All experiments involving animals were performed in accordance with the guidance of the Animal Care and Use of Laboratory Animals. The Ethics Committee of Experimental Animals and Use of Laboratory Animals of Zunyi Medical University approved all animal experiments. Healthy adult male Sprague-Dawley rats (specific-pathogen-free grade, weighing 250 ± 50 g) were purchased from Hunan Slack Jingda Experimental Animal Co., Ltd., with the license number SCXK (Xiang) 2019-0004. Rats were maintained at 22 ± 2℃ and humidity (45‑55%) on a 12-h light/dark cycle, and housed under controlled conditions: Food and water supplied ad libitum.
Grouping and modeling
The rats were randomly assigned into four groups: (i) Sham group(n = 12), underwent GL stem puncture and catheterization without clamping the superior mesenteric artery (SMA), and collected GLF for testing, without other interventions; (ii) GIRI group (n = 12), based on step (i), clamped SMA for 60 minutes, then loosened for another 120 minutes; (iii) GIRI + GLD group (n = 12), the operation method is the same as that of the GIRI group, but the collected GLF has been infused back into the body through the jugular vein; (iv) GIRI + GLP group (n = 12), suffered the same operation method as the GIRI + GLD group, but the collected GLF was treated with the oXiris GLP and then infused into the body.
Adsorption column based on oXiris biofilm technology
The fabrication details of the adsorption column: The coats were sawed neatly along a 0.25 mL scale using two 1 mL injectors with the core rod removed. The rubber chips adhering to the coats were washed with saline solution and dried. After combining the broken ends of both coats, the oXiris filter wires were filled in. The joint of the coats of the two syringes were pasted across using a pre-cut 1.5 cm wide disposable medical transparent dressing for compactness. The oXiris adsorption column was sterilized with ethylene oxide, sealed and stored at room temperature.
Jugular venipuncture catheterization for re-infusion of GLF
Jugular venipuncture catheterization: after establishing the GLD model, the skin from the right sternoclavicular connection from the midpoint of the neck was incised vertically to 1.5 cm to expose the subcutaneous connective tissue. After gradual blunt separation, the jugular vein was visualized and isolated. Subsequently, we induced congestion and swelling by clamping the proximal and distal vein with a noninvasive vascular clamp, then cut a small aperture in the middle of this section, which was slightly smaller than the external diameter of the silica gel catheter that was pre-flushed with heparin saline. Finally, within the vein orifice, the vascular clamp was loosened, the silica gel catheter was sutured and fixed, and the connective tissue and skin were sutured; GLF reperfusion: Fill the tubing of the peristaltic pump with saline in advance and connect the peristaltic pump to the jugular vein of the rats.
Cell separation and extraction
Monocyte separation. Dilute 5 ml of the whole blood of the modeled mouse with 5 ml equal volume of PBS, drop the diluted blood onto the surface of the separation solution, and then centrifugation was carried out at 2000 rpm for 20 minutes. Take the middle buffy coat cells into a clean centrifuge tube and add PBS to wash. After centrifugation, the supernatant was discarded, the cell suspension was added to the prepared culture plate. Add 50 ng/ml PMA to the isolated mononuclear cell culture medium and continue culturing for 48 hours to induce macrophages and DCs.
Lymphocyte separation. After collecting the lymph fluid, centrifuge at 1500 rpm for 3 minutes, discard the supernatant, add medium to resuspend the cells, and add the cell suspension to a culture dish for culture.
Alveolar epithelial cell separation. Take out the rat lung tissue and add it to a buffer solution containing 10% bi-antibody. Inject 0.25% pancreatin for digestion at 37°C for 25 minutes. The lung tissues are slightly cut and complete medium is added to terminate the digestion. Collect all the liquid, filter with a 70uM filter, 1500 rpm, 5 min centrifugation to collect the cells and resuspend them, add them to 500 ug/ml IgG-coated petri dishes, culture and purify for half an hour, collect the purified cell suspension and add it to the prepared culture in the board. Make a mark and place the cells in an incubator for normal culture.
At room temperature, the collected lung tissue was fixed with 4% paraformaldehyde for 24 hours, then embedded in paraffin to make it dehydrated, and then cut into thin slices (thickness of 5 µm). The sections were stained with hematoxylin and 0.5% eosin for 5 and 3 min, respectively, in room temperature (~ 22˚C). Observe the tissue section under an optical microscope (CX41, OLYMPUS).
Detection of DAMPs in the GLF and plasma
The DAMPs in the GLF and plasma were detected via enzyme-linked immunosorbent assay (ELISA) per the manufacturer’s instructions.
