Ibrutinib Alleviates Sepsis-induced Acute Lung Injury Through Inhibiting NLRP3 Inflammasome-dependent Pyroptosis

Background: Sepsis-induced acute lung injury is a leading cause of death in critically ill patients,mainly manifested by pulmonary edema,pulmonary hemorrhage and dysfunction of gas exchange in the lungs.Pyroptosis is a kind of programmed death related to pro-inflammatory response.Studies have shown that macrophage pyroptosis is related to sepsis-induced acute lung injury.NLRP3 inflammasome is involved in the classic pathway of pyroptosis.It is reported that Bruton’s tyrosine kinase is involved in the activation of NLRP3 inflammasomes.However,whether BTK participates in NLRP3 inflammasome-dependent pyroptosis and aggravates sepsis-induced acute lung injury is still lacking evidence.Therefore,we speculate whether BTK inhibitor can affect the activation of NLRP3 inflammasomes,inhibit pyroptosis and protect lung tissue by inhibiting BTK in septic lung injury.Objective: To observe the effect of BTK inhibitor ibrutinib on NLRP3 inflammasome activation and macrophage pyroptosis using cecal ligation and perforation(CLP)sepsis model,and whether it can alleviate septic lung damage.Methods: The mice(C57BL/6 male mice,6-8w,weight 20-25g)were splited into three groups,which were named sham operation group(Sham group),sepsis model group(CLP group)and BTK Inhibitor treatment group(IBR group).The plasma,lung tissue and alveolar lavage fluid of the mice were collected and analysed.(1)Observing the survival status and body weight changes of mice within 3 days after operation;(2)Collecting plasma of the mice for inflammatory factor detection;(3)HE staining to observe the pathological changes of the lung tissues;(4)quantification of alveolar lavage fluid protein;(5)lung tissue homogenate for myeloperoxidase(MPO)activity determination;(6)western blot and other methods to detect the lung tissue NLRP3/Caspase-1/GSDMD/IL-1β protein;(7)In vitro cell experiment using RAW264.7 cell line,the cells were randomly divided into LPS+ATP(-)group,LPS+ATP(+)group,LPS+ATP+IBR group.After the corresponding intervention,the cells were collected to extract total protein,and Western blotting was performed to detect the expression of pyroptosis-related proteins.Results:(1)Ibrutinib can reduce the mortality of septic mice;(2)Ibrutinib can alleviate the systemic inflammatory response in septic mice;(3)The lung tissue of mice is damaged,including inflammatory cell penetration,alveolar wall thickening,local lung tissue hemorrhage,etc.Inflammatory factors IL-1β and IL-18 are expressed in large quantities in lung tissues,while pyroptosis-related proteins such as NLRP3,C-Caspase-1,GSDMD-NT are highly expressed in lung tissues;(4)Ibrutinib can effectively alleviate lung tissue damage by inhibiting NLRP3/Caspase-1/GSDMD/IL-1β axis to inhibit pyroptosis;(5)Ibrutinib can significantly inhibit pyroptosis in RAW264.7 cell line.Conclusion: Ibrutinib can effectively improve septic lung injury by inhibiting the activation of NLRP3 inflammasomes,thereby inhibiting pyroptosis in the lungs and reducing the level of systemic inflammatory factors.