Reagents
Processed scorpion (Batch No. 20100108), scolopendra (Batch No. 20200301), astragali radix (Batch No. 2010012), black soybean seed coats (Batch No. 200414), and methotrexate (MTX) were purchased from Jiangsu Integrated Traditional Chinese and Western Medicine Hospital (Nanjing, Jiangsu, China). Calycosin and protocatechuic acid were purchased from Yuanye Bio-Technology (Shanghai, China). Bovine type II collagen and Freund’s adjuvant were purchased from Chondrex (Redmond, WA, USA). An enzyme-linked immunosorbent assay (ELISA) kit for anti-bovine collagen II-specific antibodies was purchased from Chondrex (Redmond, WA, USA). Cell counting kit-8 was purchased from MCE (NJ, USA). Antibodies against interleukin-17 (IL-17) were purchased from Abcam (Cambridge, UK). ELISA kits for TNF-α, IL-6, IL-17A, and interleukin-10 (IL-10) were purchased from R&D Systems (Minneapolis, MN, USA). Antibodies against anti-mouse CD3, anti-CD4, anti-CD25, anti-Foxp3, and anti-IL-17A were purchased from BD Biosciences (Franklin Lakes, NJ, USA). Anti-mouse CD4 magnetic particles were purchased from BD Biosciences (Franklin Lakes, NJ, USA). The permeabilization solution kit was purchased from BD Biosciences (Franklin Lakes, NJ, USA). The cell stimulation cocktail was purchased from Invitrogen (Carlsbad, CA, USA). The HiScript Reverse Transcription system was purchased from Vazyme Biotech (Nanjing, Jiangsu, China). TRIzol reagent was purchased from Invitrogen (Carlsbad, CA, USA).
Preparation of SF
SF was provided by the Pharmacy of Jiangsu Province Hospital on Integration of Chinese and Western Medicine (Jiangsu, China) and was composed of scolopendra, scorpion, astragali radix and black soybean seed coats. The daily dose of SF crude drug administered by humans in the clinic was 0.85 g/kg. The processed scorpion and scolopendra were crushed through an 80-mesh sieve to form a fine powder, which was further crushed into ultrafine powder using a cryogenic ball mill. Astragali radix and black soybean seed coats were decocted twice with 600 mL of water at 100 °C for 1 h to obtain an aqueous extract, based on a previously reported procedure [20]. The aqueous extract was freeze-dried at -80 °C for 72 h to obtain a freeze-dried powder. The freeze-dried SF powder was dissolved in double distilled water and used in the in vivo and in vitro experiments.
HPLC-Q-TOF-MS analysis and quality control of SF
HPLC-Q-TOF-MS analysis was performed to identify the main compounds in SF (50 mg/mL). Chromatographic separation was performed using an Agilent C18 column (3.0 mm × 100 mm, 2.7 μm; Agilent Technologies, Santa Clara, CA, USA) at 40 °C. The mobile phase consisted of water containing 0.1% phosphoric acid (A) and acetonitrile (B). The gradient program was set as follows: 0–0.01 min, 5% B; 0.01–25 min, 5–95% B; 25–27 min, 5% B; 27–30 min, 5% B. The mobile phase flow rate was 0.3 mL/min, and the sample injection volume was 2 μL. Electrospray ionization (ESI) with positive ion modes was used for mass detection. The source parameters were set as follows: spray voltage, 4.5 kV; gas temperature, 550 °C; pressure of nebulizer gas, 55 psi; full scan range, m/z 50–1000.
The main ingredient in SF (50 mg/mL) was confirmed using an Agilent 1260 liquid chromatography system. Methanol (80%) was used as the extraction solvent for the ultrasonic treatment of SF. The SF was separated on an X-Bridge C18 column (250 mm × 4.6 mm, 5 μm) maintained at 25 °C. The mobile phase consisted of acetonitrile (A) and 0.1% phosphate buffer (B) in a gradient elution: 0–10 min, 5% A; 10–11 min, 5–15% A; 11–20 min, 15–18% A; 20–30 min, 18–20% A; 31–40 min, 20–30% A; 31–40 min, 30–37% A; 40–46 min, 37–90% A. The flow rate was 1 mL/min, the injection volume was 10 µL, and detection was set at 260 nm. Chromatographic data were acquired and analyzed using the Empower software (Agilent). The peaks of protocatechuic acid and calycosin in SF were identified by comparing peak retention times with those of the reference compounds.
