Objective: To investigate the role of Ras-related C3 botulinum toxin substrate 1/mitogen-activated protein kinase/extracellular signal-regulated kinase (Rac1/MAPK/ERK) signal pathway in rats with ventilator induced lung injury (VILI) and its mechanism.
Methods: Thirty Sprague-Dawley (SD) rats were randomly divided into spontaneous respiration group, normal tidal volume (VT) group and high VT group with 10 rats in each group. The rats in spontaneous respiration group were kept their spontaneous breathing. The rats in normal VT group and high VT group were performed tracheal intubation after tracheostomy, and underwent mechanical ventilation on bilateral lungs with 6 mL/kg and 40 mL/kg VT respectively with maintenance anesthesia. After 4-hour ventilation, heart blood, bronchoalveolar lavage fluid (BALF) and lung tissues were harvested. The levels of interleukins (IL-1β, IL-6), tumor necrosis factor-α (TNF-α), myeloperoxidase (MPO) and macrophage inflammatory protein-2 (MIP-2) in serum and BALF were determined by enzyme linked immunosorbent assay (ELISA). Lung wet/dry radio (W/D) was determined. The lung tissues were stained with hematoxylin and eosin (HE), and pathological changes were observed, and pathological scores were evaluated. The ultra structure changes in type II alveolar epithelial cells (AEC II) were observed with transmission electron microscope. The positive expressions of phosphorylation of extracellular signal-regulated kinase (p-ERK) were determined by immunohistochemistry, and those of Rac1 and F-actin were determined by immunofluorescence. The mRNA expressions of ERK and Rac1 were determined by real-time fluorescent quantitation reverse transcription-polymerase chain reaction (RT-qPCR), and protein expressions of Rac-1, p-ERK and F-actin were determined by Western Blot.
Results: (1) Compared with spontaneous breathing group, lung W/D in both mechanical ventilation groups was significantly increased, with more significant increase in the high VT group (6.64±0.88 vs. 1.79±0.36, P < 0.01). (2) There was no obvious pathological changes in the lung tissue and AEC II of the spontaneously breathing group. In the normal VT group, there was slight edema and infiltration of inflammatory cells; AEC II had less lamellar bodies and uniform distribution of the villi of the alveolar epithelium. In the high VT group, the edema of the lung tissue, the widening of the pulmonary septum, the alveolus congestion, the infiltration of inflammatory cells, and alveolar structure disorder were found; and AEC II was irregular, the number of lamellar bodies in the plastids was decreased and was unevenly distributed. The pulmonary histopathological score in the high VT group was significantly higher than that in the spontaneous breathing group and the normal VT group (12.00±2.00 vs. 6.00±1.51, 8.50±0.53, both P < 0.01). (3) Compared with spontaneous breathing group, IL-1β, IL-6, TNF-α, MPO, and MIP-2 in serum and BALF in both mechanical ventilation groups were significantly increased, with more significant increase in the high VT group [serum IL-1β (ng/L): 104.2±15.1 vs. 20.3±8.3, IL-6 (ng/L): 46.6±11.5 vs. 22.7±7.5, TNF-α (ng/L): 39.4±6.5 vs. 5.4±1.9, MPO (ng/L): 0.66±0.24 vs. 0.06±0.03, MIP-2 (ng/L): 109.2±25.8 vs. 22.8±8.4; BALF IL-1β (ng/L): 121.5±25.6 vs. 24.0±7.5, IL-6 (ng/L): 136.7±32.7 vs. 31.4±10.5, TNF-α (ng/L): 98.0±14.8 vs. 10.1±2.6, MPO (ng/L): 0.80±0.31 vs. 0.08±0.04, MIP-2 (ng/L): 144.4±28.9 vs. 41.2±20.7; all P < 0.01]. (4) There were only a few p-ERK, Rac1 and F-actin positive expressions in the spontaneous breathing group. The positive expressions in normal VT group were increased. In high VT group, the positive expression of p-ERK was significantly increased; Rac1 and F-actin were mainly distributed in the cell membrane and cytoplasm respectively, the positive expressions were further enhanced. (5) The gene expressions of ERK and Rac1, and protein expressions of p-ERK, Rac1 and F-actin in the high VT group were significantly higher than those in the spontaneous breathing group and normal VT group [ERK mRNA (2-ΔΔCt): 8.23±2.83 vs. 1, 3.02±1.38, p-ERK protein (gray value): 1.15±0.36 vs. 0.61±0.23, 0.88±0.22; Rac1 mRNA (2-ΔΔCt): 4.45±2.26 vs. 1, 1.22±0.39, Rac1 protein (gray value): 0.91±0.16 vs. 0.48±0.11, 0.55±0.10; F-actin protein (gray value): 0.70±0.09 vs. 0.49±0.08, 0.55±0.04; all P < 0.01].
Conclusions: F-actin expression in lung tissue was up-regulated in rats with VILI, which resulted in reconstruction of AEC II cyto-skeleton, and variation of cell membrane permeability through Rac1/MAPK/ERK signaling pathway during VILI.