Role and mechanisms of MLKL-NLRP3-mediated necroinflammation in mice with sepsis-induced acute lung injury / 中华危重病急救医学

ABSTRACT

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 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). Conclusion:MLKL-NLRP3-mediated necroinflammation was significantly up-regulatedin the lung tissue of septic mice, which could be attenuated by specific inhibitor Necrostatin-1.