肺炎克雷伯菌肺炎对小鼠NLRP3炎症通路的影响

Effect of Klebsiella pneumoniae pneumonia on mice NLRP3 inflammatory pathway

目的建立肺炎克雷伯菌肺炎小鼠模型,并探究肺炎克雷伯菌感染对核苷酸结合寡聚化结构域样受体蛋白3(NLRP3)炎症通路的影响。方法给小鼠气管内接种肺炎克雷伯菌,观察小鼠死亡率;接种肺炎克雷伯菌48 h后,取小鼠肺部匀浆涂板检查菌落数;取小鼠支气管肺泡灌洗液(BALF)离心,酶联免疫吸附实验(ELISA)检测无细胞上清中白细胞介素(IL)-1β、肿瘤坏死因子(TNF-α)、IL-6、趋化因子配体1(KC)的水平,迈格林华-姬姆萨染色计数细胞沉淀中的中性粒细胞数;取肺部病理切片并进行苏木精-伊红(HE)染色观察肺部病变;提取肺部总蛋白,蛋白质印迹法检测NLRP3、核因子(NF)-κB p65、凋亡相关微粒蛋白(ASC)、天冬氨酸特异性半胱氨酸蛋白水解酶(caspase-1)、人白介素-1β前体(pro-IL-1β)表达水平。结果成功建立肺炎克雷伯菌肺炎小鼠模型,小鼠在144 h内死亡率达到百分之百,肺部有大量细菌负载,肺部中性粒细胞浸润增加,支气管肺泡灌洗液中IL-1β、TNF-α、IL-6、KC分泌升高,肺组织IL-1β、p-p65表达升高。结论通过将肺炎克雷伯菌液接种到小鼠肺部的方法所建立的肺炎小鼠模型稳定性高、可重复性好,肺部NLRP3炎症通路反应强烈。

Objective To build the murine model of Klebsiella pneumoniae pneumonia and to explore the effect of Klebsiella pneumoniae infection on NLRP3 inflammasome. Methods Mice were challenged via intratracheal instillation with Klebsiella pneumoniae and to observe their mortality. After 48 h of Klebsiella pneumoniae inoculation, the mice lung was homogenized and the supernatants were spread on Luria-Bertani agar plates. The levels of interleukin 1 beta,tumor necrosis factor, chemokines and interleukin 6 in the cell-free supernatants of bronchoalveolar lavage fluid were detected by ELISA.The cells numbers in the bronchoalveolar lavage fluid were stained and counted by May-Grunwald-Giemsa. The sections of the lungs were cut and stained with HE and histology images were evaluated. The expression of NLRP3, nuclear factor kappa-B p65, apoptosis-associated speck-like protein containing CARD, caspase-1 and pro-IL-1β were detected by westernblot. Results The mouse model of Klebsiella pneumonia was successfully established and the mortality rate of mice reached 100% within 144 h, the lung CFU reached to a very high level, and the inflammatory cell recruitment were increased in the lung. The inflammatory cytokine IL-1β, TNF-α, IL-6 and KC were increased in the bronchoalveolar lavage fluid, and the expression of IL-1β and p-p65 were increased in the lung. Conclusion The mouse model of pneumonia established by inoculating Klebsiella pneumoniae into the lung of mice is highly stable and reproducible, and the NLRP3 inflammatory pathway in the lung is strongly activated.