We used chemically synthesized 2-carba-cPA 18:1 (2ccPA)
[4, 7]. In the in vivo experiments, 2ccPA was dissolved in saline, and saline was used as vehicle. In the in vitro experiments, 2ccPA was dissolved in phosphate-buffered saline (PBS) containing 0.1% fatty acid-free bovine serum albumin (BSA), and PBS containing 0.1% BSA solution was used as vehicle.
A rabbit model was used to investigate the effects of 2ccPA on the pathogenesis of OA. The design of this animal study was approved by the ethics committee of KAC Corporation (Ethics approval number: 12–0218), a contract research organization (Shiga Japan). All animals were purchased from KITAYAMA LABES Co., Ltd. (Japan), and all animal experiments were performed by KAC Corporation using 11- or 12-week-old male SPF New Zealand white rabbits (n = 12, body weight 2.1–2.3 kg). Animals were anesthetized with intravenous (i.v.) pentobarbital (32.4 mg/kg) prior to 1–4% isoflurane followed by subcutaneous infusion of lidocaine (approx. 3 mL) during surgery.
The meniscus of the right leg was totally removed. Briefly, the boundary between the patellar ligament and articular capsule of the right hind leg and the lateral-collateral ligament were dissected. Then, the articular capsule was removed to expose the interior meniscus, and the meniscus was completely removed. Following total meniscectomy of the right knee joint, the rabbits were randomly divided into vehicle- or 2ccPA-treated group. Intra-articular treatment was initiated 7 days after surgery. Vehicle (200 μL saline; Otsuka Pharmaceutical Co., Ltd., Tokyo, Japan) or 50 μg/mL 2ccPA (200 μL) was injected into the joint cavity twice per week over five consecutive weeks (at 7, 11, 14, 18, 21, 25, 28, 32, 35, and 39 days after surgery). The animals were individually caged (48.5 × 30 × 35 cm3), received tap water ad libitum, and were fed a standard diet (CR-3, 150 g/day; CLEA, Japan Inc.) throughout the trials. During the experiments, the SPF room temperature was 18°C with unidirectional airflow systems; lighting was provided for 12 h daily (7:00 AM–19:00 PM). The experimental schedule is shown in Figure
Measurement of pain
Changes in hind paw weight distribution between the right (OA model) and left (contralateral control) limbs were measured as a pain index
]. Hind-paw weight distribution was measured once a week (at 7, 14, 21, 28, 32, and 35 days after surgery). The percentage of weight distribution of the right hind paw was calculated with the following equation:
Measurement of swelling
At 42 days after meniscectomy, articular swelling was measured with a digital vernier caliper. The maximum widths of the right and left hind paw were measured and recorded. The percentage of swelling was calculated as follows:
Histopathological assessment of OA
At 42 days after meniscectomy, the rabbits were euthanized by exsanguination immediately after pentobarbital (32.4 mg/kg) administration. The femoral condyle and tibial plateau were resected and immediately fixed in 10% formalin buffer. After decalcification with ethylenediaminetetraacetic acid (EDTA), the samples were cut into 4-μm sections, then stained with HE for general morphology, or Saf-O for proteoglycan, and were observed using a 2.0 MP microscope (H-Micron, Hyogo, Japan, Figure
3A) and an optical microscope BX51TF (OLYMPUS, Tokyo, Japan, Figure
3C2). Images of 3–6 microscopic fields were incorporated into one image for Figure
Cell culture and measurement of hyaluronic acid, IL-6, and MMP-1, -3, and -13 produced by synoviocytes and chondrosarcoma SW1353 cells
All procedures were specifically approved by the ethics committee of Ochanomizu University (Ethics approval number: 24–12) and the National Institute of Biomedical Innovation, Japanese Collection of Research Bioresources Cell Bank (previously Health Science Research Resources Bank, Ethics approval number: 36); the patient gave full written informed consent for tissue donation. Synovial tissue was excised from the knee joint of a 60-year-old female patient with OA during replacement surgery. The patient-derived synoviocytes (Japanese Collection of Research Bioresources Cell Bank, HT91989516, Lot. 07042011) were plated at 1.5 × 104 cells/well with Dulbecco’s modified Eagle medium (DMEM) containing 10% fetal bovine serum (FBS, Life Technologies Corporation, CB) on 12-well plates and incubated overnight at 37°C in humidified 95% air and 5% CO2 atmosphere. Chondrosarcoma SW1353 cells were obtained from American Type Culture Collection (ATCC) (no. HTB-94) and were plated at 1.5 × 104 cells/well with DMEM containing 10% FBS on 12-well plates and incubated overnight as well as synoviocytes. The medium was replaced with serum-free DMEM, and the cells were serum-starved for 16 h. To measure hyaluronic acid, 2ccPA was added after serum starvation at final concentrations of 1, 3, and 10 μM and incubated. The culture media were collected at 24, 48, and 72 h, then the concentration of hyaluronic acid was measured by ELISA (R&D Systems, Inc. MN).
