Animals
C57BL/6 mice, aged 8–12 weeks and weighed 24–28 g, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). The mice were housed in the SPF-grade Experimental Animal Center of Xuzhou Medical University with free access to food and water. All experimental procedures were complied with the ARRIVE guidelines and approved by the Ethnic Committee of Xuzhou Medical University (Xuzhou, China).
Preparation of ITP mouse model
ITP mouse model was established as described previously [24, 25]. In brief, mice were administrated with anti-platelet monoclonal antibody (rat anti-mouse integrin GPIIb/CD41 immunoglobulin, clone MWReg30) (BD Biosciences) intraperitoneally at a dose of 0.1 mg/kg body weight to prepare ITP model. After antibody injection, peripheral whole blood was collected to measure platelet count by an automatic hematology analyzer (BC-5300, Mindray, Shenzhen, China) [24, 25].
DMF treatment
DMF (MedChemExpress) (30, 60 or 90 mg/kg body weight) was intraperitoneally injected into mice and then antiplatelet antibody was administrated into mice to induce ITP model. Normal mice receiving injection of vehicle (DMSO) were served as a control group.
Plasma collection
Peripheral blood was drawn from the retro-orbital venous plexus of mice into EDTA-anticoagulated tubes followed by centrifugation at 4500 x g for 10 min at room temperature to collect the supernatant (plasma) which was stored at − 80 °C for later analysis.
Elisa
The plasma levels of IFN-γ (catalogue number: 70-EK206/3–96, MultiSciences) and TGF-β1 (catalogue number: 70-EK981–96, MultiSciences) were measured by ELISA kit according to the manufacturer’s instructions.
Isolation of spleen mononuclear cells
Mouse spleen was extracted and placed into dishes containing 4–5 ml RPMI-1640 medium followed by being cut into pieces and filtered. After that, the suspension was centrifuged to collect the spleen mononuclear cells.
Detection of Th1 cells
Spleen mononuclear cells were stimulated with PMA (Phorbol 12-myristate 13-acetate) (final concentration: 50 ng/ml) (Sigma-Aldrich, St. Louis, MO, USA), ionomycin (750 ng/ml) (Sigma-Aldrich) and BFA (Brefeldin A) (10 μg/ml) (Invitrogen, Carlsbad, CA, USA) for 4 h followed by addition of CD3-FITC (Invitrogen) and CD4-eflour450 (Invitrogen). After fixation and permeabilization, IFN-γ-Percp-Cy5.5 (Biolegend,505,822) was added to measure Th1 cells by flow cytometry. CD3+ CD4+ IFN-γ+ cells were defined as Th1 cells.
Measurement of Treg cells
Spleen mononuclear cells were washed and stained with CD4-FITC (Biolegend) and CD45-PE (BD Pharmingen). After fixation and permeablization, cells were stained with Foxp3-APC (eBioscience) to measure Treg cells by flow cytometry. CD4+ CD45+ Foxp3+ cells were defined as Treg cells.
Immunohistochemical staining
CD68 expression in spleen was measured by immunohistochemical staining as described previously. Briefly, isolated spleen was fixed, dehydrated, and sliced into sections with 4 μm thickness followed by incubation with anti-CD68 antibody (Abcam, Cambridge, MA, USA) and then with HRP-conjugated secondary antibody. The Color was developed with 3, 3′- diaminobenzidine. The macrophage number (CD68 positive expression) was counted in each filed [25].
Cell line
Macrophage cell line RAW264.7 cells were bought from the American Type Culture Collection (ATCC) and cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco, USA) supplemented with 10% fetal bovine serum (Gibco, USA).
Cell cycle analysis
After treatment, 2 × 106 RAW264.7 cells were collected and placed in a 37 °C 5% CO2 incubator for 3 h followed by addition of EdU (KeyGEN BioTECH) to each well of the six-well plate at 1/1000 volume for 2 h incubation. Then, cells were collected and stained with DAPI followed by analysis of cell cycle by flow cytometry. The cell cycle distribution was analyzed with FlowJo V10 software.
Cell apoptosis measurement
RAW264.7 cells were seeded into 24-well plates and treated with different concentrations of DMF for 5 h. Then, cells were collected and incubated with Annexin V (detection of cellular apoptosis) and PI (Propidium Iodide) staining (KeyGEN BioTECH) (detection of necrosis or late apoptosis) for 10 min at room temperature under dark followed by measuring cell apoptosis by flow cytometry (Calibur, BD, USA). The data was analyzed with FlowJo V10 software.
Western blot
Protein was isolated from cells after treatment with DMF (0, 1, 10, 50,100 μM) using RIPA lysis buffer containing PMSF, Cocktail, and phosphatase inhibitor. Then, protein was separated on 10% SDS-PAGE, transferred to PVDF membrane and blocked with 5% milk powder. Then, the membrane was incubated with antibodies against Bcl-2 (Cell Signaling Technology), Bax (Cell Signaling Technology), Cleaved Caspase3 (Cell Signaling Technology), cyclin D1 (Proteintech) and cyclin E2 (Affinity Biosciences) and subsequent with HRP-bound secondary antibody. Bound antibody was visualized after incubation with HRP-conjugated secondary antibody and subsequent enhanced chemiluminescence.
Quantitative real-time PCR
Quantitative real-time PCR for analysis of gene expression in spleen mononuclear cells was conducted as previously described. Briefly, RNA was reversely transcripted into cDNA followed by measuring the mRNA expression of T-bet (T-box expressed in T cells), Foxp3, FcγRI, FcγRIIb, FcγRIV by real-time PCR with β-actin as internal control. The relative mRNA expression was calculated using 2-ΔΔCt method. The primer sequences were shown in Table 1.
Statistical analysis
The Data was shown as mean ± standard deviation (SD). Student t-test was used to compare the difference between two groups and one-way ANOVA was used for comparison of difference among different groups using GraphPad Prism software. P < 0.05 was considered to be statistically significant.