Animals
We maintained 7-week-old male DBA/1J mice (Orient Bio, Gyeonggi-do, Korea) under specific pathogen-free conditions. The mice were fed standard laboratory mouse chow (Ralston Purina, St. Louis, MO, USA) and water ad libitum; they were housed (five mice per cage) in a room under controlled temperature (21–22°C) and lighting (12-h light/12-h dark cycle) conditions. All experimental procedures were approved by the Institutional Animal Care and Use Committee at the School of Medicine, Animal Research Ethics Committee of the Catholic University of Korea and were conducted in accordance with the Laboratory Animals Welfare Act according to the Guide for the Care and Use of Laboratory Animals.
Formation of LGNP-CoQ10
A chloroform solution containing 500 μg saturated neutral phospholipids (1,2-distearoyl-sn-glycero-3-phosphocholine [DSPC]) was dropped into a glass vial. Next, a lipid film was formed by evaporating the chloroform solution under an N2 stream. The film was maintained in a vacuum for longer than 1 h and then hydrated at 55°C by adding 0.5 mL ascorbic acid solution (C6H8O6, 600 mM, pH 5) and 0.5 mL CoQ10 solution. To obtain unilamellar liposomes, as-prepared multilamellar liposomes were extruded through a polycarbonate membrane (pore size: 100 nm) 20 times at 60°C using a mini-extruder. Unilamellar liposomes were purified by centrifugation and washed three times with deionized water to remove residues. Finally, the liposomes encoded with ascorbic acid and CoQ10 were re-suspended using a gold precursor aqueous solution (HAuCl4∙3H2O, tetrachloroauric acid trihydrate, 200 μM) and kept overnight in an agitating incubator at room temperature [24]. The LGNP-CoQ10 structure is shown in Figure 1A.
Arthritis induction and treatment
CIA was induced in DBA1/J mice (n = 5). The experiment was performed in triplicate. Type II collagen (CII) was dissolved overnight in 0.1 N acetic acid (4 mg/mL) with gentle rotation at 4°C. Male DBA/1J mice were immunized intra-dermally at the base of the tail with 100 µg chicken CII (Chondrex, Inc., Remosa, WA, USA) in complete Freund’s adjuvant (Chondrex Inc.). In experiments conducted to investigate preventive effects, mice were boosted with 100 µg CII emulsified with incomplete Freund’s adjuvant (Chondrex Inc.), injected intradermally at the base of the tail on day 17 after primary immunization. These arthritis model mice were treated orally with Gold Lipo CoQ10 (0.1 mg/mouse) or CoQ10 (0.1 mg/mouse) starting on day 17 after the first immunization. The mice were examined visually twice weekly for the appearance of arthritis in the peripheral joints. All mice were sacrificed on week 10 for histological analyses of splenocytes and determination of protein expression.
Clinical assessment of arthritis
Arthritis severity was recorded using the mean arthritis index on a scale of 0 to 4, as follows: (0) no evidence of erythema or swelling; (1) erythema and mild swelling confined to the midfoot (tarsals) or ankle joint; (2) erythema and mild swelling extending from the ankle to the midfoot; (3) erythema and moderate swelling extending from the ankle to the metatarsal joints; and (4) erythema and severe swelling encompassing the ankle, foot, and digits. Arthritis severity was determined as the sum of the scores of all legs, as assessed by two independent observers with no knowledge of the experimental groups.
Histological analyses
Mouse joint tissues were fixed in 4% paraformaldehyde (Sigma-Aldrich, St. Louis, MO, USA), decalcified in histological decalcifying agent (Calci-Clear Rapid; National Diagnostics, Atlanta, GA, USA), trimmed, and embedded in paraffin wax. Sections (7 μm) were prepared and stained with hematoxylin (YD Diagnostics, Yongin, Korea), eosin (Muto Pure Chemicals Co., Ltd., Tokyo, Japan), and Safranin O (Sigma-Aldrich). Cartilage damage was scored as described previously [25].
Immunohistopathological analyses
Joint tissues were first incubated with primary antibodies against IL-1β (R & D Systems, Minneapolis, MN, USA), IL-6 (R & D Systems), TNF-α (R & D Systems), IL-17 (R & D Systems), and RANK ligand (RANKL; R & D Systems) overnight at 4°C. Samples were incubated with a biotinylated secondary antibody, followed by incubation with a streptavidin–peroxidase complex for 1 h. Samples were then developed using chromogen 3,3′-diaminobenzidine (Thermo Scientific, Rockford, IL, USA). The sections were examined using a photomicroscope (Olympus, Tokyo, Japan).
Confocal microscopy of immunostaining
Spleen tissues were obtained at 10 weeks after the first immunization. Tissues were snap-frozen in liquid nitrogen and stored at –80°C. Tissue sections were fixed in 4% paraformaldehyde and stained with phycoerythrin (PE)-conjugated anti-CD4, fluorescein isothiocyanate (FITC)-conjugated anti-IL-17, FITC-conjugated anti-pSTAT3 705, and FITC-conjugated anti-pSTAT3 727 (all from eBiosciences, San Diego, CA, USA). Stained sections were analyzed microscopically (LSM510Meta; Carl Zeiss, Oberkochen, Germany).
Cell culture
Splenocytes were prepared from the spleens of normal C57NL/6 mice. Splenocytes were maintained in RPMI1640 medium supplemented with 5% fetal bovine serum (Gibco, Grand Island, NY, USA) and stimulated with anti-CD3 (0.5 µg/mL; BD Biosciences, San Jose, CA, USA) for 3 days and subjected to enzyme-linked immunosorbent assay (ELISA).
Statistical analyses
Statistical analyses were conducted using the nonparametric Mann–Whitney U test for comparisons between two groups, and one-way analysis of variance (ANOVA) with Bonferroni’s post hoc test for multiple comparisons. We used the GraphPad Prism v. 5.01 software (GraphPad Software Inc., San Diego, CA, USA) for all analyses. The threshold for statistical significance was P < 0.05. All data are presented as means ± standard deviation (SD).