Adult male Sprague-Dawley (SD) rats (220–250 g) were purchased from the Animal Center of Guangxi Medical University (Nanning, China). Rats were kept in conditions free of specific pathogens and had free access to sterilized water and food. The study was conducted in strict accordance with animal experiment protocols approved by the Institutional Animal Care and Use Committee of Guangxi Medical University (Nanning, China). All animal protocols complied with the Animal Guide of the Institutional Animal Care and Use Committee of Guangxi Medical University.
2.2. Animals model of LIRI
BMS202 (PD-1 inhibitor), SC79 (Akt activator), and NSC87877 (SHP1/2 inhibitor) were used in this study. The rats in the control group breathed autonomously and were killed after cervical dislocation without treatment, while rats in I/R models were established as in our previous study. Tracheal intubation was performed after anesthesia in rats. During mechanical ventilation, the ALC-V9A small animal ventilator provided a 1:1 inspiratory: expiratory ratio and a respiration rate of 60 breaths per minute. The tidal volume was 4–6 ml/kg, and the inhaled oxygen content was 100%. Rats in the BMS202 + I/R group were given BMS202 (5 mg/kg) by gavage 60 minutes before the start of surgery. Rats in the NSC87877 + I/R group were given NSC87877 (10 mg/kg) by gavage 60 minutes before the start of surgery. Rats in the SC79 + I/R group were given SC79 (40 mg/kg) by intraperitoneal injection 60 minutes before the start of surgery. Rats in the I/R group received thoracotomy, then the left hilus pulmonis was clamped with avascular forceps for 1 h, and the chest was closed and the skin was sutured two hours after reperfusion. Finally, rats were sacrificed by bloodletting through the carotid artery. Left lung tissues were collected and stored in liquid nitrogen for subsequent testing.
2.3. AMs model of OGD/R
NR8383 cells (a rat alveolar macrophage cell line) were purchased from ScienCell Research Laboratories (San Diego, CA, USA). The cells were routinely cultured in Ham's F-12K (Kaighn's) medium, containing 15% fetal bovine serum (FBS), at 37°C in 95% air and 5% CO2. To mimic the hypoxic-ischemic conditions of the LIRI in vitro experiment, we subjected NR8383 cells to OGD/R as described. Briefly, NR8383 cells were washed three times with pre-warmed (37°C) glucose-free balanced salt solution (PBS), fed serum-free and glucose-free Ham's F-12K medium, and then cultured for 1 h at 37°C in a Whitley H35 Hypoxystation (Don Whitley Scientific, West Yorkshire, UK) in a hypoxic atmosphere of 1% O2, 5% CO2, and 94% N2. Then, cells were recovered oxygen for 2 h at 37°C in the glucose-containing medium in an atmosphere of 5% CO2 and 95% O2. The normoxic control cells were incubated at 37°C in a humidified atmosphere of 95% air/5% CO2. In some experimental groups, cells were respectively pretreated with BMS202 (1 µM) for 30 minutes, SC79 (10 µM) for 60 minutes, and NSC87877 (10 µM) for 30 minutes and cultured in Ham's F-12K medium under normoxic condition or in serum-free and glucose-free Ham's F-12K medium under the hypoxic condition as mentioned above.
2.4. Reagents and Antibodies.
BMS202, SC79, and NSC87877 were purchased from Selleck Chemicals (Houston, TX, USA); TNF-α, IL-6, and IL-1β enzyme-linked immunosorbent assay (ELISA) kits from Elabscience Biotechnology (Wuhan, China); PD-1 ELISA kits from Meimian (Jiangsu, China); HIF-1α, SHP1, SHP2, p-Akt, Akt, and β-actin from Cell Signaling Technology (Danvers, MA, USA); PD-1, CD11c, CD16, CD86, Anti-CD86/PE, CD206, Anti-CD206/FITC, and Arg-1 antibodies from Bioss (Beijing, China); Dectin-1 from Abcam (Cambridge, UK); fluorophore-labeled goat anti-rabbit secondary antibody (Alexa Fluor 594) and goat anti-rabbit immunoglobulin horseradish peroxidase (IgG-HRP), BSA, and bicinchoninic acid assay (BCA) protein detection kits from Beyotime (Shanghai, China); SP (mouse/rabbit IgG)-POD kit from Solarbio Life Sciences (Beijing, China); FBS and Ham's F-12K (Kaighn's) medium from Gibco (Carlsbad, CA, USA).
2.5. Hematoxylin-eosin staining
Lung tissue specimens were fixed with 4% paraformaldehyde, embedded in paraffin, cut into slices, stained with hematoxylin and eosin (HE), and observed under a light microscope. Degree of lung tissue injury was scored as previously described, based on aggregation or infiltration of inflammatory cells in vessel walls or air spaces [1 point = only wall, 2 = rare cells in air space, 3 = intermediate, and 4 = severe (air space congested)], hyaline membrane formation and interstitial congestion in the lung [1 point = normal lung, 2 = moderate (> 25% of the lung section), 3 = intermediate (25–50% of the lung section), and 4 = severe (> 50% of the lung section)] and presence (1) or absence (0) of hemorrhage. The scores for each criterion were summed to obtain the score for each sample.
