高氧暴露BPD小鼠模型中巨噬细胞焦亡的检测及意义  

Detection and significance of macrophage pyroptosis in a hyperoxiaexposed BPD mouse model

作　　者：涂子坤 高雅静 韩晓 郭海艳[1,2] 周玉峰 TU Zi-kun;GAO Ya-jing;HAN Xiao;GUO Hai-yan;ZHOU Yu-feng(Institute of Pediatrics,Children's Hospital/National Children's Medical Center,Fudan University,Shanghai 201102,China;National Health Commission Key Laboratory of Neonatal Diseases(Fudan University),Shanghai 201102,China;Institutes of Biomedical Sciences,Fudan University,Shanghai 200032,China)

机构地区：[1]国家儿童医学中心/复旦大学附属儿科医院儿科研究院,上海201102 [2]国家卫生健康委员会新生儿疾病重点实验室(复旦大学),上海201102 [3]复旦大学生物医学研究院,上海200032

出　　处：《复旦学报（医学版）》2023年第6期791-801,共11页Fudan University Journal of Medical Sciences

基　　金：国家重点研发计划(2021YFC2701800,2021YFC2701802);国家自然科学基金面上项目(82241038,81974248)。

摘　　要：目的 探讨巨噬细胞焦亡是否参与支气管肺发育不良(bronchopulmonary dysplasia,BPD)的肺病理损伤。方法 40只足月新生小鼠随机分为空气(room air,RA)组和高氧(hyperoxia,HO)组,每组20只。建立慢性高氧诱导的BPD小鼠模型。HE染色评估小鼠的肺泡化情况;CD31免疫组化检测肺血管发育状况;Masson染色分析肺组织的纤维化程度;qRT-PCR检测肺组织中的炎症因子和趋化因子(Il6、Il1b、Mmp12、Ccl2、Ccl5、Il8)基因表达水平;ELISA检测血清和肺组织匀浆中的IL-1β水平;Western blot检测细胞焦亡通路的关键蛋白NLRP3、Caspase-1 p20、N-消皮素D(gasdermin D,GSDMD)、IL-1β表达;流式分析支气管肺泡灌洗液中巨噬细胞比例;免疫荧光检测F4/80与NLRP3/Caspase-1/IL-1β的共定位;免疫组化分析AQP5的表达水平。结果 与RA组相比,HO组小鼠的肺泡化阻滞和肺血管生成障碍,肺纤维化显著增加;肺组织中促炎细胞因子的表达显著上调,细胞焦亡相关蛋白表达水平显著增加,血清和肺组织匀浆中IL-1β水平显著升高;支气管肺泡灌洗液中巨噬细胞比例显著升高,肺组织中F4/80与NLRP3、Caspase-1、IL-1β均存在显著的免疫荧光共定位现象,AQP5阳性的肺泡Ⅰ型上皮细胞严重受损。结论 高氧诱导的BPD小鼠模型中,NLRP3/Caspase-1/GSDMD细胞焦亡通路过度活化,可能导致BPD的肺部病理损伤。Objective To investigate whether macrophage pyroptosis is involved in the lung injury of bronchopulmonary dysplasia(BPD).Methods Forty full-term newborn mice were divided into room air(RA)group and hyperoxia(HO)group(n=20 for each group).BPD mouse model was induced by chronic hyperoxia.Alveolarization was assessed by HE staining.Pulmonary vascular development was detected by CD31 immunohistochemistry.The degree of fibrosis was analyzed by Masson staining.The mRNA expression levels of pro-inflammatory cytokines and chemokines in lung tissue were quantified by qRT-PCR for Il6,Il1b,Mmp12,Ccl2,Ccl5 and Il8.Concentrations of IL-1βwas detected by ELISA in serum and lung homogenates.The expressions of pyroptosis-associated proteins,NLRP3,Caspase-1 p20,N-gasdermin D(GSDMD)and mature IL-1βwere evaluated by Western blot.The proportion of macrophages in bronchoalveolar lavage fluid was detected by flow cytometry.Immunofluorescence co-localization of F4/80 with NLRP3/Caspase-1/IL-1βwas performed.AQP5 protein expression was analyzed by immunohistochemistry.Results Compared with the RA group,alveolar block and pulmonary angiogenesis disorder were observed in the HO group,and pulmonary fibrosis was significantly increased.In lung tissue of HO group,the expression of pro-inflammatory cytokines was significantly up-regulated,the expression levels of pyroptosis-associated proteins were significantly increased;the levels of IL-1βin serum and lung homogenate were significantly increased;the proportion of macrophages in bronchoalveolar lavage fluid was significantly increased,and there was a significant immunofluorescence co-localization of F4/80 with NLRP3,Caspase-1 and IL-1βin lung tissue;AQP5-positive alveolar typeⅠepithelial cells were severely damaged.Conclusion Hyperoxia-induced hyperactivation of the NLRP3/Caspase-1/GSDMD mediated pyroptosis may lead to lung injury in a hyperoxia-exposed BPD mouse model.

关 键 词：支气管肺发育不良(BPD) 消皮素D(GSDMD) 巨噬细胞 焦亡 小鼠 

分 类 号：R722[医药卫生—儿科]