Reagents and materials

Freund's complete adjuvant and Thiazolyl blue tetrazolium bromide (MTT) was purchased from Sigma; Cell Counting Kit-8(CCK-8) were purchased from Beyotime; DMEM high glucose culture medium was purchased from Hyclone company (United States); recombinant human tumor necrosis factor (TNF)-α was obtained from Peprotech (United States); neonatal bovine serum and trypsin were obtained from GIBCO (United States); and 5× protein sample buffer was obtained from Novex (United States). Bovine type II collagen was purchased from Xinbosheng Biotechnology Co., Ltd.; 4% polyformaldehyde, TEMED, Dimethyl sulfoxide (DMSO) and Ethylenediaminetetraacetic acid (EDTA) were purchased from Beijing Dingguo Changsheng Biotechnology Co., Ltd; SDS, β-actin and SFRP4 were purchased from Biotoped; and a BCA kit, PMSF, RIPA lysate, and skimmed milk powder were obtained from Shanghai Biyuntian Biotechnology Co., Ltd. Protein Marker (10–180 kDa) was obtained from Beijing Solarbio Science and Technology Co., Ltd.; β-Catenin and c-myc were obtained from CST. We also purchased a range of ELISA kits to detect rat IL-1, rat IL-4, rat IL-6, and rat IL-10, and a range of ELISA kits to detect human IL-1β, human IL-6, human IL-10, and human TNF-α; these ELISA kits were purchased from Shanghai Langton Biotechnology Co., Ltd.

Animal and cell culture

We obtained 80 Specific-Pathogen-Free Wistar rats (> 220 g; 40 males and 40 females). The rats were held in separate cages and acclimatized for one week prior to experimentation; during this time, the rats were given ad libitum access to normal rat food and distilled water. The photoperiod was set to 12L:12D, the room temperature was 22 °C, and relative humidity was 50–65%.

Human fibroblast-like synoviocytes of rheumatoid arthritis (HFLS-RA) cells (Beijing Beina Chuanglian Biotechnology Research Institute) were cultured in an incubator at a constant temperature of 37 °C in high humidity and 5% CO2. Cells were cultured in DMEM high glucose medium with 20% FBS. The cells were digested with 0.25% trypsin when they reached 80–90% confluency, and a cell suspension was created for further culture. All cells used in the subsequent experiments were obtained from passages 3–7.

Preparation of DBD decoction

Astragalus membranaceus and Angelica (Tongrentang pharmacy, Beijing, identified by the school’s Chinese medicine identification teacher) were weighed and placed into a beaker in a ratio of 5:1. Distilled water was then added, and the materials were allowed to soak for 1 h. The decoction was then performed (When decocting, first fire and then light the fire). The solution produced was then filtered and decocted for a second time. Finally, the two decoctions were combined and concentrated in a constant temperature water bath to obtain a final concentration of 1.296 g of crude drug/mL; this represented a high concentration of DBD in water. We also prepared medium and low concentrations of DBD in water: 0.648 g crude drug/mL and 0.324 g crude drug/mL, respectively. These three decoctions were then sealed and stored in the dark at 4 °C.

The preparation of drug-containing serum of DBD

Wistar rats were randomly divided into five groups: a blank group, a tripterygium glycoside group (8.75 mg/kg), a DBD low dose group (3.24 g/kg), a DBD medium dose group (6.48 g/kg), and a DBD high dose group (12.96 g/kg). Rats in each group were given different concentrations of DBD water decoction by gavage at a dose of 1 mL/100 g; rats in the blank group were given the same volume of distilled water once a day. One hour after administration, on the morning of the fourth day, we injected 20% urethane into the abdominal aorta of anaesthetized rats at a dose of 1 g/kg in order to take blood samples. All blood samples were left at room temperature for 2 h and then centrifuged for 10 min at 3000 rpm. After centrifugation, the supernatant was gently aspirated and placed into a clean microcentrifuge tube; this provided us with samples of the drug-containing serum. The drug-containing serum from rats in the same experimental group was mixed; this allowed us to create different groups of serum drugs. The serum was inactivated in a constant temperature water bath at 56 °C for 30 min and a microporous filter membrane (0.22 μm) was used to sterilize the sera. Finally, sera were sealed and frozen in a refrigerator at -20 °C to await subsequent experiments.

Cell proliferation assays

MTT and CCK-8 detection of the effect of drug-containing serum on the proliferation of HFLS-RA cells. HFLS-RA cells were digested and made into a suspension. The cell density was then adjusted to 1 × 104/mL; we then used 100 μL/well to inoculate a 96-well plate. Each experimental group was allocated with 8 multiple wells; there were 6 groups in total. Once the 96-well plates had been placed in an incubator for 4 h, the culture medium was carefully discarded, and each group of cells was treated with the appropriate drug, as shown in Table 1. After that, the cells were cultured for 24 h and 48 h. For MTT assay, we then added 20 μL MTT to the culture and cultured it for 4 h at 37 °C. Subsequently, we discarded the supernatant and added DMSO to each well. The optical density (OD) was measured at a wavelength of 490 nm. For CCK-8 assay, we added 10 μL CCK-8 to the culture and cultured it for 1 h at 37 °C. The OD was measured at a wavelength of 450 nm. Cell proliferation inhibition rate calculation formula: (Model group – DBD groups)/(Model group – Blank group) × 100%.