Animals
Six-to-eight-week-old female C57BL/6 and DBA/1J mice were purchased from Changzhou Cavens Experimental Animal Co., Ltd. (Jiangsu, China) and maintained under specific pathogen-free conditions at the Animal Center of Jiangsu Province Academy of Traditional Chinese Medicine. All mice raised in circumstances that alternated between 12 h of light and 12 h of darkness at the temperature of 20–25 °C and relative humidity of 50%–70%. All animal experimental procedures were performed in accordance with the national and international guidelines and regulations, and were approved by the Animal Ethics Committee of Jiangsu Province Academy of Traditional Chinese Medicine.
CIA induction and drug administration
CIA mice were immunized twice using bovine type II collagen. In the first immunization, bovine collagen II and Freund’s complete adjuvant were mixed and administered as an intradermal injection at the base of the tail in each mouse (100 μg/mouse). On day 21, a booster injection was given using bovine collagen II and Freund’s incomplete adjuvant. DBA/1J female mice were randomly divided into five groups: normal group (Normal, n=5), CIA vehicle group (CIA vehicle, n=5), methotrexate group (MTX, n=5), SF low-dose group (SF-L, 183 mg/kg, n=5), and SF high-dose group (SF-H, 550 mg/kg, n=5). The doses of SF and MTX used in this study were determined from the recommended dosages for humans. SF low-dose was calculated based on a person’s daily administration of 1 g of scorpion and scolopendra, and SF high-dose was calculated based on a person’s daily administration of 3 g of scorpion and scolopendra. Oral administration of SF (183 or 550 mg/kg/d) began 28 d after the first immunization, and MTX was administered at a dose of 920 mg/kg twice a week. Mice in the normal and vehicle groups were administered an equal volume of deionized water at the same time points.
Evaluation of arthritis
The incidence of arthritis was evaluated every 3–4 d following immunization. The severity of arthritis was assessed on a scale of 0–4, based on the following previously described criteria [21]: 0 = no evidence of erythema or swelling; 1 = erythema and mild swelling extending to the tarsals or ankle joint; 2 = erythema and mild swelling extending from the ankle to the tarsals; 3 = erythema and moderate swelling extending from the ankle to metatarsal joints; 4 = erythema and severe swelling encompassing the ankle, foot, and digits, or ankylosis of the limb. The arthritis score for each mouse was expressed as the sum of the scores of all four limbs. The highest arthritis score that a mouse could have was 16. Hind paw swelling was measured using a paw volume meter (Woodland Hills, CA, USA).
Histological evaluation
The mice were sacrificed after 30 d of SF treatment and the hind limbs (including paws and ankles) were collected. The hind limbs were fixed in 4% paraformaldehyde solution, which was then decalcified with 10% EDTA for 1 month. After that, the hind limbs were paraffin-embedded, and tissues sectioned and stained with hematoxylin and eosin (H&E). The degree of histopathological damage was evaluated based on previously described criteria [22].
Measurement of cytokine and bovine collagen-specific IgG levels
Blood was collected from each mouse on day 30 of treatment and clotted at 25 °C for 1 h. Blood was centrifuged at 4000 rpm for 15 min to obtain serum, which was then stored at -80 °C until use. The levels of cytokines and bovine collagen-specific IgG were measured using ELISA kits according to the manufacturers’ instructions.
Immunohistochemistry
The hind limb tissue sections were incubated with an anti-IL-17 antibody. After incubation with HRP-conjugated goat anti-rabbit IgG antibody, the expression of IL-17 was visualized using a DAB kit (Servicebio, Wuhan, China). The quantitative analysis of IL-17 in hind limb sections was performed using Image J 1.37v. The results are expressed as the mean region of interest, and the average optical density was used for statistical analysis.
