Li L, Lin J T
Graduate School of Peking Union Medical College and Chinese Academy of Medical Sciences, Beijing 100730, China.
Zhonghua Yi Xue Za Zhi. 2017 Apr 25;97(16):1259-1264. doi: 10.3760/cma.j.issn.0376-2491.2017.16.017.
To develop a mouse model of eosinophilic bronchitis, and explore the effects of eosinophil activation on airway hyperresponsiveness. A total of 60 female specific pathogen free BALB/c female mice were divided randomly into four groups: polymyxin B group, normal saline group, polymyxin B+ N-methionine leucine phenylalanine (fMLP) group, normal saline+ fMLP group. All the groups were given corresponding nasal drops for 21 days. The former two groups were given 12 μl 0.5% polymyxin B or normal saline once a day by transnasal administration respectively. Besides the above, the latter two groups were given 10 μl fMLP (0.05 mg/ml, dissolved in acetic acid) once by transnasal administration 3 hours after polymyxin B or normal saline administration on the 21st day. Within 24 hours after the last transnasal administration, cells in bronchoalveolar lavage fluid (BALF) were collected and analyzed by absolute different cell counts. Airway responsiveness (inspiratory and expiratory airway resistance and lung compliance) to acetyl choline (Ach) were measured 3 hours after the last transnasal administration in the former two groups and 24 hours after the last transnasal administration in the latter two groups. Eosinophil peroxidase (EPX), eosinophil cationic protein (ECP) and eosinophil chemotactic factor (ECF) levels in serum, BALF and lung tissue were tested by emzyme linked immunosorbent assay (ELISA). HE and Chromotrope 2R staining of lung tissue were used to observe the infiltration of inflammatory cells. The morphology of low-density eosinophils was observed under electron microscope. The total cell counts of BALF in polymyxin B group were significantly higher than the normal saline group [29.50 (3.25)×10(4)/ml vs 15.25 (2.25)×10(4)/ml, <0.001], especially eosinophil counts [11.76 (6.02)×10(4)/ml vs 0.12 (1.08)×10(4)/ml, <0.001]. However, no significant differences of inspiratory and expiratory airway resistance and lung compliance existed in the two groups. Inspiratory and expiratory airway resistance in polymyxin B+ fMLP group were significantly higher, lung compliance significantly lower than those in the other three groups (all <0.001), and the EPX, ECP, ECF levels in serum, BALF and lung tissue of the polymyxin B+ fMLP group were significantly higher than the other three groups (all <0.001). There were more inflammatory cells, especially eosinophils in lung tissue of polymyxin B and polymyxin B+ fMLP group. Meanwhile, more activated eosinophils in polymyxin B+ fMLP group were observed by electron microscopy. A mouse model of eosinophilic bronchitis can be successfully developed by transnasal administration of polymyxin B, and the eosinophil activation plays an important role in the occurrence of airway hyperresponsiveness.
建立嗜酸性粒细胞性支气管炎小鼠模型，探讨嗜酸性粒细胞活化对气道高反应性的影响。将60只雌性无特定病原体BALB/c小鼠随机分为四组：多粘菌素B组、生理盐水组、多粘菌素B + N-甲硫氨酰-亮氨酰-苯丙氨酸（fMLP）组、生理盐水 + fMLP组。所有组均给予相应滴鼻剂，持续21天。前两组分别经鼻给予12 μl 0.5%多粘菌素B或生理盐水，每日1次。除此之外，后两组在第21天多粘菌素B或生理盐水给药3小时后经鼻给予10 μl fMLP（0.05 mg/ml，溶于乙酸）1次。在末次经鼻给药后24小时内，收集支气管肺泡灌洗液（BALF）中的细胞，通过绝对细胞计数进行分析。前两组在末次经鼻给药后3小时测量对乙酰胆碱（Ach）的气道反应性（吸气和呼气气道阻力以及肺顺应性），后两组在末次经鼻给药后24小时测量。采用酶联免疫吸附测定（ELISA）检测血清、BALF和肺组织中嗜酸性粒细胞过氧化物酶（EPX）、嗜酸性粒细胞阳离子蛋白（ECP）和嗜酸性粒细胞趋化因子（ECF）水平。采用苏木精-伊红（HE）染色和变色酸2R染色观察肺组织中炎症细胞浸润情况。在电子显微镜下观察低密度嗜酸性粒细胞的形态。多粘菌素B组BALF中的总细胞计数显著高于生理盐水组[29.50（3.25）×10⁴/ml对15.25（2.25）×10⁴/ml，<0.001]，尤其是嗜酸性粒细胞计数[11.76（6.02）×10⁴/ml对0.12（1.08）×10⁴/ml，<0.001]。然而，两组的吸气和呼气气道阻力以及肺顺应性无显著差异。多粘菌素B + fMLP组的吸气和呼气气道阻力显著更高，肺顺应性显著低于其他三组（均<0.001），多粘菌素B + fMLP组血清、BALF和肺组织中的EPX、ECP、ECF水平显著高于其他三组（均<0.001）。多粘菌素B组和多粘菌素B + fMLP组肺组织中有更多炎症细胞，尤其是嗜酸性粒细胞。同时，在电子显微镜下观察到多粘菌素B + fMLP组中有更多活化的嗜酸性粒细胞。经鼻给予多粘菌素B可成功建立嗜酸性粒细胞性支气管炎小鼠模型，嗜酸性粒细胞活化在气道高反应性的发生中起重要作用。
