Animals
Male SD rats weighing 180-220 g were purchased from Zhejiang Experimental Animal Center (Zhejiang, China). Male DBA/1J mice weighing 18-22 g were purchased from the Cavens Laboratory Animal Company (Changzhou, China). All animals were maintained under specific pathogen-free conditions at the Nanjing University of Chinese Medicine Experimental Animal Center. The experimental animals were acclimated for one week before the beginning of the study with free access to rodent diet and water. All experiment processes were in strict accordance with the regulations on the use and management of experimental animals of Nanjing University of Chinese Medicine.
AIA model preparation
The AIA rat model was prepared as previously described[25]. Complete Freund's adjuvant (CFA) (including Bacillus Calmette-Guerin (Beijing Institute of Biological Products Co. Ltd., Beijing, China) 7.5 mg/ml) was prepared, and the model of AIA was established by intradermal injection of CFA (0.05 ml) into the right hind foot of SD rats. All rats were continuously fed for 28 days and the changes of joint swelling were observed. The peripheral blood of the rats were collected on the 28th day. Then the rats were sacrificed and the synovial tissues of the joint were separated for the following experiment.
Establishment of co-culture system of peripheral blood monocytes and synovial fibroblasts from AIA rats to induce osteoclast-like cells
Osteoclast-like cells were induced according to the method which we have founded in the previous study [25].
The fresh anticoagulant blood of AIA rats was isolated with lymphocyte separation solution, transferred into the cell bottle and cultured for about 12h. Discarded the upper lymphocytes, and the lower adherent cells were mononuclear cells. The monocytes were then collected. The synovial tissues were washed three times with phosphate-buffered saline, and pieces of the superficial layer of synovium of about 1-2 mm3 were cut and placed in Dulbecco’s modification of Eagle’s medium. The tissue blocks were further divided with scissors in a 100 mm petri dish and were then incubated for 3 or 4h in 2 ml of medium (containing 0.5 ml fetal calf serum and 0.5 ml culture solution) at 37°C. The culture medium was changed every three days. After the synovial fibroblasts gradually grew into sheets, the tissues were removed. The cells were then cultured and transmitted to 3-5 generations.
2 ml of monocytes (1×106 mL-1) were added to the 6-well plates. Synovial fibroblasts in the logarithmic growth phase were prepared into 1×105 mL-1 cell suspension, which were added to the hanging transwell chamber in the 6-well plates. The cells were co-cultured in 5% CO2 incubator at 37°C to induce osteoclast-like cells. The culture medium was changed every other day. After 21 days, the cells were identified by tartrate-resistant acid phosphatase (TRAP) (Nanjing Jiancheng Bioengineering Institute, China) staining. TRAP-positive multinucleated cells having three or more nuclei were counted as osteoclast-like cells under a microscope. On the other hand, the co-cultured osteoclast-like cells (co-culture for 14 days) (104 /mL) were added to the 96-well plate with preset ivory slices (Immunodiagnostic Systems Limited, Bolton, UK). The bone fragments were taken out and observed by scanning electron microscope after 7 days.
Detection of the activity of osteoclast-like cells
After 21 days of co-culture, the cells were treated with different doses of TP (1, 10, 100 μg/L) in 5% CO2 incubator at 37°C for 48h. The transwell was taken out and the absorbance was detected by MTT (3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl-tetrazolium bromide) method.
Detection of the TRAP level and the bone absorptive capacity of osteoclast-like cells[26]
The co-culture cells (day 21 of co-culture) were treated with different doses of TP (1, 10, 100 μg/L) for 48h, and the level of TRAP in the supernatants were detected.
After co-culture for 14 days, the transwell was taken out, the lower osteoclast-like cells were digested. Then, the osteoclast-like cells (104 /mL) were collected and added to the 96-well plate with preset ivory slices (Immunodiagnostic Systems Limited, Bolton, UK). After 24h of culture, the cells were treated with different doses of TP (1, 10, 100 μg/L) for 48h. Then, after changing the culture medium and continuing to culture for 3 days, the bone slices were taken out and the absorption of osteoclast-like cells in the lacunae was observed by scanning electron microscope, and the bone resorption area in the lacunae was calculated by Leica.
Detection of IL-1, IL-34, RANKL, M-CSF and OPN levels in culture supernatant of co-culture cells using enzyme-linked immunosorbent assay (ELISA)
The synovial fibroblasts were co-cultured with monocytes, and treated with different doses of TP (1, 10, 100 μg/L) for 48h on the 7th and 21st days of co-culture, respectively. The culture supernatants were collected to detect the levels of IL-1 (Nanjing Jiancheng Bioengineering Institute, China), IL-34 (Nanjing Jiancheng Bioengineering Institute, China), RANKL (Shanghai yuanye Bio-Technology Co., Ltd., Shanghai, China), M-CSF (Shanghai yuanye Bio-Technology Co., Ltd., Shanghai, China) and OPN (Shanghai yuanye Bio-Technology Co., Ltd., Shanghai, China) by ELISA.
