Materials
PEA (Levagen) was kindly provided by Gencor Pacific Ltd. Diclofenac sodium salt, MIA, 4',6-diamidino-2-phenylindole (DAPI), and hematoxylin and eosin (H&E) were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA), enzyme-linked immunosorbent assay (ELISA) kits for prostaglandin E2 (PGE2), leukotriene B4 (LTB4), tumor necrosis factor-α (TNF)-α, and interleukin (IL)-1β were purchased from R&D Systems (Minneapolis, MN, USA), fluorochrome-conjugated secondary antibodies (Alexa 488 and 564) were purchased from Thermo Fisher Scientific (Waltham, MA, USA), Safranin O was purchased from ScienCell Research Laboratories (Carlsbad, CA, USA), and anti-aggrecan and anti-collagen type II alpha 1 (COL2A1) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
Animals
All experiments using animals reported herein were performed according to protocols approved by the Institutional Animal Care and Use Committee of Hallym University (HallymR1 2018-75). Six-week-old male Sprague Dawley (SD) rats were purchased from DooYeol Biotech (Seoul, Korea). The rats were housed in specific pathogen-free conditions with a temperature of 23 ± 3 °C, relative humidity of 50 ± 10 %, 10–15 times ventilation, 150–200 lux illumination, and a 12 h light/dark cycle. The rats were fed a commercial non-purified rodent diet (Cargill Agri Purina, Seongnam, Republic of Korea) with free access to water.
Induction of MIA-induced OA in rats
After acclimation for 1 week, MIA-induced OA rat models were generated as described previously (Udo et al. 2016). Rats were anesthetized with 2 %–3 % isoflurane/N2O/O2 mixture vapor using an inhalation anesthesia apparatus and ventilator (Fluovac System, Harvard Apparatus, Holliston, MA, USA). After anesthetization, 3 mg of MIA in 50 µL saline was injected into the intra-articular joint of the right knee in all rats except those in the normal control group. Rats in the control group received an injection of saline instead of the MIA solution. One day after the injection of MIA, all rats were randomly divided into five groups: (1) normal control group (injected with saline + treated with phosphate-buffered saline (PBS; vehicle) termed NOR, n = 10); (2) control group (injected with MIA + treated with PBS, termed CON, n = 10), (3) 50 mg/kg body weight (BW)/day PEA-treated group (injected with MIA + treated with 50 mg of PEA/kg BW/day termed PEA50, n = 10); (4) 100 mg/kg BW/day PEA-treated group (injected with MIA + treated with 100 mg of PEA/kg BW/day termed PEA100, n = 10), and (5) positive control group (injected with MIA + treated with 6 mg of diclofenac/kg BW/day termed DiC, n = 10). The rats in each group were orally administered PEA or diclofenac once daily for 4 weeks. At the end of the experiment, isoflurane was used to anesthetize the animals and the thickness of both knees was measured with a digital caliper, and then blood samples were collected from the heart. The rats were sacrificed by carbon dioxide asphyxiation, and the knee joints and synovia were extracted for further analysis.
Biochemical analyses of serum
Serum levels of total cholesterol, triglyceride, glucose, blood urea nitrogen (BUN), aspartate aminotransferase (AST), and alanine aminotransferase (ALT) activities were measured using a blood chemistry autoanalyzer (KoneLab 20XT, Thermo Fisher Scientific, Vantaa, Finland).
Micro-computed tomography (micro-CT) analysis
To evaluate changes in the knee joint microarchitecture, the femorotibial joint was scanned with a micro-CT scanner (VivaCT 80, Scanco Medical AG, Brüttisellen, Switzerland) with a source voltage of 70 keV, current of 114 μA, and 20 μm isotropic resolution at the Chuncheon Center of the Korea Basic Science Institute. Scans were integrated into three-dimensional images, and the three-dimensional morphometric parameters were calculated using micro-CT scanner image analysis software provided with the micro-CT scanner. The bone surface/bone volume (BS/BV, %) of the subchondral bone in the femorotibial joint was analyzed to evaluate the degree of bone erosion. The bone volume fraction (bone volume/total volume, BV/TV, %), trabecular thickness (Tb.Th, mm), and trabecular number (Tb.N, 1/mm) of the metaphysis of the tibia were analyzed to evaluate structural changes caused by various treatments.
Histological analysis
The knee joints were fixed with 4 % paraformaldehyde, decalcified, embedded in paraffin, and cut into 5-µm-thick sections. The sectioned tissues were stained with safranin O and H&E. A light microscope (Axio Imager, Carl Zeiss Meditec AG, Jena, Germany) was used to observe the stained tissues. The randomly selected fields of the slides were photographed and examined in a blinded manner.
Measurement of nitric oxide (NO)
As an indicator of NO production in sera, the nitrite concentrations in sera were determined using the Griess Reagent System (Promega, Madison, WI, USA) in accordance with the manufacturer’s instructions.
ELISA
The levels of PGE2, LTB4, TNF-α, and IL-1β in sera were measured using ELISA kits according to the manufacturer’s instructions.
Immunofluorescence (IF) staining
Paraffin-embedded knee joint tissues were sectioned at a thickness of 5 µm, deparaffinized, and blocked using 5 % bovine serum albumin. The indicated antibodies and fluorochrome-conjugated secondary antibodies (Alexa 488 or 564) were used to perform IF staining. DAPI was used to counterstain the nuclei. The randomly selected fields of the slides were photographed at 400× magnification and examined in a blinded manner. The Axio Imager microscope and AxioVision Software (Carl Zeiss) were used to quantify the immune-positive cells.
Quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR)
Total RNA was extracted from the synovia of the knee joints using TRIzol Reagent (Invitrogen Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s instructions. A Micro-volume UV-Vis Spectrophotometer (BioSpec-nano, Shimadzu, Kyoto, Japan) was used to determine the content and purity of the total RNA. Total RNA (2 µg) was reverse transcribed to complementary single stranded DNA using the HyperScript RT master mix kit (GeneAll Biotechnology, Seoul, Korea). Real-time PCR was performed with Rotor-Gene SYBR Green PCR Kit (Qiagen, Valencia, CA, USA) and Rotor-Gene 3000 instrument (Corbett Research, Mortlake, Australia) according to the manufacturer’s instructions. Table 1 shows the nucleic acid sequences of the primers used in this study. The results were analyzed using the Rotor-Gene 6000 Series software program (Corbett Research, version 6). The relative expression levels of target genes were normalized to those of glyceraldehyde 3-phosphate dehydrogenase (Gapdh).
Statistical analysis
All data are expressed as the mean ± standard error of the mean of at least three independent experiments. GraphPad Prism 5.0 (GraphPad Software, San Diego, CA, USA) was used for statistical analysis. Differences between groups were analyzed by Student’s t-test or one-way analysis of variance, and statistical significance was set at P < 0.05.