MLKL-NLRP3炎症小体介导的坏死性炎症反应在脓毒症急性肺损伤小鼠中的作用及其机制  

Role and mechanisms of MLKL-NLRP3-mediated necroinflammation in mice with sepsis-induced acute lung injury

作　　者：李娜 朱雄 程远 何庆 Li Na;Zhu Xiong;Cheng Yuan;He Qing(Department of General Practice,Chengdu Third People's Hospital/Southwest Jiaotong University Affiliated Hospital,Chengdu 610031,Sichuan,China;Department of Critical Care Medicine,Zhuhai People's Hospital,Zhuhai 519000,Guangdong,China)

机构地区：[1]成都市第三人民医院/西南交通大学附属医院全科医学科,四川成都610031 [2]珠海市人民医院重症医学科,广东珠海519000

出　　处：《中华危重病急救医学》2021年第5期523-528,共6页Chinese Critical Care Medicine

基　　金：睿E(睿意)急诊医学专项研究基金资助项目(R2019009)。

摘　　要：目的探讨混合谱系激酶结构域(MLKL)介导的肺脏细胞坏死激活NOD样受体蛋白3(NLRP3)炎症小体引发的炎症反应在脓毒症小鼠急性肺损伤(ALI)中的作用及可能的致病机制。方法采用随机数字表法将18只BALB/c小鼠分为假手术组(Sham组)、盲肠结扎穿孔术(CLP)致脓毒症模型组(CLP组)和特异性抑制剂坏死抑制素-1(Necrostatin-1)干预组(CLP+Nec-1组,制模前10 min经尾静脉注射Necrostatin-1溶液20 mg/kg),每组6只。术后2 d,经眼眶取血并断颈处死小鼠取肺组织,采用苏木素-伊红(HE)染色观察肺组织形态学改变,干湿重法检测肺组织含水率,伊文思蓝(EB)法检测肺血管通透性,蛋白质免疫印迹试验(Western blotting)检测肺组织MLKL和NLRP3的蛋白表达,酶联免疫吸附试验(ELISA)检测血清白细胞介素-1β(IL-1β)水平。结果HE染色显示,Sham组肺组织形态学正常;CLP组肺泡腔和肺间质充血水肿、中性粒细胞侵润,肺泡壁明显增厚;CLP+Nec-1组肺组织形态学改变较CLP组明显好转。与Sham组比较,CLP组肺组织含水率明显增加〔(88.00±0.00)%比(78.00±0.01)%〕,肺血管通透性明显增加〔EB含量(mg/L):11.82±1.15比4.00±0.71〕,坏死性炎症反应相关分子,即肺组织磷酸化MLKL(p-MLKL)和NLRP3的蛋白表达及血清IL-1β水平明显升高〔p-MLKL/GAPDH:0.34±0.04比0.12±0.01,NLRP3/GAPDH:0.47±0.07比0.16±0.04,IL-1β(ng/L):183.56±9.61比44.14±6.95〕,差异均有统计学意义(均P<0.01)。与CLP组比较,CLP+Nec-1组肺组织含水率明显下降〔(81.00±0.01)%比(88.00±0.00)%〕,肺血管通透性降低〔EB含量(mg/L):7.90±0.00比11.82±1.15〕,肺组织p-MLKL和NLRP3的蛋白表达及血清IL-1β水平明显下降〔p-MLKL/GAPDH:0.13±0.03比0.34±0.04,NLRP3/GAPDH:0.18±0.04比0.47±0.07,IL-1β(ng/L):113.81±6.62比183.56±9.61〕,差异均有统计学意义(均P<0.01)。结论坏死性炎症反应信号通路蛋白MLKL和NLRP3的表达水平在脓毒症小鼠肺组织中显著上调;特异性抑制�Objective To investigate the roles and underlying mechanisms of mixed lineage kinase domain like(MLKL)-mediated inflammatory response induced by NOD-like receptor protein 3(NLRP3)inflammatory corpuscles in the acute lung injury(ALI)after sepsis.Methods Eighteen BALB/c mice were randomly divided into sham operation group(Sham group),cecal ligation and perforation(CLP)-induced sepsis model group(CLP group)and specific inhibitor Necrostatin-1 intervention group[CLP+Nec-1 group,Necrostatin-1 solution(20 mg/kg)was injected intravenously 10 minutes before modeling],with 6 mice in each group.The mice were sacrificed by neck amputation at the 2nd day after operation,and the serum and lung tissue samples were collected.The morphological changes of lung tissue were observed by hematoxylin-eosin(HE)staining.The water content of lung tissue was detected by dry-wet weight method.The pulmonary vascular permeability was measured by Evans blue(EB)staining.The protein expressions of MLKL and NLRP3 in the lung tissue were detected by Western blotting,and the level of serum interleukin-1β(IL-1β)was detected by enzyme linked immunosorbent assay(ELISA).Results HE staining showed that the lung morphological structure in Sham group was normal.In CLP group,congestion and edema in the alveolar cavity and interstitium,infiltration of neutrophils and thickening of alveolar wall were observed.The histopathological changes of lung tissue in CLP+Nec-1 group were better than those in CLP group.Compared with Sham group,the water content of lung tissue[(88.00±0.00)%vs.(78.00±0.01)%],pulmonary vascular permeability[EB content(mg/L):11.82±1.15 vs.4.00±0.71],the protein expressions of phosphorylated MLKL(p-MLKL)and NLRP3 in lung tissue(p-MLKL/GAPDH:0.34±0.04 vs.0.12±0.01,NLRP3/GAPDH:0.47±0.07 vs.0.16±0.04),and the level of serum IL-1β(ng/L:183.56±9.61 vs.44.14±6.95)in CLP group were all significantly increased(all P<0.01).Compared with CLP group,the water content of lung tissue[(81.00±0.01)%vs.(88.00±0.00)%],pulmonary vascular permea

关 键 词：脓毒症 急性肺损伤 程序性坏死 炎症 坏死性炎症 

分 类 号：R459.7[医药卫生—急诊医学] R563[医药卫生—治疗学]