To measure IL-6 and MMPs, the cells were treated with either 10 μM of ibuprofen (IBF), diclofenac sodium (DCF) (Wako Pure Chemical Industries, Ltd. Osaka, Japan), or various concentrations of 2ccPA for 24 h in the presence of 10 ng/mL of IL-1β (R&D Systems, Inc.). Culture media were collected at 24 h and the concentrations of IL-6 and MMPs were measured by ELISA kit according to the manufacturer’s instructions (RayBiotech, Inc., GA). For treatment with Ki16425 (Cayman Chemicals, MI), the selective antagonist for LPA1R and LPA3R, the cells were plated at 1.5 × 104 cells/well on 12-well plates and incubated for 30 min with 10 μM of Ki16425 before adding 2ccPA (1, 3, or 10 μM) in the presence of 10 ng/mL IL-1β. Culture media were collected at 24 h and the concentration of each MMP was measured by ELISA kit.
Quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR)
To quantitate the mRNA levels of inflammatory cytokines (IL-1β, IL-6, IL-8, TNF-α), MMPs (MMP-1, -3, -13), and LPA receptors, real-time RT-PCR was performed with SYBR Premix Ex Taq (Takara Bio, Inc., Shiga, Japan). Total RNA was extracted from cultured synoviocytes and SW1353 cells using ISOGEN reagent (Nippon Gene, Tokyo, Japan) according to the manufacturer’s instructions. cDNA was synthesized with the PrimeScript RT reagent kit (Takara Bio, Inc.). mRNA levels were quantified on a lightCycler 96 system (Roche) instrument. Gene-specific primer sets for IL-1β, IL-6, IL-8, MMP-3, and -13 were described in
. Gene-specific primer sets for TNF-α and MMP-1 were described in
. Gene-specific primer sets for LPA1R, LPA4R, LPA5R, LPA6R, and p2y10 were described in
. The following primer sets were used: GAPDH, 5′-GTGAAGGTCGGAGTCAACG-3′ (F) and 5′-TGAGGTCAATGAAGGGGTC-3′ (R); LPA2R, 5′-GAGGCCAACTCACTGGTCA-3′ (F) and 5′-GGCGCATCTCAGCATCTC-3′ (R); LPA3R, 5′-GAAGCTAATGAAGACGGTGATGA-3′ (F) and 5′-AGCAGGAACCACCTTTTCAC-3′ (R); and GPR87, 5′-AAATCCAGCAGGCAATTCAT-3′ (F) and 5′-CCCTGATGCTCTGGTTATGTT-3′ (R).
The data were calculated based on the Cq values, and the expression of each gene was normalized to GAPDH.
All values are reported as means ± standard error. The data were analyzed using the Student’s t-test. A P value less than 0.05 was considered statistically significant.