Reagents and cells
PFD was purchased from Shanghai Adamas Reagents Co. Ltd.; hUC-MSCs were donated by Chongqing Stem Cell Therapy Engineering Technology Research Center; β-actin, type I collagen a1(Col1a1), type I collagen a2(Col1a2), α-smooth muscle actin(α-SMA), RGS2, calcium adhesion protein E(E-cad) and fibronectin(FN) primers were synthesized by Chengdu Kengke Zixi Biotechnology Co. Ltd.; ultrafiltration tubes were purchased from Millipore; other reagents were all domestic analytical purity. The mouse embryonic fibroblasts (NIH3T3) and human Embryonic Lung Fibroblasts(HLF-9) were purchased from Cell Resource Center, Shanghai Institute of Biological Sciences, Chinese Academy of Sciences. Cell culture and drug treatments Cells were routinely cultured at 37 °C with 5 % CO2 in 1:1 mixture of Dulbecco's Modified Eagle's Medium (DMEM) and Ham's F-12 Nutrient Mixture (F12) supplemented with 10 % fetal bovine serum(FBS) and were used at < passage 6(P6) for experiments. For dose–response experiments, human lung fibroblast cells were seeded into 12-well plates, starved in serum-free DMEM/F12 medium for 24 h, and then treated with the indicated concentrations of PFD for the indicated times. 6-well plate co-culture dishes were purchased from Thermo Fisher Scientific.
Animal grouping and treatment
SPF-grade 7-week-old C57BL/6 male rats were purchased from the Animal Experiment Center of Chongqing Medical University and housed in an SPF-grade breeding room at 22-26 ℃, 55-60% humidity, and 12 h/day light rotation, and modeling was started after 1 week of adaptation.
Thirty C57BL/6 were divided into 6 groups using the random number method: control group (N), bleomycin model group (B), P10 group (10 mg/kg PFD, P10), P30 group (30 mg/kg PFD, P30), P100 group (100 mg/kg PFD, P100), and P300 group (300 mg/kg PFD, P300). Pulmonary fibrosis model was established by dripping bleomycin 3mg/kg into the lungs of mice at an equal rate through tracheal intubation, and the control group was given an equal amount of saline at an equal rate. After successful modeling, each PFD dose group was given 10 mg/kg, 30 mg/kg, 100 mg/kg, 300 mg/kg PFD suspension by gavage from day 7 after modeling, and the control group was given equal volume of saline once a day until day 21 after modeling.
Another batch of 30 C57BL/6 was divided into 6 groups using the random number method: control group (N), hUC-MSCs control group (N+M), bleomycin model group (B), hUC-MSCs treatment group (B+M), P100 group(100 mg/kg PFD, P100) and hUC-MSCs+P30 group (hUC-MSCs+30 mg/kg PFD, B+M+P30). Modeling was performed by tracheal intubation with 3 mg/kg bleomycin drip, and on day 7 after modeling, hUC-MSCs control and hUC-MSCs treatment groups were injected with 5×105/200 μL P4 generation hUC-MSCs via the tail vein of mice, and the non-MSCs group was given an equal amount of saline. Starting from day 7 after modeling, the P100 groups and the hUC-MSCs+P30 group (B+M+P30) were given 100 mg/kg, and 30 mg/kg PFD suspension by gavage, respectively, and the control group was given an equal volume of saline once daily until day 21 after modeling. The survival of mice was observed and recorded, and lung tissues were collected on day 21. The mice survival curves, analysis of lung histopathology, determination of collagen content in the lungs, detection of mRNA, the markers of lung fibrosis, and detection of RGS2 expression will be briefly described in the following.
The survival of each group of mice was observed and recorded, and survival curves were plotted using Graphpad Prism 5.0.
Lung histological analysis
Paraformaldehyde-fixed mouse lung sections were analyzed with hematoxylin-eosin (HE) or Masson`s trichrome staining to assess fibrotic changes in the lungs. Three anterior, middle and posterior sections of each lung specimen of mice were taken for HE and Masson staining, respectively, and then five high magnification views of each section were selected for observation and scored separately using the modified Ashcroft method (scale range 0 to 8). The histopathological score of pulmonary fibrosis in each mouse was expressed as the mean score.
