2.1 Materials and reagents
The murine bone marrow-derived MSCs (mBMSCs) were purchased from Wuhan Fine Biotech company. The murine bone marrow-derived macrophage cell line RAW264.7 cells were purchased from American Type Culture Collection (ATCC). According to our team’s previous research results, we choose WKYMVm peptide as the activator of FPR2 pathway. The WKYMVm peptide with purity more than 95% was synthesized by GL Biochem company and dissolved in acetonitrile. However, WRW4 peptide, an inhibitor of FPR2 pathway, with purity more than 95% was also synthesized by GL Biochem and dissolved in acetonitrile. F–12 medium, Dulbecco’s Modified Eagle’s Medium (DMEM) and 0.25% trypsin were purchased from Hyclone company. Fetal Bovine Serum (FBS) without exosomes was purchased from Lonsera company. DMSO was purchased from Sigma-Aldrich company. Penicillin and streptomycin were purchased from Beyotime Biotechology company. Phosphate Buffered Saline (PBS) solution, 4% paraformaldehyde, Triton X–100 solution and fluorescent mounting media were purchased from Solarbio company. PCR prime and Trizol buffer were purchased from Invitrogen company. RNA reverse transcription kit and Fluorescence quantitative PCR kit were purchased from Takara company. RIPA dissociation solution, PMSF, WB suit and sealing solution were purchased from Bioss company. All kinds of primary antibody and secondary antibody were purchased from Abcam company. Exosomes extraction kit was purchased from QIAGEN company. In addition, cell counterkit–8 assay (CCK–8) was purchased from Beyotime Biotechology company.
2.2 Cell culture
mBMSCs were cultured in a T25 culture flask filled with 90% F–12 medium and 10% FBS supplemented with penicillin (100 units/mL) and streptomycin (100 mg/mL) at incubation environment of 37.0℃ and 5% CO2. RAW 264.7 cells were also cultured in a T25 culture flask containing 90% of DMEM medium and 10% FBS supplemented with penicillin (100 units/mL) and streptomycin (100 mg/mL) in an incubator set of 37.8℃ and 5% CO2. 24 hours later, the non-adherent cells were removed. After reaching confluence, the adherent cells would continue to be cultured. The whole incubation process remained unchanged in 37.0℃ and 5% CO2 environment for mBMSCs and 37.8℃ and 5% CO2 environment for RAW 264.7 cells. All operation procedures were strictly in accordance with the operation manual proposed by Army Medical University.
2.3 Cytotoxicity assay
In this study, we used WKYMVm peptide as an activator for FPR2 pathway, so it was necessary to detect the toxicity of WKYMVm peptide. We used CCK–8 to detect the toxicity of WKYMVm peptide on mBMSCs. According to previous studies, we choose 1μmol/L as the concentration of WKYMVm peptide and 10μmol/L as the concentration of WRW4 peptide. mBMSCs were seeded in 96-well plates with a density of 5*103 per well, and after one night of culture, the mBMSCs were tested with WKYMVm peptide and observed for 24, 48 and 72 hours. Next, we added CCK–8 solution (100μl/well) into the 96-well plates and leave it in the dark for another one hour. Finally, we detected and recorded the absorbance at 450nm with a microplate reader and compared with the control group without WKYMVm peptide.
2.4 Western blotting
To detect the protein expression, western blotting was used. β-Actin served as an internal control. According to the experimental design, mBMSCs, exosomes or M2 macrophages were dissolved in a cell lysate containing PMSF. The proteins were transferred to PVDF membrane by electrophoresis. The PVDF membrane was placed in the sealing liquid and shaken at low speed for 1 hour. Next, the sealed PVDF membrane was putted into the antibody box of the primary antibody and placed in the environment of 4℃ overnight. Then the PVDF membrane was washed with TBST for three times, after that PVDF membrane was putted into the antibody box containing the secondary antibody and incubated for 2 hours at room temperature. We also washed PVDF membrane three times with TBST. At last, we used the Chemi Doc XRS Imaging System (Bio-Rad) to capture images of protein bands and analyzed the image with Image J software.
2.5 RNA isolation and Qrt-PCR
To investigate gene expression, RNA isolation and Qrt-PCR were used. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) served as an internal control. mBMSCs were lysed with Trizol buffer. Complementary DNA was synthesized from 1 μg total RNA by reverse transcription kit, and the gene expression was detected by Qrt-PCR.
2.6 Exosomes extraction
To collect exosomes secreted by mBMSCs, exosomes extraction kit was used. In the continuous centrifugation stage, the first centrifugation was performed at 300 g for 5 minutes to remove cells. The second centrifugation was performed at 1200 g for 10 minutes and the third centrifugation was performed at 10000 g for 30 minutes to remove cellular debris. The exosomes were obtained from the supernatant according to the instructions of the exosomes extraction kit.
