The present study was performed in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health (Bethesda, MA, USA) (19). The protocol was approved by the Chinese Association for Laboratory Animal Sciences (Beijing, China), Animal Health Products (Beijing, China), Committee on the Ethics of Animal Experiments Defense Research (Beijing, China) and Development China (Beijing, China), and Animal Experiments of Shanghai Sixth People's Hospital (approval no. SCXK-2014-1243). All surgery and euthanasia was performed under sodium pentobarbital anesthesia, and all efforts were made to minimize suffering.

Hela cells were purchased from the American Type Culture Collection (Manassas, VA, USA). Mouse RA synoviocytes were donated by Harbin Medical University (Harbin, China). Culture flasks, medium, chemicals and reagents were purchased from recognized commercial suppliers.

Mouse RA synoviocytes were cultured in Dulbecco's modified Eagle's medium (DMEM; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) with 10% fetal bovine serum (FBS) in 6-well plates. At 90% confluence, cells were stimulated with 50 µl/ml PRP (Sigma-Aldrich; Merck KGaA), or 50 µl/ml, dexamethasone (DEX; Sigma-Aldrich; Merck KGaA) or PBS for 12 h at 37°C in a 5% CO₂ humidified atmosphere. RA synoviocytes without antigen and antibody were used as negative controls. Hela cells incubated with DEX, PRP or PBS for 12 h were used as positive controls. The cells were collected following 12 h of incubation, and total RNA was extracted to detect the expression of mRNA for IL-6 and IL-8 by using the reverse transcription-quantitative polymerase chain reaction (RT-qPCR).

PMBCs were prepared by Ficoll-Hypaque density gradient (Sigma-Aldrich; Merck KGaA) centrifugation (2,000 × g at 4°C for 10 min). ADCC and CDC activities of PRP were measured by lactate dehydrogenase (LDH) assay (Merck KGaA, Darmstadt, Germany), which measures the activity of LDH released from the cytosol of damaged cells. Hela cells stably expressing transmembrane TNF-α were incubated with different concentrations (0.2–35 µg/ml) of anti-human TNF antibody for 1 h in assay medium (DMEM + 5% FBS) in a 5% CO₂ incubator at 37°C, followed by the addition of human PBMCs (Shanghai Sixth People's Hospital, Shanghai, China) as effector cells (ADCC assay, effector to target ratio = 20:1) or human complement-rich serum (Quidel Corporation, San Diego, CA, USA) (CDC assay, 1.25% v/v). Following an additional incubation at 37°C for 16 h for the ADCC assay and 5 h for the CDC assay, 100 µl supernatant from each well was transferred into a flat-bottomed 96-well plate. LDH substrate (100 µl) was added to each well and incubated for 30 min at room temperature in the dark. The absorbance of the samples was measure at 490 nm with a microplate reader.

Total RNA was extracted from RA synoviocytes or synovial tissue using RNAzol (Sigma-Aldrich; Merck KGaA), and RNase-free DNase (Sigma-Aldrich; Merck KGaA) was used to digest total RNA at 37°C for 15 min. An RNeasy kit (Sigma-Aldrich; Merck KGaA) was used to purify RNA and adjust its concentration to 1 µg/µl. A total of 1 µg RNA was reverse-transcribed using the Thermo Script RT-qPCR system (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA). The concentration of RNA was quantified using spectrophotometry at 260 nm (Smart Spec TM 3000; Bio-Rad Laboratories, Inc., Hercules, CA, USA). Total RNA in the RA synoviocytes and synovial tissue of CIA mice was denatured by incubating for 5 min at 70°C with 4 µM oligo(dT) primer and reverse-transcribed using a final concentration of 0.5 mM dNTP, 40 U/µl RNaseOUT (Sigma-Aldrich; Merck KGaA), 0.01 M dithiothreitol, and 10 U/µl Thermo Scriptreverse transcriptase (mRNA expression in RA synoviocytes or synovial tissue). Reverse transcription was performed by incubating at 42°C for 180 min. The obtained cDNA was diluted 10-fold with distilled H₂O and 10 µl was used for amplification. Specific primer sets for interleukin (IL)-6, IL-8, matrix metalloproteinase (MMP)-3, IL-17A, IL-1β, TNF-α, receptor activator for nuclear factor-κB ligand (RANKL), VEGF, PDGF, IGF-1, TGF-β and interferon-γ (IFN-γ) were generated. All PCR primers are listed in Table I. The PCR was performed using the Light Cycler Fast Start DNA SYBR Green I kit (Roche Diagnostics, Basel, Switzerland) according to the protocol provided with the parameter-specific kits (45 amplification cycles, denaturation at 96°C, primer annealing at 68°C with touchdown to 58°C, and applicant extension at 72°C) (20).