T-cell isolation and cell viability assay
CD4+ T cells were isolated from the splenocytes of six-to-eight-week-old female C57BL/6 mice using anti-mouse CD4 magnetic particles and an IMag Cell Separation Magnet. To evaluate the effect of SF on the viability of CD4+ T cells, the cells were treated with different concentrations of SF (0.1–1 mg/mL) for 72 h, and cell viability was detected with a cell counting kit-8.
In vitro Th17 cell differentiation
The polarization of Th17 cells was performed as previously described [23], CD4+ T cells were stimulated with plate-bound anti-CD3 (1 µg/mL), anti-CD28 (1 μg/mL), anti-interleukin -4 (2 μg/mL), 2 μg/mL anti-interferon-γ (IFN-γ), 2 ng/mL transforming growth factor-β (TGF-β), 30 ng/mL IL-6, 20 ng/mL interleukin-23 (IL-23), and 10 ng/mL interleukin-1β (IL-1β) for 72 h. After 3 d, the cells were re-stimulated with a cell stimulation cocktail and then stained with anti-mouse CD4 and anti-IL-17A antibodies. The level of IL-17A in the cell supernatant was detected using an ELISA kit.
Flow cytometry
After oral administration of SF for 30 d, all mice were sacrificed, and the spleen and lymph nodes extracted, mashed and washed with phosphate-buffered saline (PBS). For intracellular IL-17A and Foxp3 staining, the splenocytes and lymph node cells were stimulated with a leukocyte activation cocktail for 5 h. Next, cells were stained with surface FITC-conjugated anti-CD4 antibody or stained with FITC-conjugated anti-CD4 and APC-conjugated anti-CD25 antibodies. After fixation and permeabilization, cells were stained with PE–conjugated anti–Foxp3 or PE–conjugated anti-IL-17A antibodies. Finally, the cells were analyzed via flow cytometry (BD Biosciences, Franklin Lakes, NJ, USA). Flow Jo v10 was used to further analyze the levels of Treg and Th17 cells.
RNA extraction and real-time qPCR
CD4+ T cells were cultured for 3 d with or without SF (0.1, 0.5, and 1 mg/mL) under Th17-polarizing conditions. Total RNA was extracted from CD4+ T cells using TRIzol reagent according to the manufacturer’s protocol. cDNA was synthesized using the HiScript Reverse Transcription system. A 7500 real-time PCR system (Applied Biosystems, Waltham, MA, USA) was used for PCR amplification. All reactions were performed using the ChamQ Universal SYBR qPCR Master Green kit (Vazyme Biotech, Nanjing, China). The 2−ΔΔCt method was used for data analysis. The primer sequences used are listed in Table 1.
Table 1. Primers used for real-time qPCR in the present study.
Gene
|
Forward
|
Reverse
|
Mouse RORγt
|
5'-GACCCACACCTCACAAATTGA-3'
|
5'-AGTAGGCCACATTACACTGCT-3'
|
Mouse IL-17A
|
5'-TTTAACTCCCTTGGCGCAAAA-3'
|
5'-CTTTCCCTCCGCATTGACAC-3'
|
Mouse IRF4
|
5'-TCCGACAGTGGTTGATCGAC-3'
|
5'-CCTCACGATTGTAGTCCTGCTT-3'
|
Mouse GAPDH
|
5'-TGTGGATGGCCCCTCTGGAA-3'
|
5'-TGACCTTGCCCACAGCCTTG-3'
|
Statistical analysis
GraphPad Prism (GraphPad Software version 7.0, San Diego, CA, USA) was used for statistical analysis. Data are expressed as the mean ± SEM. One-way ANOVA with Dunnett’s post-hoc multiple comparison tests and two-way ANOVA with Bonferroni post-tests were used to determine statistical significance. Statistical significance was established at P<0.05.