Twenty pathogen-free male DBA/1 mice at 8 weeks of age were purchased from Samtako (Osan, Korea). The mice were housed in a temperature (22–24°C) and humidity (55–60%) controlled environment with a 12-h light/dark cycle and were maintained on standard laboratory chow ad libitum. All experiments were performed in accordance with the guidelines for animal experimentation of the Institute Committee of Wonkwang University (WKU15-78).
Induction of CIA in DBA/1 mice
DBA/1 mice were immunized with 150 µL of bovine type II collagen (CII) emulsified with an equal volume of complete Freund’s adjuvant (CFA; Chondrex, Redmond, WA, USA). The point of initial immunization was designated day 0. The mice were then boosted with an equal amount of bovine type II collagen emulsified in Freund’s incomplete adjuvant (IFA; Chondrex) on day 21 and by an intraperitoneal injection of lipopolysaccharide (LPS, 30 µg; Sigma, St. Louis, MO, USA) was on day 28 . To evaluate the preventive or therapeutic effect of HAR (Sigma) on CIA progression, HAR (10 mg/kg) or PBS was orally administered every other day. The experiment consisted of four groups: group 1, control with no disease (n = 5); group 2, CIA with vehicle treatment (n = 5); group 3, CIA with HAR treatment at 5 days before second immunization (Prevention, n = 5); group 4, CIA with HAR treatment at 1 day after the second immunization (Therapy, n = 5) (Figure. 1A). The mice were monitored daily in a blinded manner for signs of arthritis. The symptoms were graded and scored as previously described . Briefly, all four limbs of the mice were evaluated and scored from 0 to 4 according to the following scale: 0 = no swelling; 1 = slight swelling and erythema confined to either the ankle or midfoot; 2 = slight swelling extending from the ankle to midfoot; 3 = moderate swelling from the ankle to metatarsal joints; and 4 = severe swelling in the ankle, foot, and digits.
Micro-computed tomography (micro-CT) analyses
Micro-CT images of the hind paw and femur of the mice in all four groups were acquired on day 42, using a high-resolution micro-CT (NFR-Polaris-S160; Nanofocus Ray, Iksan, Korea) with a source voltage of 45 kVp, current of 90 µA, and an isotropic resolution of 133 mA. To verify bone destruction, 3-dimensional models of the hind paw and calcaneus and 2-dimensional models of the femur were reconstructed using INFINITT-Xelis software (INFINITT Healthcare, Seoul, Korea). The structural parameters included total porosity, trabecular pattern factor (Tb·Pf), trabecular bone volume/total volume (BV/TV, %), trabecular thickness (Tb·Th, mm), trabecular separation (Tb·Sp, mm), and trabecular number (Tb·N, 1/mm).
Joint tissues and femurs were fixed with 10% formalin for 24 h, decalcified for 3 weeks in 12% EDTA, and then embedded in paraffin. Section slides (5 µm thick) of ankle joints prepared using a Leica microtome RM2145 (Leica Microsystems, Bannockburn, IL, USA) were stained with hematoxylin and eosin (H&E), safranin O, or toluidine blue. The histological arthritis score was determined in a blinded fashion for changes in synovial proliferation, inflammation, cartilage damage, and bone erosion and scored on a scale of 0–4 . To investigate the effect of osteoclasts on bone erosion in CIA mice, tissue sections were stained with tartrate-resistant acid phosphatase (TRAP). The number of TRAP-positive cells per field of tissue were quantified using Image Pro-Plus software version 4.0 (Media Cybernetics, Silver Spring, MD, USA).
Preparation of mouse bone marrow macrophages (BMMs) and osteoclast differentiation
Bone marrow cells (BMCs) from 5-week-old ICR mice were isolated by flushing the femurs and tibiae with α-minimum essential medium (α-MEM) and then suspended in α-MEM supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin as described previously . Non-adherent cells were collected and cultured for 3 days in the presence of macrophage colony-stimulating factor (M-CSF, 30 ng/mL). Floating cells were discarded, and cells adhering to the bottom of the culture dish were classified as BMMs. BMMs were seeded at 3.5 × 104 cells/well and cultured in the presence of M-CSF (30 ng/mL) and RANKL (50 ng/mL) for 4 days in the presence of various concentrations of HAR. The cells were fixed in 3.7% formalin for 10 min, permeabilized with 0.1% Triton X-100, and stained with TRAP solution. TRAP-positive multinucleated cells with more than five nuclei were counted as osteoclasts.
