2.1. Reagents
KLHTT (batch number: 0503-2-403-01) was supplied by Sun Ten Pharmaceutical Corporation, New Taipei City, Taiwan. Chicken collagen type II (CII) was purchased from Chondrex, Inc., WA, USA. Mycobacterium tuberculosis H37RA was bought from Difco Laboratories Inc., MI, USA. Methotrexate (MTX) and EDTA were ordered from Bio Basic Inc., Toronto, Canada. Enzyme-linked immunosorbent assay (ELISA) kits (for IL-1β, IL-6, and TNF-α) were obtained from eBioscience, San Diego, CA, USA. An IL-17A ELISA kit was purchased from R&D Systems Inc., Minneapolis, MN, USA. A hydrogen peroxide assay kit was purchased from Cell Biolabs, Inc, San Diego, CA, USA. Goat anti-mouse IgG1 and goat anti-mouse IgG2a secondary antibodies were purchased from Jackson ImmunoResearch Laboratories, PA, USA. ABTS substrate solution was ordered form Roche Diagnostic Systems, CA, USA. A 3H labelled compound was purchased from Amersham Pharmacia Biotech, Arlington Heights, IL, USA. Brefeldin A and Freund’s adjuvant were bought from Sigma-Aldrich, St. Louis, MO, USA. PE-conjugated anti-mouse CD4 (clone GK1.5), FITC-conjugated anti-mouse IL-17A (clone TC11-18H10.1), and FITC-conjugated anti-mouse interferon (IFN)-γ (clone XMG1.2) antibodies were ordered from Biolegend, San Diego, CA, USA. Formalin was bought from AVANTOR, Center Valley, PA. Bovine serum albumin (BSA) was purchased from EMD Millipore, Billerica, MA, USA. Tween 20 was obtained from EMD Millipore, France.
2.2. KLHTT preparation
The herbs of KLHTT were purchased and identified by Sun Ten Pharmaceutical Corporation, New Taipei City, Taiwan. A total of 27.93 g of herbs (6.25 g Soapstone, 4.58 g Artemisia capillaris Thunb. (seedling), 4.17 g Scutellaria baicalensis Georgi (root), 2.50 g Acorus gramineus Soland. (rhizome), 2.08 g Clematis armandii Franch. (rattan and stem), 2.08 g Fritillaria cirrhosa D. Don (bulb), 1.67 g Pogostemon cablin (Blanco) Benth. (plant shoot), 1.67 g Forsythia suspensa (Thunb.) Vahl (fruits), 1.67 g Amomum kravanh Pierre ex Gagnep. (fruits), 1.67 g Mentha haplocalyx Briq. (stem and leaf plot), and 1.67 g Belamcanda chinensis (L.) DC (rhizome)) was extracted by boiling water (12 times the weight of the herbs) for 1 h, and then concentrated to a voucher specimen (CGU_KLHTT-01) by the freeze dryer (LABCONCO, USA) [25]. The voucher specimen complied with Chang Gung University guidelines.
2.3. Ultra-performance liquid chromatography-tandem mass spectrometry
The chemical profile of KLHTT extract was obtained using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) comprising a LC-30AD pump, SIL-30AC auto-sampler, CTO-20AC column, and SPD-M20A Photodiode Array Detector (Nexera X2, Shimazu, Kyoto, Japan). Prior to being loaded onto the UPLC column, 1 mg of KLHTT extract was first dissolved in 1 mL of methanol and filtered through a 0.45 μm membrane. Sample injections of 1 μL were then performed automatically. Liquid chromatography was performed using a CORTECS UPLC C18 column (90Å, 1.6 µm, 2.1 mm x 100 mm) (Waters, MA, USA). The mobile phase was a mixture of MeCN (A) and water (W, containing 0.1 % formic acid). A gradient sequence was executed as follows: 0–10 min, 10–20 % A; 10–14 min, 20–25 % A; 14–24 min, 25–30 % A; 24–28 min, 30–40 % A; 28–33 min, 40–50 % A; 33–38 min, 50–75 % A; 38–40 min, 75–100 % A; and 40–43 min, 100 % A. The column temperature was set at 35 °C. The flow rate was at 0.4 mL/min. The range of detection wavelengths was fixed in the 190-500 nm.
Multiple reaction monitoring (MRM) experiments (in negative) were carried out using Shimazu LCMS-8045 triple quadrupole mass spectrometry to identify the constituents of KLHTT extract. The precursor ion settings of the corresponding profiling peaks were determined using the full scan experiment (50-1000 amu). The product ions were settled according to previously reported data. The dwell time was fixed at 100 ms and the collision energy was set at 25–45 eV. All MS data were acquired and processed using LCMS LabSolutions software Version 5.93 (Shimazu, Kyoto, Japan).
2.4. Experimental animals
DBA/1J mice (male, six- to eight-week old, weight 20–22 g) were purchased from Jackson Laboratories (Bar Harbor, ME, USA) and maintained at 20–25 °C with half day light/dark cycle under a specific pathogen-free condition. All mice were treated according to the guidelines of the Institutional Animal Care and Use Committee of National Chung Hsing University (NCHU). The study protocol was approved by NCHU ethics committee.
