苯亚甲基丙二腈脂类衍生物AG490减轻中性粒细胞性哮喘模型小鼠气道炎症

Lipid derivative of benzylidene malononitrile AG490 attenuates airway inflammation of mice with neutrophilic asthma

目的观察苯亚甲基丙二腈脂类衍生物AG490对中性粒细胞性哮喘模型小鼠气道炎症的影响。方法无特定病原体(SPF)级C57BL雌性小鼠54只,按随机数字表法分成中性粒细胞性哮喘(NA)组、AG490处理的NA(NAAG)组和正常(NC)组,每组18只。NA、NAAG组小鼠于第0、6、13天经气道滴卵清蛋白(OVA)、脂多糖(LPS)致敏。NAAG组小鼠于实验第0天起经腹腔注射AG490每只500μg,3次/周,连续3周。于第21天连续2 d雾化吸入10 g/L OVA激发,每次1 h。最后1次雾化结束后24 h时,收集小鼠标本并检测相关指标。收集支气管肺泡灌洗液(BALF)并检测其有核细胞总数及分类比例;分离肺组织行HE染色及过碘酸希夫(PAS)染色,光镜下观察肺组织病理变化和杯状细胞增生;采用流式细胞术检测肺脏Th17细胞和调节性T细胞(Treg)的频数;采用ELISA检测小鼠BALF中IL-17的水平。结果与NA组相比,NAAG组BALF有核细胞计数、中性粒细胞百分比、嗜酸性粒细胞百分比均明显降低,NAAG组肺组织病理学改变及杯状细胞增生较NA组明显减轻。与NA组相比,NAAG组BALF IL-17水平降低、肺组织Th17细胞比例减少、Treg比例增加。结论 NA小鼠致敏阶段给予AG490处理,可增加小鼠激发阶段肺组织Treg数量、减少Th17细胞数量,减轻NA小鼠气道炎症。

Objective To observe the effect of lipid derivative of benzylidene malononitrile AG490 on the airway inflammation in a mouse model of neutrophilic asthma（ NA）. Methods Fifty-four specific pathogen-free（ SPF） female C57 BL/6 mice were randomly divided into 3 groups： NA group,AG490-treated NA（ NAAG） group,and normal control（ NC）group,18 mice in each group. The NA group and the NAAG group were sensitized by airway instillation of ovalbumin（ OVA）and lipopolysaccharide（ LPS） on day 0,6 and 13. The NAAG group was injected with AG490（ 500 μg/mouse,i. p.） three times a week,from day 0 after the first sensitization,for 3 weeks. Mice were challenged on day 21,22 for 1 hour/time with an aerosol of 10 g/L OVA. At 24 hours after the final challenge,bronchoalveolar lavage fluid（ BALF） was collected. The total number and differential counts of nucleated cells and the percentage of each type were determined. HE staining and PAS staining was employed for observing the lung pathological changes. The percentages of Th17 cells and regulatory T cells（ Treg） in the lung issue were determined by flow cytometry. The level of interleukin-17（ IL-17） in BALF was measured using ELISA. Results Compared with the NA group,the total number of nucleated cells,the percentage of neutrophils and the percentage of eosinophils in BALF in the NAAG group were obviously reduced; lung tissue pathologic changes were improved in the NAAG group; goblet cell hyperplasia and the level of IL-17 in BALF in the NAAG group were significantly down-regulated; the proportion of Treg in the lung increased and the proportion of Th17 cells in the lung decreased in the NAAG group. Conclusion After NA mice are treated with AG490 during the sensitization phase,the proportion of Treg in the lung would increase and the proportion of Th17 cells in the lung would decrease. AG490 could attenuate the airway inflammation in the mouse model of NA.