Animals
All procedures in this study were approved by the Laboratory Animal Care and Use Committee of Nanfang Hospital, Southern Medical University, China. Male Sprague-Dawley rats (250–300 g) were housed (4 per cage) with food and water ad libitum, and were raised in standard conditions (temperature: 22°C, humidity: 55%, 12/12 h cycle).
Animal experimental design
The rats were randomly divided into the following three experimental groups: a sham-operated (Sham) group, a standard diet (SD) group, and a ketogenic diet (KD) group. SD was provided by the Experimental Animal Center of Nanfang Hospital, Southern Medical University, and KD with a ratio of carbohydrate and protein to fat of 1:4 was provided by Trophic Animal Feed High-Tech Co., Ltd., China, as previously described [13].
After being acclimated for 7 days, the rats underwent knee anterior cruciate ligament transaction (ACLT) together with partial medial meniscectomy (PMM) on the left knee joint to establish the model of OA in both the SD and KD groups, and surgery without damage to joint integrity in the Sham group.
The blood ketone βOHB concentration (mM) was measured by an electrochemical blood ketone monitor T-1 (Yicheng, China), and body weight was measured at different times.
Micro-computed tomography (CT) analysis
To investigate the microstructures of rat knee joints, after modeling for 8 weeks, the specimens were taken for a Micro-CT (µCT 80, Scanco Medical, AG, Switzerland) scan. All the joint samples were scanned at an isotropic voxel size of 15 µm, and the scanner was set at a voltage of 55 kV and a current of 145 µA. SkyScan NRecon software was used to reconstruct the images, and CTvox software was used to analyze the parameters of the tibial subchondral trabecular bone. The region of interest was defined as the whole subchondral bone compartment in the metaphysis of the tibia. Trabecular bone volume per tissue volume (BV/TV), trabecular number (Tb. N), trabecular thickness (Tb. Th), and trabecular separation (Tb. Sp) were calculated.
Analysis of the general view of the knee joint
The general view of the knee joint was examined after the animals were killed. Fibrillation, erosion and ulcer formation, and osteophyte formation of the knee joint were evaluated. The macroscopic score was performed with reference to the previous literature [14].
Histological and immunohistochemistry analysis
After decalcification with Ethylene Diamine Tetraacetic Acid for at least 4 weeks, the samples were embedded in paraffin, and sectioned sagittally into 4-µm slices. Then, the slices were stained with Safranin O-fast green and H&E. Three blinded reviewers evaluated the grade of degenerative changes using the Osteoarthritis Research Society International (OARSI) Scoring System. The content of glycosaminoglycan in the cartilage was evaluated by Alcian blue staining.
For immunohistochemical staining, slices were incubated in 3% hydrogen peroxide for 30 min, and then they were subjected to antigen retrieval. The specimens were incubated with primary antibodies against NLRP3 (1:100, Abcam, MA, USA), ASC (1:100, Abcam, MA, USA), Caspase-1 p20 (1:100, Bioworld Technology, MN, USA), IL-1β (1:100, Bioworld Technology, MN, USA), IL-18 (1:100, Bioworld Technology, MN, USA), MMP-13 (1:100, Abcam, MA, USA), and COL2 (1:100, Bioworld Technology, MN, USA) at 4°C overnight. After washing in PBS, the slides were incubated with goat anti-rabbit HRP-conjugated secondary antibody (Boster Corporation, Wuhan, China). Finally, reactions were developed using 3´3-diaminobenzidine. The stained sections were photographed with a light microscope (Leica DM4000 B, Leica Microsystems Inc., Germany), and then they were semi-quantitatively analyzed by Image-Pro Plus software. We repeatedly measured the optical density of the positively stained cells three times in randomly selected sections.
Enzyme-linked immunosorbent assay (ELISA)
After the end of the experiment, the rats were killed under overdose anesthesia. The joint cavities were flushed with 1 mL 0.9% sodium chloride solution and collected as joint fluid samples. The samples were centrifuged at 4000 r/min for 5 min, and the supernatant was extracted and stored in a refrigerator sealed at − 80°C. The expression levels of IL-1βand IL-18 in the rat articular fluid were detected by ELISA kits (CUSABIO, Wuhan, China). This experiment was performed in accordance with the manufacturer’s instructions.
Western blot (WB) analysis
The knee articular cartilage tissue was homogenized in ice-cold RIPA buffer with 1 mM PMSF, and protease and phosphatase inhibitors. The protein concentration of each sample was measured using the bicinconinic acid Protein Assay Kit. After being separated by 10% or 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis, the protein samples were transferred to polyvinylidene fluoride membranes (Millipore, MA, USA). The blots were blocked with 5% skim milk in Tris-buffered saline with 0.1% Tween 20 for 1 h at room temperature and then incubated with primary antibodies against β-actin (1:1000, Abcam, MA, USA), Caspase-1 p20 (1:1000, Bioworld Technology, MN, USA), ASC (1:1000, Abcam, MA, USA), and NLRP3 (1:500, Abcam, MA, USA) overnight at 4°C. The blots were then washed with TBS-T and incubated with the secondary antibody (1:5000; Bioworld Technology, MN, USA) at room temperature for 1 h. The blots were washed again with TBS-T, and they were visualized with an enhanced chemiluminescence reagent (Millipore).
Statistical analysis
All experiments were repeated at least three times. Statistical analyses were performed with SPSS 20.0 (IBM, NY, USA) software. One-way analysis of variance (ANOVA) with the least significant difference post-hoc test was used to detect differences among multiple groups. Multivariate analysis of variance (MANOVA) with repeated measures was used to detect differences among groups at different time points. A P value lower than 0.05 was considered statistically significant.