2.6. Transmission electron microscopy (TEM)
Lung tissue samples were taken and cut into small pieces of about 1 mm, fixed in 3% glutaraldehyde for more than 2 h, fixed with 1% osmic acid for 1–2 h, and dehydrated in different concentrations of acetone, then finally embedded in resin. The sample was cut into ultrathin sections by an ultramicrotome and analyzed on a transmission electron microscope (Hitachi H-7560, Tokyo, Japan).
2.7. Determination of wet-to-dry ratios
The lung wet-to-dry (W/D) ratios were measured to assess the extent of pulmonary edema. Immediately after collecting the sample, the wet weight of the left lung tissue was weighed, the tissue was incubated at 60℃ for at least 96 h until constant weight, and W/D ratios were calculated.
2.8. Enzyme-linked immunosorbent assay (ELISA)
The concentrations of TNF-α, IL-1β, and IL-6 in lung tissues and cell culture supernatant were quantified using ELISA kits according to the manufacturer’s instructions. The concentration of PD-1 in rat serum was measured using a PD-1 ELISA kit.
2.9. Western blot assay
Fresh lung tissue from a single rat was homogenized in a RIPA buffer containing a protease inhibitor mixture and a phosphatase inhibitor mixture. Different groups of AMs were lysed in RIPA lysis buffer. After quantifying the protein concentration with a BCA protein assay kit, each lysate sample was separated on a 10–15% sodium dodecyl sulfate-polyacrylamide gel and transferred to a 0.22-µm polyvinylidene fluoride (PVDF) membrane at low temperature. After the membrane was treated with 5% bovine serum albumin (BSA) for 1 h, the membrane was incubated on a shaker at 4℃ overnight with specific primary antibodies against PD-1 (1:500), HIF-1α (1:1000), SHP1 (1:1000), SHP2 (1:1000), p-Akt (1:2000), Akt (1:2000), and β-actin. After washing, the membrane was incubated with horseradish peroxidase (HRP)-conjugated anti-rabbit secondary anti-IgG for 1 h at room temperature. The membrane was visualized using an enhanced chemiluminescent reagent and then imaged using a Bio-Rad imaging system. The results obtained under different experimental conditions were normalized to the average value of the corresponding control.
2.10. Immunofluorescence staining
NR8383 cells were fixed with 4% paraformaldehyde at 4 ℃ for 1 h, washed with phosphate buffer (PBS) 3 times, permeated with 0.2% Triton X-100 for 25 min, and then sealed with 5% BSA for 20 min. The samples were incubated with CD86 (1:500) and CD206 (1:500) antibodies at 4°C for at least 12 h and then washed with PBS 3 times (3 min each time). Then the cells were stained with fluorescence-labeled secondary antibody Alexa Fluor 546, incubated at 25 ℃ in the dark for 50 min, and then washed with PBS for 3 times (3 min each time). Finally, the nucleus was stained with 4-diimide-2-phenylindole (DAPI). The staining of the cells was observed under the fluorescence microscope (Olympus BX51, Tokyo, Japan).
2.11. Immunohistochemical staining
After paraffin embedding, lung tissues were sliced and the sections were dewaxed with xylene, dehydrated through graded ethanol, treated with hydrogen peroxide to inactivate endogenous peroxidases, and subjected to antigen retrieval at pH 6.0 and high pressure. The sections were blocked with 3% hydrogen peroxide for 10 min and then incubated overnight at 4℃ with primary antibodies against the following polarization markers for M1: CD11c (1:250), CD16 (1:500), and CD86 (1:500). Other sections were incubated with primary antibodies against the following polarization markers for M2: Arg-1 (1:250), Dectin-1 (1:500), and CD206 (1:250). The sections were exposed to biotin-labeling goat anti-rabbit IgG as a secondary antibody from an SP (mouse/rabbit IgG)-POD kit following the manufacturer's instructions. Expression of the macrophage surface markers was visualized by a light microscope and quantified from five randomly selected fields of view using Image-Pro-Plus (Media Cybernetics, Silver Spring, MD, USA). The staining reaction was scored using the immunoreactive score = staining intensity × percentage of positive cells: a staining intensity value of 0 was negative; 1, weak; 2, moderate; and 3, strong; a percentage of positive cells value of 0 was negative; 1, 10% positive cells; 2, 11–50% positive cells; 3, 51–80% positive cells; and 4, more than 80% positive cells. Five visual fields from different areas of each section were used to calculate the average immunoreactive score.
2.12. Flow cytometry
The macrophages from the left lung tissue were extracted and made into single-cell suspension, stained with CD86/PE and CD206/FITC fluorescent conjugated antibodies at 4˚C in the dark for 30 min, and then the excess antibodies were washed out with PBS. The expression of CD86, CD206 in macrophages was detected by FACSCalibur™ flow cytometry (BD Biosciences, San Jose, CA, USA), and the data were analyzed by Flow Jo software (Tree Star, San Carlos, CA, USA).
2.13. Statistical analysis
Experimental data were expressed as mean ± SD and analyzed using SPSS v22.0 (IBM, Armonk, NY, USA). Data were plotted using GraphPad Prism v5.0 (GraphPad Software Inc., San Diego, CA, USA) Comparisons among multiple groups were analyzed by one-way analysis of variance, followed by the Tukey test for intergroup comparisons. Differences associated with p < 0.05 were considered statistically significant.