Determination of intrapulmonary collagen content
80mg of lung tissue from the right lung was taken and determined according to the instructions of the Sircol method for measuring soluble collagen kit. A standard curve was made using collagen standards, and then the collagen concentration was calculated from the standard curve. Soluble collagen content was calculated according to the following formula: soluble collagen content = calculated collagen concentration × total volume of hydrolysate (1 mL)/80 mg × total wet weight of right lung tissue (mg).
Fluorescence quantitative PCR
Total RNA was extracted from each group of lung tissues using the TRIZOL method, and the RNA was reverse transcribed into cDNA using a reverse transcription kit. cDNA obtained was used for fluorescence quantitative PCR, and the expression of Col1a1, Col1a2 and α-SMA/ACTA2, RGS2, E-cad, and FN was detected using the β-actin gene as the internal reference gene levels. The genes, mRNA or protein are referred to using offcial gene symbols as provided by Te National Center for Biotechnology Information (NCBI;https:// www.ncbi.nlm.nih.gov/).
The expression of G protein signaling regulator 2 in lung tissues was detected in each group. Samples were electrophoresed and subjected to western blot using primary antibodies against RGS2 and β-actin.
Acquisition and identification of hUC-MSCs
P2 generation hUC-MSCs were obtained from Chongqing Stem Cell Therapy Engineering Technology Research Center. hUC-MSCs were cultured and identified according to the methods reported in the literature. hUC-MSCs were grown to P4 generation, and cell suspensions were collected after trypsin digestion, centrifuged and resuspended with PBS and counted to a final cell concentration of 2.5×106/mL, placed on ice for use.
Acquisition, grouping and treatment of myofibroblasts
P4 generation NIH3T3 and HLF-9 were spread in six-well plates at 1×106, respectively, and when the cell fusion reached approximately 80%, they were replaced with fresh DMEM medium containing 1% fetal bovine serum and treated with 4ng/ml transforming growth factor-β1(TGF-β1) and cultured for 24 hours, i.e. myofibroblasts of both cell lines.
Different concentrations of PFD were added in MFB: 0, 2, 4, 6, 8 and 10 mM, respectively, and the cells in each group were collected after 2h of incubation, and RGS2 mRNA expression was detected byRT- PCR to find out the effective concentration of RGS2 mRNA elevation caused by PFD.The effective concentration of PFD was added to MF separately, and the cells in each group were collected after 24h of culture separately, and the mRNA expression of fibrosis markers was detected by RT-PCR.
The cells were divided into three groups according to different culture methods: NIH3T3 group (N) or HLF-9 group(H), hUC-MSCs group (M), and NIH3T3+hUC-MSCs group (N+M) or HLF-9+hUC-MSCs group (H+M). NIH3T3 or HLF-9 were cultured in the lower chamber of one well of the co-culture six-well plate alone (N or H). The P4 generation hUC-MSCs were cultured in the upper chamber of the other well of the co-culture six-well plate alone (M). MFB were cultured in the lower chamber of the co-culture six-well plate while P4 generation hUC-MSCs were cultured in the upper chamber of the co-culture six-well plate (N+M or H+M). All were cultured using DMEM/F12 containing 5% fetal bovine serum. When the cell fusion of each group reached approximately 80%, the effective concentration of PFD or equal amount of PBS buffer
was added, and each group of cells was collected after 24 h of culture respectively, and the mRNA expression of RGS2 and fibrosis markers were detected by PCR.
The hUC-MSCs were divided into three groups: hUC-MSCs group (M), hUC-MSCs+TGF-β1 group (M+T), and hUC-MSCs+TGF-β1+PFD group (M+T+P). hUC-MSCs were cultured using DMEM/F12 containing 5% fetal bovine serum, and when the fusion of cells in each group reached approximately 80%, they were replaced with fresh DMEM containing 1% DMEM medium with fetal bovine serum, treated with 4ng/ml TGF-β1 and cultured for 24h. Cells in the M+T group were collected and detected by PCR for E-cad, FN,α-SMA mRNA expression; in the M+T+P group, PFD or equal amount of PBS buffer was added after 24h of TGF-β1 treatment and cells were collected after 24 h of culture and detected by PCR for E-cad, FN, α-SMA mRNA expression.
Data were expressed as mean±SEM. Comparisons between groups were made using Student's t test for unpaired observations or two-factor ANOVA and Bonferroni correction for multiple comparisons. p < 0.05 was considered statistically significant.