PAD4依赖的中性粒细胞胞外诱捕网加重包虫囊液所致过敏性休克  

PAD4-dependent neutrophil extracellular traps exacerbates allergic shock induced by hydatid cyst fluid

作　　者：何金盼 崔键 戴庆[1] HE Jinpan;CUI Jian;DAI Qing(Department of Anesthesia,First Affiliated Hospital of Xinjiang Medical University,Xinjiang Key Laboratory of Perioperative Organ Protection(XJDX1411),Urumqi 830054,China)

机构地区：[1]新疆医科大学第一附属医院麻醉科,新疆围术期器官保护重点实验室(XJDX1411),乌鲁木齐830054

出　　处：《山西医科大学学报》2023年第10期1389-1396,共8页Journal of Shanxi Medical University

基　　金：新疆维吾尔自治区自然科学基金项目(2021D01C309)。

摘　　要：目的探讨精氨酸脱亚胺酶4(PAD4)依赖的中性粒细胞胞外诱捕网(NETs)在包虫囊液所致过敏性休克(AS)中的作用和相关机制。方法将野生型(WT)和PAD4敲除(PAD4^(-/-))的C57/B6小鼠分为4组:WT组、WT模型组、PAD4^(-/-)组和PAD4^(-/-)模型组,每组10只。WT模型组和PAD4^(-/-)模型组腹腔注射包虫囊液建立包虫感染小鼠模型。1个月后,两个模型组小鼠再次注射包虫囊液,监测各组小鼠休克和死亡情况。采用qRT-PCR检测各组小鼠脾脏中中性粒细胞PAD4 mRNA表达水平,采用绿菁染色法检测各组小鼠脾脏NETs形成水平,采用流式细胞术检测各组小鼠外周血单核细胞(PBMC)中Th1/Th2细胞比例,采用ELISA检测各组小鼠血清中IL-4、IL-5和IgE水平。体外将NETs与小鼠PBMC共孵育,分为对照组和NETs组,采用流式细胞术检测各组细胞中Th1/Th2细胞比例,ELISA检测各组细胞上清中IL-4、IL-5和IgE水平。结果与WT组比较,WT模型组小鼠休克率和死亡率升高,脾脏中中性粒细胞PAD4 mRNA表达和NETs形成水平升高(P<0.05),PBMC中Th1/Th2细胞比例降低(P<0.05),IL-4、IL-5和IgE水平升高(P<0.05)。与WT模型组比较,PAD4^(-/-)模型组小鼠休克率和死亡率降低,脾脏中中性粒细胞PAD4 mRNA表达和NETs形成水平降低(P<0.05),PBMC中Th1/Th2细胞比例升高(P<0.05),IL-4、IL-5和IgE水平降低(P<0.05)。与PAD4^(-/-)组比较,PAD4^(-/-)模型组小鼠脾脏中中性粒细胞PAD4 mRNA表达和NETs形成水平、PBMC中Th1/Th2细胞比例以及IL-4、IL-5和IgE水平无显著性差异(P>0.05)。与对照组比较,NETs组Th1/Th2细胞比例降低(P<0.05),上清中IL-4、IL-5和IgE水平升高(P<0.05)。结论包虫囊液感染可诱导PAD4依赖的NETs形成,加重小鼠过敏性休克。敲除PAD4可抑制NETs形成,并通过恢复失衡的Th1/Th2细胞比例最终缓解包虫囊液所致的过敏性休克。Objective To investigate the role and its underlying mechanisms of peptidyl arginine deiminase 4(PAD4)-dependent neutrophil extracellular traps(NETs)in anaphylactic shock(AS)induced by hydatid cyst fluid.Methods Wild-type(WT)and PAD4 knockout(PAD4^(-/-))C57/B6 mice were divided into four groups:WT group,WT model group,PAD4^(-/-)group,and PAD4^(-/-)model group,with 10 mice per group.The mice in WT model group and PAD4^(-/-)model group were intraperitoneally injected with hydatid cystic fluid to establish a mouse model of echinococcosis infection.One month after initial injection,a second round of cystic fluid injections was administered in both model groups,and the incidences of shock and death were observed.RT-qPCR was used to detect PAD4 mRNA expression level in neutrophils from the spleen of mice in each group.Green fluorescence staining was performed to detect the NETs formation in the spleen of mice.Flow cytometry was used to detect Th1/Th2 cell ratio in peripheral blood mononuclear cell(PBMC)of mice,and ELISA was used to detect the levels of IL-4,IL-5,and IgE in the serum of mice.In vitro,NETs were co-cultured with PBMC,and the cells were divided into control group and NETs group.Flow cytometry was employed to detected Th1/Th2 cell ratio in PBMC,and ELISA was used to measure the levels of IL-4,IL-5,and IgE in the cellular supernatants.Results Compared to WT group,the incidences of shock and death in mice were increased in WT model group,PAD4 mRNA expression in neutrophils from the spleen was increased(P<0.05),NETs formation levels were elevated(P<0.05),and the Th1/Th2 cell ratio in PBMC was decreased(P<0.05).Compared to WT model group,the incidences of shock and death in mice were decreased in PAD4^(-/-)model group,PAD4 mRNA expression in neutrophils from the spleen was decreased(P<0.05),NETs formation levels were decreased(P<0.05),and the Th1/Th2 ratio in PBMC was elevated(P<0.05).There were no significant differences in the expression of PAD4 mRNA and NETs formation level of neutrophils in spleen,the propo

关 键 词：包虫囊液 过敏性休克 精氨酸脱亚胺酶4 中性粒细胞胞外诱捕网 TH1/TH2失衡 

分 类 号：R532.32[医药卫生—内科学]