Western Blot (WB)
The sample is lysed with 400 µl lysis buffer (P0013, Beyotime Institute of Biotechnology) containing 100 mmol/L PMSF (ST506, Beyotime Institute of Biotechnology) for 30 minutes, then transfer to the EP tube. Centrifuge for 10 min in a high-speed centrifuge at 2000 r/min. The protein concentration was determined according to the BCA kit. The protein was denatured, loaded, and subjected to sodium dodecylbenzene sulfonate gel electrophoresis (SDS-PAGE) for 2 hours, and then transferred to the membrane with a constant current of 300 mA for 80 min. Incubate the primary antibody solution at 4°C overnight; incubate the secondary antibody solution at room temperature for 2 hours. Drop the ECL luminescent liquid on the film and expose it in the gel imaging system (Chemi DocTM XRS+, Bo Le Biomedical Products (Shanghai) Co., Ltd.). Use "ImageJ" software to analyze the gray value of each band.
Quantitative real-time polymerase chain reaction (qRT-PCR)
Trizon lysate was added to the sample to extract miRNA. Measure the concentration and purity of RNA (OD260/OD280) with UV-Visible spectrophotometer. The RNA was used to synthesize cDNA with an RNA reverse transcription kit. Use RT-qPCR instrument for quantification (CFX Connect™ Real Time, Bole Life Medical Products (Shanghai) Co., Ltd.). The reaction system is as follows: RNase Free dH2O 9.5 µl, cDNA 1 µl, upstream primer 1 µl, downstream primer 1 µl, 2×SYBR Green PCR Master Mix 12.5 µl. The reaction steps are as follows: pre-denaturation 95°C, 10 min; denaturation 95°C, 10 s; annealing 58°C, 30 s; extension 72°C, 30 s; 40 cycles. The primer sequence is provided by General Biosystems (Anhui) Co., Ltd., and the sequence is shown in the table below. β-actin F was used as an internal control, and the relative expression levels of bcl-2, Bax, FAS, and FASL were calculated according to the 2−△△Ct method.
The primer sequences were as follows: bcl-2, forward 5'-GCGTCAACAGGGAGATGTCA‑3' and reverse 5' TTCCACAAAGGCATCCCAGC‑3'; Bax, forward 5'-GCGATGAACTGGACAACAAC‑3' and reverse 5' GCAAAGTAGAAAAGGGCAACC‑3'; Fas, forward 5'-GCCCATTTTGCTGTCAACCG‑3' and reverse 5' GTCTTCAAGTCCACACGAGGT‑3'; Fas-L, forward 5'-CACCAACCACAGCCTTAGAGT‑3'and reverse 5' GAGCGGGGGTTCCCTGTTAAG‑3'; β-actin, forward 5'-GCCATGTACGTAGCCATCCA‑3' and reverse 5' GAACCGCTCATTGCCGATAG‑3'.
Function detection of MPs
The state and function of MPs were detected, and lymphocytes were separated from the collected GLF and cultured with MPs, and then their proliferation, apoptosis and secretion functions were tested. CCK8 measured its proliferation rate, flow cytometry (Novocyte 2060R, Aisen Biology (Hangzhou) Co., Ltd.) measured its apoptosis rate and ELISA measured its inflammatory factor secretion function.
Detection of the dendritic cells’ (DCs) antigen-presenting function
The number of DCs was adjusted to 5×104 /mL, and 1.5 mL of DCs suspension was added to each hole in the 6-well plate. Next, 150 µL of GLF was added to each well and cultured in a 5% CO2 incubator at 37℃ for 24 hours. Cells were scraped and suspended with 5 mL of PBS, then centrifuged at 250 g for 10 minutes. The precipitate was resuspended with 5 mL of PBS and centrifuged for another 10 minutes, then the cells in PBS were resuspended, and 1 µL of OX-6 and 2 µL of OX-42 were added to avoid light for up-flow detection within 1 hour.
AECII cells were identified by transmission electron microscopy (TEM)
AECII was identified by TEM. AECII was incubated for 48 hours and digested with 0.125% trypsin. Collect the cell suspension and centrifuge at 100 × g at 4˚C for 10 min. The supernatant was removed and the cells were fixed to 4% glutaraldehyde for 24 hours at room temperature. The cell pellets were rinsed three times for 10 minutes at 4˚C in PBS and fixed at 4˚C with 1% osmium tetroxide for 30 minutes, then rinsed three times with PBS and observed by TEM (JEM-1230(80KV), JEOL).
Statistical analysis was performed using SPSS software (version 25.0; IBM, Corp.). The data are expressed as mean ± standard deviation. In order to compare the differences between groups, a one-way analysis of variance and post hoc Dunnett's T test were performed. P < 0.05 was considered to indicate a statistically significant difference.