LY294002干预对哮喘小鼠M2巨噬细胞极化及气道炎症的机制研究

Mechanism of LY294002 intervention on M2 macrophage polarization and airway inflammation in asthmatic mice

摘要:

目的 探讨LY294002干预对哮喘小鼠M2巨噬细胞极化及气道炎症的机制研究。方法 将24只雄性BALB/C小鼠随机分为空白对照组、哮喘模型组、LY294002组、地塞米松组，每组6只。采用腹腔注射卵清蛋白致敏及雾化吸入激发法复制小鼠哮喘模型，连续7 d腹腔注射给药后，观察小鼠肺组织中气道炎症、杯状细胞增生情况，酶联免疫吸附试验检测血清免疫球蛋白E（IgE）、白细胞介素-4（IL-4）、IL-25及支气管肺泡灌洗液中炎症细胞分类计数，实时荧光聚合酶链反应检测肺组织炎症因子IL-4、IL-13、IL-25R及M2标志物ARG-1、Ym-1 mRNA表达，Western blotting检测肺组织PI3K、p-PI3K、Akt、p-Akt及M2标志物CD206蛋白表达。结果 哮喘模型组小鼠的支气管肺泡灌洗液中细胞总数、嗜酸性粒细胞、中性粒细胞、淋巴细胞均较空白对照组高（P <0.05），巨噬细胞较空白对照组低（P <0.05）；LY294002组、地塞米松组细胞总数、嗜酸性粒细胞、中性粒细胞、淋巴细胞比例均较哮喘模型组低（P <0.05），巨噬细胞较哮喘模型组高（P <0.05）。哮喘模型组血清IgE、IL-4和IL-25水平均较空白对照组高（P <0.05），LY294002组、地塞米松组血清IgE、IL-4和IL-25水平均较哮喘模型组低（P <0.05）。哮喘模型组ARG-1、YM-1、IL-4、IL-13、IL-25R mRNA相对表达量均较空白对照组高（P <0.05），LY294002组、地塞米松组ARG-1、YM-1、IL-4、IL-13、IL-25R mRNA相对表达量均较哮喘模型组低（P <0.05）。哮喘模型组p-PI3K/PI3K、p-Akt/Akt、CD206蛋白相对表达量均较空白对照组高（P <0.05），LY294002组、地塞米松组p-PI3K/PI3K、p-Akt/Akt、CD206蛋白相对表达量均较哮喘模型组低（P <0.05）。结论 LY294002可显著减低哮喘小鼠肺部组织的M2极化，减轻哮喘小鼠的气道炎症作用，其作用机制可能与PI3K/Akt信号通路有关。

Abstract:

Objective To explore the mechanism of LY294002 intervention on M2 macrophage polarization and airway inflammation in asthmatic mice.Methods The 24 male BALB/c mice were randomly divided into the blank control group, the asthma model group, the LY294002 group, and the dexamethasone group, with 6 mice in each group. The asthma mouse model was established by sensitization through intraperitoneal injection of ovalbumin and provocation via aerosol inhalation. After 7 days of continuous intraperitoneal drug administration, the airway inflammation and goblet cell hyperplasia in the lung tissues of mice were observed. Serum immunoglobulin E (IgE), interleukin-4 (IL-4), and IL-25 were detected by ELISA, and inflammatory cell counts in bronchoalveolar lavage fluid were determined. Quantitative real-time polymerase chain reaction was used to detect the mRNA expressions of inflammatory factors IL-4, IL-13, and IL-25R, as well as M2 markers ARG-1 and Ym-1 in lung tissues. Western blotting was performed to detect the protein expressions of PI3K, p-PI3K, Akt, p-Akt, and M2 marker CD206 in lung tissues.Results Compared with the blank control group, the asthma model group had a higher total number of cells and greater percentages of eosinophils, neutrophils and lymphocytes (P < 0.05), but a lower percentage of macrophages in bronchoalveolar lavage fluid (P < 0.05). The total number of cells and the percentages of eosinophils, neutrophils and lymphocytes were lower (P < 0.05), but the percentage of macrophage was higher in the LY294002 group and the dexamethasone group compared with the asthma model group (P < 0.05). The serum levels of IgE, IL-4 and IL-25 in the asthma model group were higher than those in the blank control group (P < 0.05), while they were lower in the LY294002 group and the dexamethasone group than in the asthma model group (P < 0.05). The relative mRNA expressions of ARG-1, YM-1, IL-4, IL-13 and IL-25R in the asthma model group were higher than those in the blank control group (P < 0.05), while they were lower in the LY294002 group and the dexamethasone group than in the asthma model group (P < 0.05). The relative protein expressions of p-PI3K/PI3K, p-Akt/Akt and CD206 in the asthma model group were higher than those in the blank control group (P < 0.05), while they were lower in the LY294002 group and the dexamethasone group than in the asthma model group (P < 0.05).Conclusions LY294002 can significantly reduce the M2 polarization of lung tissues and alleviate airway inflammation in asthmatic mice, and its mechanism may be related to the PI3K/Akt signaling pathway.