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. Corresponding author: He Qing, Email:
Zhonghua Wei Zhong Bing Ji Jiu Yi Xue. 2021 May;33(5):523-528. doi: 10.3760/cma.j.cn121430-20200622-00483.
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.
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).
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 permeability [EB content (mg/L): 7.90±0.00 vs. 11.82±1.15], protein expressions of p-MLKL and NLRP3 in lung tissue (p-MLKL/GAPDH: 0.13±0.03 vs. 0.34±0.04, NLRP3/GAPDH: 0.18±0.04 vs. 0.47±0.07), and the level of serum IL-1β (ng/L: 113.81±6.62 vs. 183.56±9.61) were all significantly decreased (all P < 0.01).
MLKL-NLRP3-mediated necroinflammation was significantly up-regulatedin the lung tissue of septic mice, which could be attenuated by specific inhibitor Necrostatin-1.
探讨混合谱系激酶结构域样蛋白（MLKL）介导的NOD样受体蛋白3（NLRP3）炎性小体诱导的炎症反应在脓毒症后急性肺损伤（ALI）中的作用及潜在机制。
将18只BALB/c小鼠随机分为假手术组（Sham组）、盲肠结扎穿孔（CLP）诱导的脓毒症模型组（CLP组）和特异性抑制剂Necrostatin-1干预组[CLP+Nec-1组，建模前10分钟静脉注射Necrostatin-1溶液（20 mg/kg）]，每组6只。术后第2天断头处死小鼠，采集血清和肺组织样本。采用苏木精-伊红（HE）染色观察肺组织形态学变化。采用干湿重法检测肺组织含水量。采用伊文思蓝（EB）染色检测肺血管通透性。采用蛋白质免疫印迹法检测肺组织中MLKL和NLRP3的蛋白表达，采用酶联免疫吸附测定（ELISA）法检测血清白细胞介素-1β（IL-1β）水平。
HE染色显示，Sham组肺组织形态结构正常。CLP组可见肺泡腔和间质充血、水肿，中性粒细胞浸润，肺泡壁增厚。CLP+Nec-1组肺组织的组织病理学变化较CLP组好转。与Sham组比较，CLP组肺组织含水量[（88.00±0.00）% vs.（78.00±0.01）%]、肺血管通透性[EB含量（mg/L）：11.82±1.15 vs. 4.00±0.71]、肺组织中磷酸化MLKL（p-MLKL）和NLRP3的蛋白表达（p-MLKL/GAPDH：0.34±0.04 vs. 0.12±0.01，NLRP3/GAPDH：0.47±0.07 vs. 0.16±0.04）及血清IL-1β水平（ng/L：183.56±9.61 vs. 44.14±6.95）均显著升高（均P<0.01）。与CLP组比较，CLP+Nec-1组肺组织含水量[（81.00±0.01）% vs.（88.00±0.00）%]、肺血管通透性[EB含量（mg/L）：7.90±0.00 vs. 11.82±1.15]、肺组织中p-MLKL和NLRP3的蛋白表达（p-MLKL/GAPDH：0.13±0.03 vs. 0.34±0.04，NLRP3/GAPDH：0.18±0.04 vs. 0.47±0.07）及血清IL-1β水平（ng/L：113.81±6.62 vs. 183.56±9.61）均显著降低（均P<0.01）。
MLKL-NLRP3介导的坏死性炎症在脓毒症小鼠肺组织中显著上调，特异性抑制剂Necrostatin-1可使其减轻。
