早期炎性介质释放可能启动盐酸吸入性肺炎的病理过程

Early cytokines release may trigger the pathologic process in acute lung injury model induced by hydrochloric acid inhalation on mice

探索酸性物质吸入性肺损伤的机制。120只C57BL/6J小鼠随机分为稀盐酸(HCl)吸入组和生理盐水(NS)吸入组。实验小鼠经导管滴注0.1mol/L HCl/NS(1μl/g体重)至左肺,吸入后30min、2h、6h和24h分别检测小鼠的呼吸功能(Penh)(除外30min、肺湿干重比值(W/D)、支气管肺泡灌洗液(BALF)的白细胞计数和蛋白浓度,观察肺组织病理学改变;免疫组化研究小鼠左肺炎性细胞浸润变化;免疫印迹检测小鼠左肺肺组织炎性因子水平。12hHCl组的呼吸功能(Pehn)严重受损(P<0.01vs NS组),肺W/D明显高于其它时间组(P<0.05)和对照组(P<0.01vs NS组);BALF蛋白含量和白细胞总数在6hHCl组达到峰值(P<0.05vs其它时间组,P<0.01vs NS组);炎性因子IL-1β、TNF-α、NF-κB和IL-6在左肺组织中表达随HCl刺激时间逐渐升高,12h后表达量降低,24h时继续下降;HCl刺激30min即出现嗜中性粒细胞肺组织浸润,而巨噬细胞和T淋巴细胞出现相对较晚。研究通过吸入稀盐酸模拟吸入性肺炎小鼠模型,发现吸入性肺炎早期即有大量炎性因子,随后出现白细胞升高而巨噬细胞增多出现较晚;大量炎性因子均参与了整个疾病发生发展过程。另外,实验经气管单侧滴注法获得单侧吸入性肺炎的模型,更符合临床吸入性肺炎的特点,为临床深入研究吸入性肺炎制作了很好的动物模型。

To explore the mechanism of acid-induced acute lung injury（ALI） in mice, 120 male C57BL/6J mice were randomly divided into hydrochloric acid-inhalation group（HC1） and normal saline- inhalation group（NS）. Mice were intra-tracheally in- stilled of hydrochloric acid （1 /11/g weight） or normal saline（NS, 1 /xl/g weight） into the left lung with a catheter. After inha- lation mice in each group were sacrificed at different time points of 30 rain, 2 h, 6 h, 12 h and 24 h respectively. Respiratory function was ere observed （except for 30 rain） at each time point before the mice were sacrificed. Then bronchoalveolar lavage fluid（BALF） was were harvested and different parts of lungs were isolated and kept in --80 ~C or fixed with neutral formalin for further Western blotting or hematoxylin and eosin （HE） staining. The wet/dry weight （W/D） ratio of the left lung was measured and the number of leukocytes in BALF was counted. The morphology changes of the left lung samples were observed and evaluated by two different pathologists. Meanwhile, immunohistochemistry were utilized in analyzing the inflammatory cells infiltration in the lung, and the protein expression of interleukin-6 （IL-6）, IL-1β, tumor necrosis factor-α（TNF-α） and nu- clear transcription factor kB （NF-kB） were detected by Western blotting. Pehn, an airway resistance index was severely dam-aged at 12 h in HC1 group（I^G0.01 vs NS）, and in addition, W/D ratio was significantly higher in HC group than that in the NS group（P〈0.01 vs NS） and those in the HC1 group at other time points（P〈0. 0/5） I Protein concentration and total WBC in BALF peaked at 6 h in HC1 group were significantly higher compared those at other time points （P〈0, 05 vs other HC1 groups, P〈0.01 vs NS）; Expression of IL-6, IL-113, TNF-a and NF-JcB was increased in lung tissue with inhalation of HC1, but the levels decreased after 12 h; Neutrophil infiltration occurred after 30 min, and macrophages and T-lymphocytes appeared later in the lung of HC1 groups. Large quantities of inflammatory cytokines were involved at very early stage after inhalation of HC1 and even higher levels throughout the whole pathological process. Notably, the neutrophils responded promptly while macrophages and T lymphocytes functioned later. In addition, cytokine levels decreased after 12 h indicating that preventing patients from inhalation as soon as possible may be pivotal in extinguishing inhalation induced inflammation.