小鼠肺干湿比, 取小鼠肺组织固定进行苏木精-伊红染色, 并取小鼠全血进行血细胞计数实验. 取小鼠肺组织提取蛋白并提取血清, 采用酶联免疫吸附测定 (ELISA) 检测炎症因子与趋化因子, 并使用蛋白质免疫印迹 (western blotting) 试验测定小鼠肺内小鼠含生长因子样模体粘液样激素样受体 (F4/80)、 巨噬细胞甘露糖受体 (CD206) 和诱导型一氧化氮合酶 (iNOS)的表达量. 使用免疫组化检测小鼠肺组织切片内 F4/80、 CD206 和 iNOS 的阳性细胞的比例. 提取小鼠肺组织内总信使核糖核酸 (mRNA), 使用荧光实时定量聚合酶链反应测定极化标记蛋白 CD206 和 iNOS 以及炎症因子 TNF-α 和 IL-10 的基因表达量.
结 论
多次减压可导致肺水肿、 组织结构破坏和肺功能下降, 病变程度和减压次数有关, 证明模型建立成功. DILI 可以诱导肺内和循环炎症反应的激活, 巨噬细胞可能向肺内迁移趋化并向不同亚型极化, 极化后的巨噬细胞 M1 与 M2 分别参与炎症激活与炎症抑制的过程. 吸入 HCH 可以显著改善小鼠肺损伤, 降低肺内炎症反应, 抑制巨噬细胞向 M1 亚型极化并促进其向 M2 亚型极化, 从而证明吸入 HCH 对于 DILI 具有治疗作用.
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Xue-iun SUN and Wen-wu LIU designed the study, wrote and edited the manuscript. Ke NING and Zhen-biao GUAN performed the establishment of animal models and contributed to the study design, data analysis, writing and editing of the manuscript. Hong-tao LU and Ning ZHANG contributed to the study design and data analysis. All authors have read and approved the final manuscript and, therefore, have full access to all the data in the study and take responsibility for the integrity and security of the data.
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Ke NING, Zhen-biao GUAN, Hong-tao LU, Ning ZHANG, Xue-jun SUN, and Wen-wu LIU declare that they have no conflict of interest.
All institutional and national guidelines for the care and use of laboratory animals were followed. The study was approved by the Ethics Committee of the Navy Medical University, Shanghai, China (approval No. 20170236).
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Project supported by the National Natural Science Foundation of China (No. 81772015)
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Ning, K., Guan, Zb., Lu, Ht. et al. Lung macrophages are involved in lung injury secondary to repetitive diving. J. Zhejiang Univ. Sci. B 21, 646–656 (2020). https://doi.org/10.1631/jzus.B1900687
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DOI: https://doi.org/10.1631/jzus.B1900687