In order to count the polarized number of M2 macrophages, immunofluorescence was used. The cells were fixed with 4% paraformaldehyde for 20 minutes. Next, the cells were permeated with 0.3% Triton X–100 solution for 15 minutes. Then cells were covered with 5% BSA at 37℃ for 30 minutes. After the BSA was removed, we added the primary antibody and antibody diluents in the ratio of 1:200 into the plates overnight at 4℃. The next day, we removed the primary antibody at room temperature. Then we added the secondary antibody and antibody diluents in the ratio of 1:400 into the plates. We incubated the cells at 37℃ for 30 minutes. Then we removed the secondary antibody. The cells were incubated with DAPI solution at room temperature for 5 minutes. Finally, we added the fluorescent mounting media into the plates and observed the cells under the fluorescence microscope. The quantification of M2 macrophages polarization was performed with Image J.
2.8 Preparation of WKYMVm peptide-conditioned medium and WKYMVm peptide plus WRW4 peptide-conditioned medium
One day after mBMSCs were stimulated by WKYMVm peptide or WKYMVm peptide plus WRW4 peptide, the supernatant was collected by centrifugation at 3000 rpm for 10 minutes. The collected supernatant was used as conditioned medium for growing RAW 264.7 cells in follow up study. In this study, it was called WKYMVm peptide-conditioned medium or WKYMVm peptide plus WRW4 peptide-conditioned medium.
2.9 ISG15 expression assay
mBMSCs were seeded in 6-well plates with a density of 1*106 per well. After one night of culture, WKYMVm peptide and WKYMVm peptide plus WRW4 peptide were added into different plates. After 24 hours, we used western blotting and Qrt-PCR (Forward primer: TTTCCTGGTGTCCGTGA - Reverse primer: TCTGGGCAATCTGCTTCT) to detect the ISG15 expression. The ISG15 expression of WKYMVm peptide group was compared with the control group without WKYMVm peptide and the WKYMVm peptide plus WRW4 peptide group.
2.10 TFEB expression assay
mBMSCs were seeded in 6-well plates with a density of 1*106 per well, WKYMVm peptide and WKYMVm peptide plus WRW4 peptide were added into different plates the next day. After 24 hours, we used western blotting and Qrt-PCR (Forward primer: AAGAACAGGGGTGAGGCA—Reverse primer: CCCAGGCTCAGGAGAGG) to detect the TFEB expression. The TFEB expression of WKYMVm peptide group was compared with the control group without WKYMVm peptide and the WKYMVm peptide plus WRW4 peptide group.
2.11 Exosomes content assay
It had been reported that the exosomes secreted by MSCs containing abundant miRNA–146, which could promote the polarization of M2 macrophages. mBMSCs were seeded in 6-well plates with a density of 1*106 per well. After one night of culture, WKYMVm peptide and WKYMVm peptide plus WRW4 peptide were added into different plates. After 24 hours, we centrifuged the cell culture medium continuously. After that, we used the exosomes extraction kit to collect exosomes. Then the exosomes were observed under the transmission electron microscopy (JEM–1400PLUS, Japan) and analyzed by western blotting (Marker: CD9, CD63). At last, we compared the exosomes content of WKYMVm group with the control group without WKYMVm peptide and the WKYMVm peptide plus WRW4 peptide group.
2.12 Assay of M2 macrophages polarization promoted by exosomes secreted by mBMSCs
In addition, we also needed to count the polarized number of M2 macrophages. We cultured RAW 264.7 cells in the WKYMVm peptide-conditioned medium, the medium without WKYMVm peptide and the WKYMVm peptide plus WRW4 peptide-conditioned medium. After 24 hours, we used the immunofluorescence (Marker: CD206) and western blotting (Marker: CD206, Arg–1) to count the polarized number of M2 macrophages. At first, RAW 264.7 cells were seeded in 96-well plates with a density of 5*103 per well for immunofluorescence and 6-well plates with a density of 1*106 per well for western blotting. The next day the WKYMVm peptide-conditioned medium, the medium without WKYMVm peptide and the WKYMVm peptide plus WRW4 peptide-conditioned medium were removed. Then we washed the plates with PBS twice. After immunofluorescence and western blotting were completed, the polarized number of M2 macrophages in the WKYMVm peptide-conditioned medium group were compared with the medium without WKYMVm peptide group and the WKYMVm peptide plus WRW4 peptide-conditioned medium group.
2.13 Statistical analysis
All the data came from three independent researchers. All data expressed in the form of mean ± standard deviation (SD). Student’s t test was used to compare the differences between the two groups. All statistical analysis was performed by SPSS 22.0 software, and the value of P lower than 0.05 was statistically significant. (*P<0.05, **P<0.01, ***P<0.001)