Simvastatin mitigates ventilator-induced lung injury in mice with acute respiratory distress syndrome via a mechanism partly dependent on neutrophil extracellular traps

Abstract

Background: Mechanical ventilation (MV) is an essential life support for patients with acute respiratory distress syndrome (ARDS). However, mechanical ventilation in patients with ARDS can cause ventilator-induced lung injury (VILI). Simvastatin can alleviate acute lung injury by anti-inflammatory and enhancing endothelial barrier. The present study aimed to evaluate whether simvastatin could attenuate VILI in mice with ARDS.

Methods: Mice were randomized into six groups: the sham (S), LPS (L), MV (V), LPS/MV (LV), LPS/MV/simvastatin (MS) and LPS/MV/GSK484 (MG) groups. The mice in the L group received LPS but not ventilation, the mice in the V group received only MV, and the mice in the LV, MS and MG groups received LPS and MV. Additionally, MS group were treated with simvastatin, MG group were treated with GSK484, and the other mice were injected with saline, starting three days prior to mechanical ventilation. The PaO₂/FiO₂ ratio and wet‒dry weight ratio were calculated. Histopathological changes were observed, and injury scores were calculated. Inflammatory factor levels in the bronchoalveolar lavage fluid (BALF) were detected. Peptidylarginine deiminase 4 (PAD4), neutrophil elastase (NE) and citrullinated histone 3 (Cit-H3) in the lung tissue were detected, apoptosis were also evaluated.

Results: All indices were improved in group S compared with the other groups. The lung injury score and wet‒dry weight ratio were lower, the PaO₂/FiO₂ ratio was greater, inflammatory factor levels in the BALF were lower, PAD4, NE, and Cit-H3 expression was lower, and apoptosis was decreased in the MS and MG groups compared with the LV group.

Conclusions: Simvastatin attenuated VILI in mice with ARDS, potentially via reductions in neutrophil extracellular traps (NETs) generation and apoptosis.

Keywords: Acute respiratory distress syndrome; Mechanical ventilation; Neutrophil extracellular traps; Simvastatin.

Fig. 1

Effects of simvastatin on alveolocapillary permeability. a The PaO₂/FiO₂ ratio were assessed at the end of 4 h ventilation. b The wet/dry weight ratio were assessed. c The total protein concentration in the BALF were assessed. The data are presented as the means ± SD. a P < 0.05 vs. the S group; b P < 0.05 vs. the LV group. N = 12 per group

Fig. 2

Effects of simvastatin on lung histopathology. a Lung tissue injury was evaluated by HE staining (scale bar: 50 μm). b Lung injury scores were assessed. The data are presented as the means ± SD. a P < 0.05 vs. the S group; b P < 0.05 vs. the LV group. N = 12 per group

Fig. 3

Effects of simvastatin on inflammation. a The levels of TNF-α, IL-1β, and IL-6 in BALF were determined via ELISA. The number of neutrophils in the BALF was also detected. b The levels of TNF-α, IL-1β, and IL-6 in serum were determined via ELISA. The data are presented as the means ± SDs. a P < 0.05 vs. the S group; b P < 0.05 vs. the LV group. N = 12 per group

Fig. 4

The production of NETs was reduced by simvastatin. a The expression of PAD4 in lung tissue sections was detected by immunohistochemistry (scale bar: 50 μm). b Representative immunofluorescence images of CitH3 and DAPI staining (scale bar: 50 μm). c The NET-associated proteins PAD4, NE, Cit-H3 and MPO were determined. The data are presented as the means ± SD. a P < 0.05 vs. the S group; b P < 0.05 vs. the LV group; c P < 0.05 vs. the MS group. N = 12 per group

Fig. 4

The production of NETs was reduced by simvastatin. a The expression of PAD4 in lung tissue sections was detected by immunohistochemistry (scale bar: 50 μm). b Representative immunofluorescence images of CitH3 and DAPI staining (scale bar: 50 μm). c The NET-associated proteins PAD4, NE, Cit-H3 and MPO were determined. The data are presented as the means ± SD. a P < 0.05 vs. the S group; b P < 0.05 vs. the LV group; c P < 0.05 vs. the MS group. N = 12 per group

Fig. 5

Effects of simvastatin on apoptotic cell death. a TUNEL staining was performed on lung tissues from different treatment groups (scale bar: 10 μm). Quantification of TUNEL staining was also calculated. b The apoptotic proteins Bax and Bcl-2 measured by western blotting. The data are presented as the means ± SD. a P < 0.05 vs. the S group; b P < 0.05 vs. the LV group. N = 12 per group