Isolation and culture of BMSCs
The BMSCs are a cell line obtained from rats thighbone. First, we took out the femur of rats with intact femoral head, washed the femur with 75% alcohol and double antibody, then removed the epiphyseal end and exposed the bone marrow cavity, finally the culture medium was used to wash the cells. BMSCs cells were cultured with OriCell Wistar Rat Bone Marrow Mesenchymal Stem Cell Medium supplemented with 10% OriCell Superior-Qualified Fetal Bovine Serum, Penicillin-Streptomycin and L-Glutamine (Cyagen Biosciences, China) at 37℃ in a humidified atmosphere of 5% CO2. Non-adherent cells were removed by changing the culture medium after incubation for 24 hours. The medium was replaced every 2-3 days. All the experiments in this study were carried out using 4th generation cells.
Identification of BMSCs
To identify whether they are BMSCs, Immunofluorescence was used to detect expression of CD34 and CD44 of BMSCs. The sections containing BMSCs were fixed with 4% paraformaldehyde for 30 min and blocked by 5% goat serum at 37℃ for 30 min. BMSCs were incubated with antibody CD34 and antibody CD44 at 4℃ overnight. Then BMSCs were incubation with Alexa Fluor 594 conjugated goat anti-rabbit IgG antibodies (1:50, #ZF-0516, ZSGB-BIO, Beijing, China) for visualization. The nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI). The fluorescent signal was showed using 3D scanner (3DHISTECH, Hungary).
To test the multidifferentiation potential of BMSCs toward the osteogenic and adipogenic, the BMSCs were cultured in adipogenic and osteogenic differentiation medium (Cyagen Biosciences, China). After 4 weeks of differentiation induction, adipose cells were stained with Oil Red O and osteogenic cells were were stained with Alizarin Red. The stained cells were observated by a microscope.
Animals and Experimental models
45 male C57BL/6 mice with an average body weight of 20 g at 8 weeks of age purchased from Jinan pengyue experimental animal co., Ltd were utilized for all experiments. All animal procedures were approved by the Ethics Committee of The First Affiliated Hospital of Shandong First Medical University. Totally 45 Mice were evenly divided into three groups including control group, model group and BMSCs group with 15 mice in each group. Thorax irradiation was delivered to all except the mice from control group. The mice were anesthetized by intraperitoneal injection of 10% chloral hydrate (0.04ml/10g) and exposed to chest irradiation at a dose rate of 2 Gy / min, with a total dose of 20 Gy. Within 24 hours after irradiation, the mice in BMSCs group were injected with BrdU labeled BMSCs (1×106 cells/0.1ml) via tail vein. All the mice were sacrificed 4 weeks after irradiation. The lung tissues were procured for histological and molecular biological analysis.
Animal sample collection
Four weeks after irradiation, the mice were anesthetized by intraperitoneal injection of 10% chloral hydrate (0.04ml/10g). Firstly, the eyeballs of the mice were removed and the blood was collected. The supernatant was collected by centrifugation and frozen at - 20 ℃. The left lung was cryopreserved at - 80 ℃ to prepare for the molecular experiment. The right lung was fixed in 10% formaldehyde for pathological experiment.
H&E staining was performed to evaluate the pathological changes of lung tissue. Briefly, the lung tissues were fixed in 10% formaldehyde, paraffin-embedded and then sectioned into 4 μm slices (Leica, Germany). Then the nucleus and cytoplasm were stained with hematoxylin and eosin respectively, the neutral gum was used for sealing. All sections were observed under light microscope. Immunohistochemistry staining was used to detect expression of inflammatory factors in lung tissues. The paraffin-embedded lung tissue sections were dewaxed to water, repaired by EDTA for 15 min at 100℃, then incubated with 5% BSA (Solarbio, Beijing, China) for 30 min at 37℃. The slides were incubated with Interleukin 6 (IL-6) (1:100, #bs-0782R, Bioss, Beijing, China) antibody and Tumor necrosis factor α (TNF-α) (1;100, #bs-10802R, Bioss, Beijing, China) at 4 ℃ overnight. Subsequently, the slides were incubated with secondary antibody (1:200, #bs-0295D-HRP, Bioss, Beijing, China) for 60 min at 37℃. The slides were visualized by 3,3-diaminobenzidin (DAB). Finally, the slices were stained with hematoxylin, dehydrated with gradient alcohol, transparent with xylene and sealed with neutral gum. The positive signal was determined by a 3D scanner (3DHISTECH, Hungary).