DBA/1J male mice at 8–10 weeks of age (30–35 g) were purchased from Shanghai SLAC Laboratory Animals Co., Ltd. (Shanghai, China). Mice were maintained in a climate-controlled 20–25°C environment with a 12-h light/dark cycle. The induction of type II CIA was achieved as previously described (21), by the subcutaneous injection of 2 mg collagen (ModiQuest Research, Oss, Netherlands) in each mouse (n=9/group) (22). Clinical arthritis scores were evaluated using a scale of 0–2 for each paw, for a total score of 8. Paws were assigned a clinical score based on the index (ModiQuest Research scoring method): 0=normal; 0.25=1–2 swollen toes; 0.5=3–4 swollen toes; 0.75=slightly swollen footpad/ankle; 1=swollen footpad/ankle; 1.25=1–2 swollen toes and swollen footpad/ankle; 2.0=swollen toes, footpad and ankle. At days 30 and 70 following the first injection, blood samples were taken from each mouse and anti-CII immunoglobulin (Ig)G antibodies in sera were detected using a mouse anti-type II collagen IgG assay kit (CDT31102; Cayman Chemical Company, Ann Arbor, MI, USA) according to the manufacturer's protocol. The treatment was started on day 30 following the initial injection. Mice were administered 10 mg/kg PRP or PBS by subcutaneous injection every 4 days for a total of 9 doses, and were sacrificed on day 72 (14).

The RA mice were sacrificed on day 72, and the knee joints (condyle) were obtained and subsequently fixed in 10% formalin for 2 h at 37°C. The joints were decalcified and embedded in paraffin. The ankle joints were stained with hematoxylin and eosin (1%) for 10 h at 37°C to analyze the efficacy of PRP for CIA RA mice. The severity of arthritis in the joints was scored on a scale of 0–5 as described in a previous study (23).

All date were expressed as mean and standard deviation of triplicate dependent experiments. Statistical significance was determined using the Student's t-test and Microsoft Excel software (2010; Microsoft Corporation, Redmond, WA, USA). One-way analysis of variance (ANOVA), two-way ANOVA, Kaplan-Meier estimators and log rank statistical analyses were performed using SPSS software (version 19.0; IBM SPSS, Armonk, NY, USA). P<0.05 was considered to indicate a statistically significant difference.

The in vitro function of PRP in regulating inflammatory factors in synovial cells was assessed. Gene expression of IL-6 and IL-8 in Hela cells and human fibroblast-like synovial cells was measured by RT-qPCR. The results in Fig. 1 demonstrated that PRP stimulated Hela and fibroblast-like synovial cells to increase gene expression of IL-6 and IL-8, while IL-1β, IL-17A, TNF-α and IFN-γ mRNA expression was downregulated compared with the DEX and control groups. The results of the present study indicated that treatment with PRP decreased cytokine production in vitro.

A previous study demonstrated that inflammatory factors may be able to affect the progression of RA through ADCC or CDC mechanisms, and that this may lead to various clinical effects (24). In the ADCC assay, the results presented in Fig. 2A demonstrated that ~34% of the IL-1R-expressing Hela target cells were lysed by PRP at a concentration of 10 µg/ml. In the CDC assay, the result in Fig. 2B demonstrated that PRP was also capable of lysing IL-1R-expressing Hela cells in the presence of human complement. These data indicated that PRP was able to mediate ADCC and CDC effects upon binding to transmembrane IL-1R expressed on the cell surface, and therefore exhibits the potential to be developed into a more effective IL-1R-neutralizing agent in RA therapy. In addition, all synovial specimens in RA mice treated by PRP, DEX or PBS were analyzed. The results demonstrated that hypertrophy and leukocyte infiltration were suppressed following 4 weeks of treatment with PRP, compared with DEX- and PBS-treated groups (Fig. 2C and D).

In order to assess the therapeutic effects of PRP on RA development, the CIA mouse model was used. The experimental mice were recorded, and the arthritis score was evaluated every 4 days. As presented in Fig. 3A, treatment with PRP ameliorated the clinical symptoms and hind paw swelling, with reduced arthritis scores compared with DEX- and PBS-treated CIA mice. In addition, the therapeutic effects of PRP were assessed by analyzing the humoral anti-collagen response. The serum concentration levels of anti-collagen type II IgG on day 72 were measured. The results in Fig. 3B demonstrated that a significant decrease in the serum level of anti-CII IgG was exhibited in PRP-treated RA mice compared with PBS- and DEX-treated groups (P<0.01 vs. control groups). Vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), insulin-like growth factor (IGF)-1 and transforming growth factor (TGF)-β expression was assessed at the mRNA level in whole blood samples from RA mice, using RT-qPCR. The results of the present study indicated that VEGF, PDGF, IGF-1 and TGF-β expression in peripheral whole blood was upregulated following treatment with PRP compared with PBS and DEX groups (Fig. 3C-F).

The therapeutic mechanism of PRP in RA synovial cells was analyzed in the present study. Target mRNA and protein expression levels were measured in synovial tissue from independent experiments following treatment with PRP. The results in Fig. 4A and B demonstrated that IL-6, IL-8, IL-17A, IL-1β mRNA expression was decreased in synovial tissue in PRP-treated mice compared with PBS-treated and DEX-treated groups. In addition, in order to observe alterations in RA synovial tissue following treatment with PRP, histological staining was used to analyze the therapeutic effects of PRP in mice with RA. As presented in Fig. 4C-E, the PRP-treated mice with RA exhibited marked reduction of synovial hyperplasia, inflammatory cell infiltration and destruction of cartilage and bone. In addition, it was observed that fewer infiltrating cells presented in the joints of the PRP-treated mice compared with PBS-treated and DEX-treated mice with CIA. The pathological symptoms of the PRP-treated CIA mice were attenuated. These results indicated that PRP may be an effective drug for RA therapy.