Table 1 Drug components in each groupDetection the of TNF-α, IL-1β, IL-6 and IL-10 by ELISA

When the HFLS-RA cells reached 80% confluency, the cells were divided into different groups and stimulated with different medicinal regimens, as described in Table 1. Cells were then cultured for 24 h, after which, the supernatant was removed and stored at 4 °C. The levels of TNF-α, IL-1β, IL-6 and IL-10 in the supernatant were then detected using appropriate ELISA kits according to the manufacturer’s instructions. The level of TNF-α, IL-1β, IL-6 and IL-10 in the supernatant was determined as same as above described.

Establishment of collagen-induced arthritis (CIA) rat model

We prepared a rat model of CIA with bovine type II collagen, as described in the previous literature, but with a few modifications [10]. Briefly, we prepared bovine type II collagen emulsion at a concentration of 1 g/L by emulsifying a mixture of glacial acetic acid solution and bovine type II collagen in an aseptic environment. We established a blank group and allocated the CIA rats into different groups: a model group, a Tripterygium glycoside group, a DBD high group, a DBD medium group, and a DBD low dose group. Wistar rats were immunized for the first time one week after adaptive feeding. When immunized for the first time, the rats were injected with 0.2 mL of type II collagen emulsion into the toe, tail root (1.5–2.0 cm from tail root), or subcutaneously, depending on the dosage. One week after the first immunization, we used the same method to strengthen the second immunization response but with a dose of 0.1 mL of type II collagen emulsion. The rats were divided into two groups and then, treated with drugs on the 12th day. Each group was administered with DBD decoction by gavage according at a dose of 1 mL/100 g; the blank group and the model group were given an equivalent volume of distilled water. The Tripterygium glycoside group was given Tripterygium glycoside suspension by gavage for 28 days at a dose of 8.75 mg/kg. During this time, we observed the general condition of each rat (mental state, appetite, and fur color). We also recorded any evidence of joint swelling. We made efforts to measure the rate of any joint swelling. At the beginning of the experiment, we recorded the peripheral diameter of the right ankle joint of each rat; we then re-measured this area once a day before the first immunization; these measurements were used as a baseline. Following the first immunization, we measured the right ankle joint once a week. We then calculated the swelling rate as follows: Swelling rate = (the peri-ankle diameter after inflammation—peri-ankle diameter before inflammation)/peri-ankle diameter before inflammation × 100%. We also calculated the arthritis index (AI) for each joint, using the following scoring criteria: no swelling of joint, 0 points; slight swelling of the small toe joint, 1 point; swelling of toe joint and toe, 2 points; swelling of the foot below ankle joint, 3 points; swelling of all feet including the ankle joints, 4 points. According to these scoring criteria, we scored the four toes of each rat and created an overall score by adding the score for each foot; the maximum score was 16 points. An AI value greater than 4 points indicated successful CIA modeling [11]. Paw swelling and arthritis scores are used to evaluate the anti-arthritic effects of drugs [12].

Hematoxylin–eosin (H&E) staining to observe the morphological changes of ankle joint

Ankle joint tissues were first fixed with paraformaldehyde, decalcified, dehydrated, cleared and embedded in paraffin wax. Thin Sects. (4–6 microns) were then prepared, unfolded in water, baked and placed into a fixed frame on a slide for 10 h at 62 °C. For staining, sections were baked, dewaxed, stained in hematoxylin and eosin, dehydrated, fixed onto glass slides and sealed. Five visual fields were randomly selected and assessed for immunoreactive areas at × 200 magnification using a light microscope.

Western blot analysis of β-Catenin, SFRP4 and c-myc in the synovium

The synovial tissue protein was extracted, and the protein content was determined by the BCA protein detection kit. Protein concentration was quantified with the Easy Protein Quantitative Kit. Protein was subjected to 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and then, transferred onto poly-vinylidene fluoride membranes. Membranes were incubated with primary antibodies such as rabbit polyclonal antibody against β-actin (1:1000), rabbit polyclonal antibody against β-catenin (1:5000), and rabbit polyclonal antibody against sfrp4 (1:500) and rabbit monoclonal antibody against c-myc (1:1000) overnight at 4 °C. Beta-actin was used as an internal reference. The next day, the membranes were washed with PBST and then incubated with horseradish peroxidase labeled anti-rabbit IgG (1:5000, Bioworld company) for 1 h at room temperature. Protein bands were visualized using a chemiluminescent imaging system.

Statistical analysis

All experimental data were processed and analyzed by SPSS version 13.0 software (SPSS Inc., Chicago, IL). Data were expressed as means ± standard deviation. Differences between groups were tested for significance by one-way analysis of variance (ANOVA). P < 0.05 was considered statistically significant.