Protective effect and mechanism of down-regulating NLRP3 inflammasome expression on acute lung injury in septic mice
Objective To investigate the protective effect of down-regulating the expression of nucleotide-binding oligomerization domain like receptors family pyrin domain containing 3 (NLRP3) inflammasome on acute lung injury of septic mice and its mechanism of modulating mitophagy. Methods Eighteen BALB/c male mice were randomly divided into sham group,cecal ligation and puncture (CLP) group and CLP+MCC950 (NLRP3-specific inhibitor) group using a random number table method,with six mice in each group. The CLP procedure was used to establish the sepsis model. Mice in the CLP+MCC950 group were intraperitoneally injected with 50 mg/kg MCC950 (diluted to 0.5 mL with 0.9％ NaCl solution) 2 h before the operation,while the sham and CLP groups were given equal amounts of 0.9％ NaCl solution. Bronchoalveolar lavage fluid (BALF) and lung tissue were collected 24 h after CLP operation. Another 45 mice were divided into sham group,CLP group,and CLP+MCC950 group using the random number table method,and the 7 d survival rate of mice was recorded. HE staining was used to observe the severity of lung injury and the wet weight/dry weight (W/D) ratio was measured. Enzyme-linked immunosorbent assay was used to measure the protein levels of IL-1β,IL-6,TNF-α,and IL-10 in BALF;Western blot was used to detect the expressions of lung tissue inflammation associated proteins[NLRP3,phosphorylated NF-κB (p-NF-κB) p65,and NF-κB-p65],apoptosis associated proteins[cleaved-cysteine-aspartic acid protease 3 (Cleaved-Caspase-3),B-cell lymphoma-2 (Bcl-2),B-cell lymphoma-2 associated X protein (Bax)],and autophagy associated proteins[PTEN induced putative kinase 1 (PINK1),E3 ubiquitin ligase (Parkin),microtubule-associated protein 1 light chain 3Ⅱ/Ⅰ(LC3 Ⅱ/Ⅰ),and selective autophagy adaptor protein p62]. Results The 7 d survival rate of mice in the sham group was 100.0％. Eleven mice in the CLP group died cumulatively at 7 d,with the 7 d survival rate of 26.7％ (4/15). Compared with the sham group,the 7 d survival rate of mice in the CLP group was significantly lower (P<0.05),the lung W/D ratio was elevated (P<0.05),and the lung tissues experienced severe destruction of alveolar structure and thickening of alveolar wall,accompanied by obvious pulmonary edema and inflammatory cell infiltration;the levels of IL-1β,IL-6 and TNF-αproteins were elevated in the BALF,accompanied by elevated level of IL-10 protein (all P<0.05);the levels of NLRP3,p-NF-κB p65,Cleaved-Caspase-3,Bax,PINK1,Parkin,and LC3 Ⅱ/Ⅰ proteins were increased,while the levels of Bcl-2 and p62 proteins were decreased (all P<0.05). Eight mice in the CLP+MCC950 group died consecutively during 7 d,with a 7 d survival rate of 46.7％ (7/15). Compared with the CLP group,the 7 d survival rate of mice in the CLP+MCC950 group was not statistically different (P>0.05),and lung W/D ratio was decreased (P<0.05);the destruction of alveolar structure in lung tissues was slightly reduced,the thickening of alveolar wall was alleviated,and the pulmonary edema and inflammatory cell infiltration were also improved;the levels of IL-1β,IL-6 and TNF-αproteins in the BALF were reduced,while the level of IL-10 protein was further elevated (all P<0.05);the levels of NLRP3,p-NF-κB p65,Cleaved-Caspase-3,Bax proteins were decreased,while the level of Bcl-2 protein increased in lung tissues (all P<0.05);the levels of PINK1,Parkin,and LC3 Ⅱ/Ⅰ protein expressions were further elevated,while the level of p62 protein was further decreased (all P<0.05). Conclusion Down-regulation of NLRP3 inflammasome ameliorates acute lung injury of septic mice,and its effect may depend on regulation of the PINK1/Parkin-mediated mitochondrial autophagy pathway.
SepsisNucleotide-binding oligomerization domain like receptors family pyrin domain containing 3Acute lung injuryMitophagy
万方数据
维普