Pit formation assay
BMCs (1 × 107 cells) and primary osteoblasts (1 × 106 cells) were seeded on collagen gel-coated culture dishes and cultured for 7 days in the presence of 10− 8 M 1,25-dihydroxyvitamin D3 (Sigma) and 10− 6 M prostaglandin E2 (Sigma) as described previously . The co-cultured cells were detached by 0.1% collagenase treatment at 37°C for 10 min and then re-plated on hydroxyapatite-coated plates (Corning, Corning, NY, USA). The cells were incubated in the presence of various concentrations of HAR. After 12 h, the cells were removed, and the total resorption pits were photographed and analyzed using Image-Pro Plus version 4.0 (Media Cybernetics, Silver Spring, MD, USA).
Quantitative real-time reverse transcription polymerase chain reaction (Real-Time RT-PCR) analyses
Total RNA was isolated using QIAzol reagent (QIAGEN, Valencia, CA, USA) following the manufacturer’s instructions. To obtain cDNA, equal amounts of total RNA were reverse-transcribed into cDNA using the Reverse Transcription System cDNA synthesis kit (Promega, Madison, WI, USA). Real-time RT-PCR was performed in a 20 µL reaction mixture containing 10 µL of SYBR Green Premix (Bioneer Co., Daejeon, Korea), 10 pmol of forward primer, 10 pmol of reverse primer, and 1 µg of cDNA using an Exicycler™ 96 Real-Time Quantitative Thermal Block (Bioneer Co.) as described previously . The follwing mouse-specific primers used: TRAP, forward 5′-TCATGGGTGGTGCTGCT-3′ and reverse 5′-GCCCACAGCCACAAATCT-3′; osteoclast-associated receptor (OSCAR), forward 5′-GGAATGGTCCTCATCTGCTT-3′ and reverse 5′-GGAATGGTCCTCATCTGCTT-3′; calcitonin receptor (CTR), forward 5′-TCCAACAAGGTGCTTGGGAA-3′ and reverse 5′-CTTGAACTGCGTCCACTGGC-3′; Cathepsin K, forward 5′-CACTGCTCTCTTCAGGGCTT-3′ and reverse 5′-ACGGAGGCATTGACTCTGAA-3′; and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), forward 5′-TCAAGAAGGTGGTGAAGCAG-3′ and reverse 5′-AGTGGGAGTTGCTGTTGAAGT-3′. The mouse GAPDH gene was used as the internal control. The amplification parameters consisted of an initial denaturation step at 95°C for 5 min, followed by 40 cycles of denaturation at 95°C for 1 min, annealing at 60°C for 30 s, and extension at 72°C for 1 min. Expression data were analyzed using the 2-ΔΔCT method.
Western blot analyses
Whole-cell lysates were prepared using lysis buffer containing 50 mM Tris-HCl, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, 1 mM sodium fluoride, 1 mM sodium vanadate, 1% deoxycholate, and protease inhibitors as described previously . Equal amounts of protein (20 µg) were run on 8–10% SDS-PAGE gels and were transferred by electroblotting onto polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA). Non-specific interactions were blocked with 5% skim milk for 1 h, and the membranes were incubated for 2 h with the following primary antibodies: anti-p38, anti-phospho-p38, anti-JNK, anti-phospho-JNK, anti-Akt, anti-phospho-Akt, anti-ERK, anti-phospho-ERK, anti-IκB, anti-phospho-IκB, anti-p65-NF-κB, anti-phospho-p65-NF-κB (Cell Signaling Technology, Beverly, MA, USA); anti-c-Fos, anti-NFATc1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA); and β-actin (Sigma). The membranes were washed in tris-buffered saline contacting 0.1% Tween-20 and incubated for 1 h with horseradish peroxidase-conjugated sheep anti-mouse or donkey anti-rabbit immunoglobulin antibodies (Enzo Life Sciences, New York, USA). Specific signals were detected using the Western Chemiluminescent HRP substrate kit (Millipore).
To determine cytokine levels in CIA mice, serum was obtained on day 42. Serum RANKL, osteoprotegerin (OPG), TNF-α, IL-6, and IL-1β were measured using an enzyme-linked immunosorbent assay (ELISA) kit (R&D systems, Minneapolis, MN, USA) according to the manufacturer’s guidelines.
Each experiment was performed at least three times, and the data are expressed as the mean ± standard deviation (SD) or the mean ± standard error (SE). All data were analyzed by one-way ANOVA, followed by the multiple comparisons Tukey’s post-hoc test, using the Statistical Package for the Social Sciences software (SPSS; Korean version 14.0). A p-value less than 0.05 was considered statistivally significant.