Role and mechanism of the NOD-like receptor 3 inflammasome in ventilator-induced lung injury in rats

Abstract

To investigate the role and its mechanism of the NOD-like receptor 3 (NLRP3) inflammasome in alveolar macrophages in ventilator-induced lung injury (VILI) in rats. Thirty adult male Sprague-Dawley (SD) rats were randomly divided into three groups, with 10 rats in each group: spontaneous breathing control group, normal tidal volume (V(T)) group (V(T) 8 mL/kg) and high V(T) group (V(T) 40 ml/kg). All of the rats underwent tracheotomy. Then rats in spontaneous breathing control group were kept to have spontaneous breathing, while rats in normal V(T) group and high V(T) group received mechanical ventilation. After 4 hours, the rats were sacrificed by carotid artery bleeding, and the bronchoalveolar lavage fluid (BALF), blood serum and lung tissue were collected. Lung wet/dry ratios (W/D) were measured. Light microscopy and electron microscopy were performed to observe the pathological changes in lung tissue, and the ultrastructural changes in alveolar macrophages. Enzyme linked immunosorbent assay (ELISA) was performed to measure the total protein content in the BALF and the interleukins (IL-1β and IL-18) in the serum and BALF. The mRNA expressions and protein levels of NLRP3, apoptosis-associated speck-like protein containing CARD (ASC), caspase-1. and nuclear factor-κB (NF-κB) in alveolar macrophages were assayed by real-time fluorescent quantization reverse transcription-polymerase chain reaction (RT-PCR) and Western Blot. The structure of lung tissue and alveolar macrophages of rats in spontaneous breathing control group and normal V(T) group appeared normal, while obvious inflammatory changes were found in high V(T) group. Compared with spontaneous breathing control group and normal V(T) group, the ratio of W/D (8.89 ± 0.90 vs 5.18 ± 0.86, 5.71 ± 0.82, both P < 0.05), contents of total protein, IL-1β, IL-18 in BALF were significantly increased [total protein (g/L): 2.34 0.41 vs. 1.77 ± 0.14, 1.81 ± 0.06, IL-1β (ng/L): 133.48 ± 10.48 vs 81.54 ± 3.12, 83.80 ± 5.22, IL-18 (μg/L): 4.57 ± 0.45 vs 3.04 ± 0.51, 3.43 ± 0.43, aa P < 0.05], and IL-1β and IL-18 in serum were also increased [IL-1β (ng/L); 105.06 ± 10.18 vs 65.11 ± 8.58, 75.30 ± 10.62, IL-18 (μg/L); 2.27 ± 0.09 vs 1.18 ± 0.34, 1.43 ± 0.15, all P < 0.05]. The mRNA and protein expressions of NLRP3, ASC, caspase-1 and NF-κB in alveolar macrophages of high V(T) group were significantly increased compared with that of spontaneous breathing control group and normal V(T) group. The mRNA expressions of NLRPs, ASC, caspase-1 and NF-κB in high V(T) group were (8.53 ± 2.21), (5.75 ± 1.17), (7.47 ± 1.23) and (10.86 ± 2.38) folds of those in spontaneous breathing control group, and the protein expressions of NLRP3, ASC, caspase-1 and NF-κB were (1.50 ± 0.14), (1.49 ± 0.04), 1.53 ± 0.15) and (1.51 ± 0.110 folds of those in spontaneous breathing control group (all P < 0.01). There were no significant differences in all the indexes between normal V(T) group and spontaneous breathing control group. NLRP3 inflammasome in alveolar macrophages may be involved in the mechanism of occurrence of VILI.