The clinical and pathological diagnosis of OLP conforms to World Health Organization diagnostic criteria . The summary of each participant information is shown in Table 1. The study was approved by the Peking University Third Hospital Medical Science Research Ethics Committee (M2020092), and every participant has written informed consent.
Male CD1 and BALB/c mice (8 -12 weeks) were obtained from animal laboratory center in a specific pathogen-free condition (Vitalriver, Beijing, China). The mice were housed under constant temperature (23±2 °C) and humidity (50±5%) and maintained on a 12 h/12 h light/dark cycle with free access to food and water. All of the procedures were performed with approval from the Biomedical Ethics Committee for Animal Use and Protection of Peking University and in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.
Cell isolation and culture
OLP tissues were biopsied from the oral mucosa, and normal oral mucosal tissues were adjacent to benign mass, obtained from patients who underwent surgical resection (Table 1). The collected tissues were treated with 2 mg/ml dispase (Sigma–Aldrich) to separate the subepithelial lamina propria from the epithelial. The mesenchymal tissues were then minced into 1 mm3 fragments and digested with type I collagenase (3 mg/ml) and dispase (4 mg/ml) for 1 h. The cell suspensions were filtered and plated on dishes in a-modified Eagle’s minimum essential medium (a-MEM; Gibco) with 10% fetal bovine serum (FBS; Hyclone). The cells at passages 2 to 4 were used in subsequent experiments. All experiments were performed thrice and repeated at least twice.
Cell proliferation assay
MSCs from OLP lesions (OLP-MSCs) were seeded into the 96-well tissue culture plate at the density of 1 × 103 cells. Then, the cells were treated with various concentrations of paeoniflorin (0, 0.3, 3, 30, 300 μM) (Ningbo Liwah Pharmaceutical Co., Ltd.) for 11 days. The cell proliferation was assessed by a Cell Counting Kit-8 (CCK8; Dojindo Laboratory). The absorbance microplate reader (ELx808) was used to measure the optical density at 450 nm, and then the cell proliferation rates were calculated. To assess the potential immunomodulation role of paeoniflorin on MSCs, the allogeneic T lymphocytes were assessed [28, 29]. Human peripheral blood mononuclear cells (PBMC) were obtained from four healthy controls and extracted via Ficoll gradient separation. Different numbers of human OLP-MSCs (2.5 × 103/ml, 5 × 103/ml, 1 × 104/ml) were plated onto 96-well plates in 100 μl in triplicates and allowed to adhere to the plates overnight. PBMCs resuspended were added to wells at 2 × 105/ml containing or lacking OLP-MSCs in the presence or absence of 10 μg/ml PHA (Sigma-Aldrich). The OLP-MSCs and PF pretreated OLP-MSCs on PHA-stimulated PBMC were then set proportionally for 3 days. The proliferations of allogeneic T lymphocytes by MSCs were also assessed.
Transwell migration assay
MSCs (1.5 × 105/ml) without FBS were seeded in the upper chamber, and PHA-stimulated PBMC was placed into the lower well to induce cell migration in 24-well Transwell plates (Corning Costar). MSCs invaded into the lower chambers, containing PHA-stimulated PBMC with 10% FBS in RPMI-1640 after 24 h. Cells were fixed with methanol for 20 min at the room temperature and stained with 0.1% crystal violet counted at least five randomly fields by the microscope at 200 x magnification.
Multilineage differentiation in vitro
MSCs were cultured in osteogenic induction medium with 10 nM dexamethasone, 0.1 mM L-ascorbic acid-2-phosphate, 2 mM glutamine and 10 mM β-glycerophosphate. Cells were fixed by staining with 2% Alizarin Red S (Sigma-Aldrich) to visualize calcium deposition after 4 weeks,. MSCs were cultured in adipogenic induction medium supplemented with 1 µM dexamethasone, 60 mM indomethain, 0.5 mM 3-isobutyl-l-methylxanthine, 10 mg/ml insulin, and 2 mM glutamine. Cells were fixed for detecting lipid droplets by staining with 0.3% Oil Red O (Sigma-Aldrich) after 21 days. MSCs were induced with 100 µM CoCl2 (Sigma-Aldrich) for neurogenic differentiation at least 3 days and fixed. They were incubated with primary antibody, rabbit polyclonal IgG for human MAP-2, and followed by FITC-labeled secondary antibody (Bioworld Technology, USA). Samples were observed with the confocal laser scanning microscope (LSM 5, Germany), according to previous studies .