Electron microscope sample preparation
The lung tissue fixed with 3% glutaraldehyde was rinsed with PBS for 5-6 times, 15min each time, and then fixed with 1% osmic acid in dark for 90min. PBS was rinsed twice, 15min each time, rinsed with ultrapure water for 3-4 times, 15min each time, and then dehydrated with gradient ethanol for 20min each stage. Pure acetone was used for 3 times, 20min each time. In order to gradually infiltrate the embedding agent into the tissue cells in the later stage, the infiltration treatment is carried out before embedding, mainly using acetone and resin 1:1 mixed and infiltrated for 1h, then acetone and resin 1:2 mixed and infiltrated for 4h or overnight, and pure resin infiltrated twice for 4H each time. After infiltration, put the tissue sample into both ends of the embedding plate, add pure resin, put it into the polymerization instrument, and perform the procedure of 37 ℃ 24h-45 ℃ 24h-60 ℃ 48h to form the resin block of the embedded sample. After that, trim the resin block, make ultra-thin sections with a thickness of 70nm, and fish the pieces to three aromatic membrane carrier nets; Dye with 2% uranyl acetate solution for 15min and lead citrate solution for 8min, wash with water, bake the sheet under infrared lamp for 10min, dry, observe with Hitachi ht-7800 transmission electron microscope at 80kV, and take photos at different magnification.
Cells and tissues Immunofluorescence
The BrdU-Labeled BMSCs and lung tissue sections were fixed with 4% neutral formaldehyde solution for 15 min, then denatured with 2m hydrochloric acid for 15 minutes, neutralized with sodium borate and washed with distilled water, followed with 0.1% Triton X-100 for 10 min and incubated with 5% BSA (Solarbio, Beijing, China) for 30 min at 37℃. The cells were then incubated with primary antibodies: mouse anti-BrdU antibody (1:100, #ZM-0013, ZSGB-BIO, Beijing, China) and rabbit anti-AQP5 (1:100; #bs-10927R, Bioss, Beijing, China) and rabbit anti-Pro-spc (1:200; #ab90716, Abcam, China) at 4 °C overnight. Subsequently, these cells were incubated with the corresponding goat anti-mouse IgG (H + L) secondary antibody, Alexa Fluor 488 ((1:50, #ZF-0512, ZSGB-BIO, Beijing, China) and goat anti-rabbit IgG(H + L) secondary antibody, Alexa Fluor 594 ((1:50, #ZF-0516, ZSGB-BIO, Beijing, China) for 60 min at 37℃. The cell nuclei were counterstained by DAPI. The cells stained with green fluorescence were counted as the BrdU-positive cell. The cells stained with red fluorescence were counted as the AQP5 and Pro-spc positive cell. BrdU positive and AQP5 positive or Pro-spc positive were positive signal. Immunofluorescence images were detected by a 3D scanner (3DHISTECH, Hungary).
Enzyme-linked immunosorbent assay (ELISA)
We used a commercially available mouse ELISA kits, according to the manufacturer’s instructions, to detect the levels of AngII (CK-E21284, MLBIO, Shanghai, China) and Ang-(1-7) (CK-E20733, MLBIO, Shanghai, China) in lung tissues. The absorbance was read at 450 nm with a microplate reader.
Total RNA was extracted from freshly isolated lung tissues using Trizol reagent (Vazyme biotech co., Nanjing, China) according to the manufacturer’s instructions. Then RNA was reverse-transcribed into cDNA with cDNA Synthesis Kit (Vazyme Biotech Co., Nanjing, China). Quantitative real-time PCR was performed with SYBR qPCR Mix (Vazyme Biotech Co., Nanjing, China). The mRNA levels of MasR in lung tissues were examined by qRT-PCR. Data were calculated using the 2- △△t method. MasR forward primer 5′-ACAACACGGGCCTCCTATCTG-3′ and reverse primer 5′-GAAGGGCACAGACGAATGCT-3′, β-actin forward primer 5′-GGCTGTATTCCCCTCCATCG-3′ and reverse primer 5′-CCAGTTGGTAACAATGCCATGT-3′.
Western blots was used to analyse the related proteins of ACE/ACE2/ Ang(1-7) and MAPK parhway in lung tissue. The lung tissues were lysed in RIPA lysis buffer (Beyotime, China) with protease inhibitors for 30 min at 4°C, followed by centrifugation at 12,000 rpm for 15 min at 4 °C. The supernatant will be collected and added to the loading buffer for denaturation. Protein samples were separated by 8-15% SDS-PAGE, then transferred to a PVDF membrane and blocked in 5% skim milk at room temperature for 90 min. The membranes were incubated with primary antibody including ACE (1:1000; #bs-0439R, Bioss, Beijing, China), ACE2 (1:1000, #21115-1-Ig, Proteintech, China ), AT1R (1:1000; #bs-0630R, Bioss, Beijing, China), overnight at 4 °C and the corresponding secondary antibody at room temperature for 1h. Finally, proteins were visualized with ECL(Milipore, USA) and analyzed with Image J.
All data are shown as mean±SEM. Data was analyzed using SPSS 25 software. The measurements for this study were performed with one-way analysis of variance (ANOVA) for multiple groups. P<0.05 was considered statistically significant.