The 6-8 weeks old, weighing 20-22 g, SPF male C57BL/6 mice were purchased from the Experimental Animal Center of Wuhan University. The mice were randomly divided into 3 per cage and with free access to food and water. All the mice were housed in the animal experiment center of the SPF environment with the humidity of 60–65%, the light-dark cycles 12/12h, and the temperature of 22-24°C. All the mice were acclimatized for one week before the start of the experiment. The experiment was performed in accordance with the Chinese Animal Research Guidelines and was approved by the Laboratory Animal Management Committee of Tongji Medical College of Huazhong University of Science and Technology.
2.2 Experimental design
The construction method of the VILI model is the same as that described in our previous literature. All experiments were repeated no less than three times.
The mice were first randomly divided into the following 4 groups (n=7): (1)The sham operation group (the Sham group): tracheal intubation was performed, but no mechanical ventilation was performed. 200 μl of saline was injected into the tail vein 30 minutes before the tracheal intubation (2) The maresin1(Cayman Chemical, Ann Arbor, MI, USA) intervention control group（the MaR1 group）: tracheal intubation was performed, but no mechanical ventilation was performed. Tail vein injection of Maresin1 1 ng(200 μl) 30 minutes before the tracheal intubation; (3) The large tidal volume mechanical ventilation group (the HVT group): tidal volume 40 ml/kg; 200 μl of saline was injected into the tail vein 30 minutes before the tracheal intubation. (4) the Maresin1 intervention group（the MaR1 + HVT group): tidal volume 40 ml/kg, tail vein injection of Maresin1 1 ng(200 μl) 30 minutes before the beginning of the tracheal intubation. After that, we used Znpp, the specific HO-1 inhibitor. The mice were randomly divided into the following six groups(n=7). (1) The Sham group(2) The MaR1 group (3) The ZnPP intervention control group（the Znpp group）: tracheal intubation was performed, but no mechanical ventilation was performed. The HO-1 antagonist ZnPP (10 mg/kg) was intraperitoneally injected and 200 μl of saline was injected into the tail vein 30 minutes before the tracheal intubation. (4) The HVT group (5) The MaR1 + HVT group (6) The HO-1 antagonist zinc protoporphyrin + Maresin1 group (the ZnPP + Maresin1 group): ZnPP (10 mg/kg) was intraperitoneally injected 30 minutes before the start of the tracheal intubation and the rest of the treatment was consistent with the MaR1 + HVT group. The mice in the sham group, the MaR1 group, the HVT group, and the MaR1 + HVT group were intraperitoneally injected additional saline(10 mg/kg) 30 minutes before starting the experiment. The respiratory rate of each group of mechanical ventilation is 80 times/min, the mechanical ventilation time is 4h, FiO2=2l%, I:E=1:2, PEEP=0 mmHg.
2.3 Histological analysis of lung tissues
The right upper lobe of the lung tissue was harvested for the pathological analysis. The lung tissue was made into slices about approximated 4 μm thick after being fixed in 4% paraformaldehyde for 24h. The slices were stained with the hematoxylin and the eosin. Then the sections were observed under the ordinary optical microscope. The pathological injury score of the lung tissue was performed by using the lung tissue injury scoring criteria published by the American Thoracic Society.
2.5 Lung wet-to-dry weight ratio
The middle lobe of the right lung tissue from each group was collected to calculate the lung wet-to-dry weight ratio. After cleaning the surface of the fresh lung tissue with absorbent paper, weigh the initial weight of the lung tissue. Then put the lung tissue into the oven until the weight of the lung tissue becomes a constant weight. The weight of the lung tissue was weighed again. Calculate the lung wet-to-dry weight ratio and assess the extent of the lung tissue edema.
2.6 Analysis of arterial blood gas
At the end of the experiment, the blood (heparin anticoagulant) of the left carotid artery was collected and the arterial partial pressure of oxygen was immediately measured by a blood gas analyzer (Radiometer, Copenhagen, Denmark).
2.6 Measurement of the markers of the oxidative stress in lung tissues
According to the manufacturer's instructions, the malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), glutathione (GSH), myeloperoxidase(MPO) and 15-F2t-isoprostane were detected by their corresponding commercial assay kits (Jiancheng Bioengineering Insti-
tute, Nanjing, China). The activities of these indicators were assessed by a spectrophotometer
2.7 Bronchial alveolar lavage fluid analysis
After the ligation of the right lung, 0.5 mL phosphate buffer saline (PBS) was utilized to lavage the left lung to obtain the BALF. The lavage process was repeated three times, and the recovery rate of more than 80% was considered effective.
The number of cells in the BALF was counted by a haematocytometer.
The protein concentration of the BALF was measured by using the BCA Protein Assay kit (Thermo Fisher Scientific).
According to the manufacturer’s instructions, the corresponding murine ELISA kits (Dakewe Bioengineering Co., Ltd., Shenzhen, China) were used to evaluate the levels of the related inflammatory mediators (IL-6, TNF-α, IL-β) in the BALF.
2.10 Western blotting analysis
According to the manufacturer’s protocol, the protein of the lung tissue was extracted by using the protein extraction reagent kit (KeyGEN BioTECH, Nanjing, China) and the nucleoprotein was extracted using the Nuclear and Cytoplasmic Protein Extraction Kit(Beyotime, China). The protein concentration was measured with the BCA Protein Assay kit(Thermo Fisher Scientific). The western blotting analysis was performed as mentioned in our previous research. The membrane was first incubated with the primary antibody: Nrf2 (Cell Signaling Technology, Danvers, MA, USA), HO-1(Cell Signaling Technology, Danvers, MA, USA), NF-κB p65(Cell Signaling Technology, Danvers, MA, USA), Histone 3 (Cell Signaling Technology, Danvers, MA, USA), GAPDH（Antgene, China）at 4℃ overnight. Then the membrane was incubated with the corresponding secondary antibody (Cell Signaling Technology, Danvers, MA, USA). Finally, the enhanced chemiluminescence reagent (Beyotime Institute of Biotechnology, Shanghai, China) and the UVP 160 imaging system (Upland, CA, USA)was used for exposure imaging. The grayscale value of the images was analyzed using the Image J software.
2.11 Statistical Analysis.
The whole data were expressed as means±SEM. The processes of the statistical analysis process and the mapping were performed by the Graphpad Prism 6.0 software. One-way analysis of variance (ANOVA) was used for comparison among groups, and the differences between groups were compared using Newman-Keuls. P < 0.05 was considered to be statistically significant.