Detection of the expressions of MAPKs andTRAF6 proteins on osteoclast-like cells using Western blot [27]
The co-cultured cells (day 21 of co-culture) were treated with different doses of TP (1, 10, 100 μg/L) for 48h. The transwell was removed, and the osteoclast-like cells were collected. The cells were washed twice with ice-cold PBS. Then, the cells were resuspended in cold lysis buffer for 30 min at 4°C and centrifuged at 12000 rpm for 10 min at 4°C. Next, the supernatants were collected and the proteins were quantified by the bicinchoninic acid (BCA) (Beyotime, Shanghai, China) method. Equal amounts of protein (25 ug) were electrophoresed on gradient 10% polyacrylamide gels, and transferred to polyvinylidene difluoride (PVDF) membrane. The membranes were blocked for 1h at room temperature in 5% nonfat dry milk in TBST and incubated with primary antibodies against p-p38MAPK, p-ERK1/2, p-JNK, p38MAPK, ERK1/2, JNK, TRAF6 and β-actin (each at 1:1000 dilution) (Cell Signaling, USA) with gentle rotation overnight at 4°C. Membranes were next incubated with horseradish peroxidase (HRP) secondary antibody (1:5000 dilution) (Santa Cruz, USA) for 2h at 37°C. The membrane was added with developer, placed at room temperature with 1 min, and exposed the protein on the X-ray film in the darkroom. Develop the image in the processor.
Detection of the expression of NFATc1 of osteoclast-like cells using immunofluorescence
Different doses of TP (1, 10, 100 μg/L) were used to treat co-cultured cells (day 21 of co-culture) for 48h. The transwell was taken out, and immunofluorescence detection was performed as previously described[28]. The cells were plated on glass coverslips, fixed with 4% paraformaldehyde for 1-5h and permeabilized with 1% Triton X-100 for 15 min at room temperature. These samples were soaked in a cold solution of 30% H2O2 for 15 min after washing with PBS, and covered with 100 μL primary antibody (anti-NFATc1 (1:50, Beijing BoAo Biological Company, China), β-actin (1:1000, Cell Signaling, USA)) for 2h. Then they were added enhancer and incubated for 2h. After soaking in PBS for 5 min and washing 3 times, the osteoclast-like cells were incubated with 50 μL Cy3-conjugated secondary antibody (1:500, Jackson, PA, USA) and counterstained with 4, 6-diamidino-2-phenylindole (DAPI) (1: 500, Abcam, USA) at 37℃. All images were taken with a fluorescence microscopy (Laika, Germany).
CIA model preparation and TP treatment
The CIA model was prepared as previously described[28]. In brief, bovine type II collagen (CII) (Redmond, WA, USA) was dissolved in acetic acid (0.1 mol/L) at a concentration of 2 mg/mL. The bovine CII and CFA were mixed at equal volumes (final concentration of 1 mg/mL). The mixed emulsion of 100 μL CII and CFA was intradermally injected into the posterior part of the caudal root of DBA/1J mice for the first time. On the 21st day after the first immunization, the posterior part of the caudal root of DBA/1J mice were intradermally injected with equal proportional mixture of 100 μL CII and incomplete Freund's adjuvant (IFA) (final concentration of 1 mg/mL) to enhance immunization.
After 7 days of accommodation, CIA mice were randomly divided into two groups (n= 6 per group) as follows: model and TP group. Another 6 DBA/1J mice were used as control group. The mice in TP group were intragastrically administered with 60 μg/kg of TP for 2 consecutive weeks from the 28th day after the first immunization. The mice in the control and model group received the same volume of saline. The effect of TP on joint swelling and arthritis index were observed on the 22th, 26th, 30th, 34th, 38th and 42th days after the first immunization. X-ray observation of the morphological changes of the knee joint were investigated on day 42.
Arthritis assessments
During the experiment, the mice were assessed regularly for signs of arthritis. Arthritis index (AI) was used as a measure of CIA. Disease severity was scored 0-4 (0=no edema or swelling, 1=swelling in toe joint, 2=swelling in metatarsophalangeal joint and foot pad, 3=swelling of the feet below the ankle joint, 4=swelling of the entire paw and joint malformation)[29]. The score was less than 16 and AI > 2 was considered as CIA. The investigator was blinded to the experimental group to avoid any bias.
Statistical analysis
Quantitative data were presented as the mean ± standard deviation (SD). Unpaired-Student’s t-tests, one-way ANOVA with Tukey's Studentized range test and two-way ANOVA for repeated measures with Bonferroni's post hoc test were used to analyze data. Date analysis was performed using GraphPad Prism 8.0. P values <0.05 were considered statistically significant.