Total RNA in the plasma of each recipient mice was extracted using TRIzol reagent (Invitrogen Life Technologies). Revert Aid First Strand cDNA Synthesis Kit was used to perform reverse-transcription reactions (Fermentas) and cDNA was used as template for each PCR. The primers were described as follows: GAPDH forward primer, 5′-TGTGTCCGTCGTGGATCTGA-3′ and reverse primer, 5′-TTGCTGTTGAAGTCGCAGGAG-3′; OPN forward primer, 5′- AGCCATGAGTCAAGTCAGCT -3′ and reverse primer, 5′- ACTCGCCTGACTGTCGATAG -3′; and LPL forward primer, 5′- AGCTGACCAGTTATGGCACC-3′ and reverse primer, 5′- ATCCTGACCCTCGTAGCCTT-3′. PCR was performed by 7500 Real-time PCR system (Applied Biosystems).
Flow Cytometry Analysis
PHA-stimulated PBMC treated with OLP-MSCs, or paeoniflorin pretreated MSCs and PHA-stimulated PBMC were subjected to a flow cytometry system (BeckmanCoulter, Fullerton, CA). Cell cycle distributions were analyzed in percentages of cells in G0, G1, S, and G2M phases.
Enzyme-Linked Immunosorbent Assay (ELISA)
OLP-MSCs, or paeoniflorin pretreated MSCs and MLR co-cultures were seeded into 96-well plates at ratios of 1:20 for 3 days. Then Th1 cytokines (TNF-α, IFN-γ, IL-1β and IL-2), Th2 cytokines (IL-4, IL-5, IL-10 and IL-13) released in the supernatants were collected for assessment by ELISA kits (Proteintech) according to the manufacturer’s instructions.
EdU labeling of OLP-MSCs
EDU was used to label cells for transplantation in vivo. OLP-MSCs were seeded into a 10-cm dish in the medium with 20 μM EdU (Invitrogen, Carlsbad, CA) for labeling cells. 24 h later, cells were washed with PBS for 3 times, then added into the culture medium for further incubation and prepared for in vivo transplantation.
MSC transplantation in vivo
Male BALB/c (8 –12 weeks old) recipients were divided randomly into 4 groups (n=6 per group) and CD1 mice served as donors. Skin grafts (15 mm2) obtained from the back of donors were transplanted into recipients and 2 × 106 MSCs were injected into the recipients. The groups were divided as: (1) Control group, non-transplantation group; (2) Skin graft group, naive group without any treatment; (3) OLP-MSCs group injection group; (4) Paeoniflorin pretreated OLP-MSCs injection group. The saline injection group was used as negative control.
The skin rejection was observed and recorded from the third day after the transplantation. Rejection was defined as eschar formation or the epidermal sloughing and served as the survival time. Survival data of mice were collected in each group after transplantation 3 weeks, and punch biopsies were fixed in neutral-buffered formalin for HE on day 14. Skin rejection after transplantation is classified and each slide was given a histological score ranging from 1 to 5 according to the previous parameters . Serum of recipient mice was harvested before surgery. The expression level of Th1 cytokines (TNF-α, IFN-γ, IL-1β and IL-2) and Th2 cytokines (IL-4, IL-5, IL-10 and IL-13) were measured using ELISA.
Tracking of transplanted OLP-MSCs
After EDU labeled MSC transplantation, tissues were harvested, fixed and embedded in paraffin at day 1. Then, 4 mm sections were prepared and washed three times with PBS. Subsequently, the sections were incubated in 3% bovine serum albumin (BSA) in PBS, and followed by 0.5% Triton® X-100 in PBS for 20 min. Freshly prepared iClick reaction cocktails, which contained 1 × iClick reaction buffer, CuSO4, Andy Flour 647 azide, and 1× reaction buffer additive (iClickTM EDU Andy Fluor 647 Imaging Kit, GeneCopoeiaTM) was used to incubate tissues for 30 min without light at room temperature. The reaction cocktail was removed and was washed with PBS. Nuclei were stained with DAPI for 5 min, washed twice with PBS, and imaged by fluorescence microscopy. At least three fields were randomly captured and the percentage of positive staining was measured with the Image J software.
Data analyses were performed by SPSS v.13.0 software and presented as Mean ± SEM of at least 3 independent experiments. One- or two-way analysis of variance (ANOVA) and the post hoc Bonferroni tests were used to compare differences among three or more groups. The survival curves were plotted by Kaplan-Meier method and log-rank analysis was used to compare the survival rates among each group. P < 0.05 was considered as statistically significant.