Role and mechanism of mitophagy in ventilator-induced lung injury in rats

Abstract

To explore the role and mechanism of mitophagy in ventilator-induced lung injury (VILI) in rats. Thirty-six adult Sprague-Dawley (SD) rats were randomly divided into three groups (each n = 12): spontaneous breathing group (CON group), normal tidal volume (VT) group (NVT group, VT was 8 mL/kg) and high VT group (HVT group, VT was 40 mL/kg). All rats received endotracheal tube by tracheostomy. The rats in CON group were maintained spontaneous breathing, while those in NVT and HVT groups received mechanical ventilation with corresponding VT. After 4 hours of ventilation, the serum, bronchoalveolar lavage fluid (BALF) and lung tissues were harvested. The lung wet/dry weight (W/D) ratio was assessed, and the histopathology changes were observed by light microscopy, and the ultra structure changes in type II alveolar epithelial cell (AEC II) were observed by electron microscopy. The levels of interleukin (IL-1β and IL-6) and tumor necrosis factor-α (TNF-α) in serum and BALF were determined by enzyme linked immunosorbent assay (ELISA). The total protein in BALF was measured by bicinchoninic acid methods, and the infiltrated cells in BALF were counted. The mRNA expressions and protein levels of microtube associated light chain 3B (LC3B), mitochondrial DNA coded cytochrome C oxidase IV (COX-IV) and nuclear factor-KB p65 (NF-KB p65) in lung tissues were determined by real-time fluorescent quantitation reverse transcription-polymerase chain reaction (RT-qPCR) and Western Blot. The histopathology of lung tissue and the ultra structure of AEC II were normal in CON group and NVT group, and the obvious inflammatory changes and pathological injury were found in HVT group. Compared with CON and NVT groups, the W/D ratio in HVT group was significantly increased (8.53±1.05 vs. 5.12±0.65, 5.57±0.55, both P < 0.05), and total protein, infiltrated cells, and IL-1β, IL-6 and TNF-α in BALF were significantly increased [total protein (g/L): 2.35±0.45 vs. 1.46±0.34, 1.76±0.51; infiltrated cells (×105/mL): 2.05±0.48 vs. 0.40±0.08, 0.60±0.23; IL-1β (ng/L): 119.82±6.56 vs. 76.15±3.32, 79.39±4.44; IL-6 (μg/L): 4.10±0.52 vs. 1.97±0.40, 2.27±0.36; TNF-α (mg/L): 1.49±0.28 vs. 0.43±0.23, 0.61±0.24; all P < 0.05], IL-1β, IL-6 and TNF-α levels in serum were also elevated [IL-1β (ng/L): 127.53±7.10 vs. 79.40±2.80, 82.95±2.25; IL-6 (μg/L): 6.28±0.82 vs. 2.96±0.35, 3.36±0.72; TNF-α (mg/L): 1.59±0.42 vs. 0.53±0.22, 0.78±0.25; all P < 0.05]. The mRNA expressions and protein levels of LC3B, COX-IV and NF-KB p65 in lung tissue of HVT group were significantly higher than those of CON group and NVT group, the mRNA expressions of LC3B-II, COX-IV and NF-KB p65 were (3.52±0.90), (3.76±1.16) and (9.54±2.06) folds of those in CON group, and the protein expressions were (1.76±0.24), (1.65±0.20) and (1.91±0.12) folds of those in CON group, with significantly statistical differences (all P < 0.05). There were no significant differences in the parameters mentioned above between NVT group and CON group. Mitophagy may be associated with VILI resulting from escaped mitochondrial DNA for activation of inflammation.