2.5. CIA model establishment
CIA was induced by active immunisation with chicken CII [26]. Briefly, 2 mg/ml CII was dissolved in 10 mM acetic acid solution and emulsified with an equal volume of complete Freund’s adjuvant containing Mycobacterium tuberculosis H37RA (250 μg/mouse). The mixture (200 μL/mouse) was intradermally injected at the base of the tail. Incomplete Freund’s adjuvant and CII were administered as booster injections to the mice 21 days after the first immunisation. ddH2O, KLHTT, or MTX was administered orally once a day from day 21 to 42. Mice were divided into four groups (n = 6/group) randomly as follows: Group I, Normal; Group II, Vehicle (ddH2O) + CII; Group III, KLHTT (50 mg/kg) + CII; Group IV, KLHTT (100 mg/kg) + CII. MTX (0.5 mg/kg) was used as positive control. Mice were euthanized with CO2 exposure (100 % CO2 for 5 min) by experienced experimenters humanely on day 42.
2.6. Assessment of clinical arthritis severity
The body weight and arthritis severity score were obtained [26]. The arthritis severity score was evaluated as: 0, no swelling nor redness; 1, mild swelling and redness restricted to the tarsals or the ankle joint; 2, mild swelling and redness from the tarsals to the ankle; 3, moderate swelling and redness extending to the metatarsal joints; 4. severe swelling and redness from the ankle to the foot and the digits, or limb ankyloses. In addition, paw volume was measured using Plethysmometer 37140 (Ugo Basile SRL, Comerio, VA, Italy).
2.7. Assessment of histological arthritis severity
After the mice were humanly sacrificed, the hind limbs were fixed in 10 % buffered formalin, decalcified in 15 % EDTA, and embedded in paraffin. Serial paraffin sections (5 μm) were stained with haematoxylin and eosin. The severity of histopathological lesions was scored [26] as follows: 0, normal appearance; 1, mild infiltration of inflammatory cells, mild pannus front, and minimal cartilage damage; 2, moderate infiltration of inflammatory cells, erosive pannus front, and moderate cartilage damage; 3: diffuse infiltration of inflammatory cells, severe cartilage damage and bone resorption.
2.8. Measurement of pro-inflammatory cytokine levels
Hind paw was dissected and homogenised in ice-cold saline using a tissue homogeniser. After being centrifuged at 3000 rpm (4 °C, 10 min, twice), the hind paw homogenates were harvested. Blood was collected from the heart. The levels of cytokines in hind paw homogenates and serum were measured by ELISA [16].
2.9. Measurement of the concentrations of oxidative markers
Malondialdehyde (MDA) concentration was determined by thiobarbituric acid reactive substances assay at 532 nm. The standard curve was obtained using 1,1,3,3-tetramethoxypropane. Hydrogen peroxide (H2O2) concentration was measured using a colorimetric OxiSelect™ hydrogen peroxide assay kit at 560 nm [16].
2.10. Anti-collagen type II antibody analysis
Serum samples were diluted 1:250 for IgG1 or 1:125 for IgG2a in Tris-buffered saline (1 % BSA and 0.5 % Tween 20, pH 8.0), and then transferred to CII (10 μg/ml) pre-coated 96-well plates (MicrotiterTM, Thermo Fisher Scientific, Roskilde, Denmark) at 4 °C overnight. The plates were washed and incubated with goat anti-mouse secondary antibodies IgG1 (1:500 dilution) or IgG2a (1:500 dilution) at 25-27 °C for 1 h. After being washed, ABTS substrate was added and the reactions were stopped by adding H2SO4. The level of IgG1 and IgG2a was measured at 450 nm by an ELISA reader (Sunrise, Tecan Inc., Switzerland) [16].
2.11. Splenocyte proliferation assay
Splenocytes (4×105 cells/well) were cultured with chicken CII (50 μg/mL) at 37 oC for 40 h, and then incubated with 3H for 8 h. Cell proliferation was evaluated by radioactive thymidine incorporation [16].
2.12. Intracellular staining
Splenocytes (1×106 cells/well) were cultured with chicken CII (50 μg/mL) at 37 oC for 48 h, and then brefeldin A (5 μg/mL) was added for 6 h. Cells were harvested and extracellularly stained with PE-conjugated anti-mouse CD4 antibodies. After being fixed and permeabilised with Cytofix/Cytoperm solution (BD Pharmingen), cells were then intracellularly labelled with FITC-conjugated anti-mouse IL-17A and anti-mouse IFN-γ antibodies. Splenocytes were detected by an Accuri C5 flow cytometer (Accuri Cytometers, Ann Arbor, MI, USA) and analysed by BD Accuri™ C6 Plus software [26].
2.13. Statistical analysis
Data are presented as mean ± SD. Statistical analyses were performed using one- or two-way ANOVA followed by Tukey’s honestly significant difference test. A P-value < 0.05 was considered statistically significant.