2.1 Ethics statement
Animal welfare and experimental procedures were carried out in accordance with the Declaration of Helsinke, and were approved by the Ethics Committee of the First Affiliated Hospital of Soochow University.
2.2 Compounds and reagents
AKBA (CAS No.: 67416-61-9, purity: 99.71%) was purchased from MCE (Shanghai, China). N-3-oxo-dodecanoyl-L-Homoserine lactone (3-O-C12-HSL, Cas No.:168982-69-2, purity: 98.00%) was purchased from Apexbio (Houston, USA).
2.3 Animals and experimental design in vivo
Approximately 24 eight-week-old Sprague Dawley (SD) male rats were raised in a standard environment and were randomly kept in cages, where water and fodder supply were sufficient. The Hulth-Telhag OA model was established. After one week of adaption feeding, SD rats were randomly divided into four groups: control, vehicle, low-AKBA, and high-AKBA groups (n = 6/group). Except for the control group, the rats in the other three groups received chloral hydrate (1 mg/kg, intraperitoneally) for anesthesia. After anesthesia, both lower extremity operation area of the rat was shaved, followed by repeated penicillin G (80000 U/rat) intramuscular administration for 3 days. The operation area was disinfected per the aseptic operation regulations, the anterior medial longitudinal skin incision was taken, the knee joint was exposed, and the medial side was cut off. The medial collateral ligament, the anterior and posterior cruciate ligaments, and the medial meniscus were removed. The articular cartilage surface was protected during the operation. The joint cavity and epidermis were sutured layer by layer using a 3-0 silk thread. The affected limb was not fixed after the operation. The experimental animals were returned to the animal room to continue feeding. One week after modelling, the rats were administered with 0.5 ml/kg saline, as well as 2 and 8 mg/kg AKBA solution by gavage every other day. All experimental animals were sacrificed after 6 weeks and samples were taken for analysis.
2.4 Micro-computed tomography (micro-CT) and reconstruction
The rats were sacrificed after 6 weeks by intraperitoneal injection of excess pentobarbital sodium, and the knee joint (n = 6/group) were collected and fixed with 10% paraformaldehyde. A SkyScan 1176 CT machine (BRUKER, Belgium) was used for CT. The scanning parameters were 60 kV source voltage and 170 µA source current, and the resolution was set at 9 µm. The 3D reconstruction used the procedure provided by the factory. Bone trabecular bone density (Tb. BMD, mg/cm3), bone volume fraction (BV/TV, %), number of trabecular bone (Tb. N, mm−1), the thickness of trabecular bone (Tb. Th, µm), trabecular bone separation (Tb. Sp, mm), and connection density (Conn. D, mm−1) were analyzed with SkyScan software.
2.5 Histologic analysis
For decalcification, samples from each group were placed into 10% ethylenediaminetetraacetic acid (EDTA) for 4 weeks, paraffin-embedded samples were cut into 6-µm slices and staining procedures, including hematoxylin and eosin (H&E), safranin O-fast green, and tartrate-resistant acid phosphatase (TRAP) staining, were performed according to the manufacturer’s protocols. An AxioCam HRc microscope (Carl Zeiss, Jena, Germany) was used to capture the images. Morphological scores of H&E and safranin O-fast green staining were given to sections under a micro-imaging system according to the International Academy of Osteoarthritis Research (OARSI) scores, and three readers were arranged for each section to obtain the average as the final score. After TRAP staining was completed, 5 fields of vision under the cartilage or above the tide line were randomly selected, the number of TRAP-positive cells, and the proportion of osteoclast surface (OcS/BS, %) were counted using the BIOQUANT OSTEO (BIOQUANT, USA).
2.6 Immunohistochemical (IHC) analysis
Expression levels of IL-1β, IL-6, TNF-α, MMP13, col Ⅰ, col Ⅱ, IKKα/β, IκBα, and p65 in the knee joint was examined using IHC analysis with corresponding primary antibodies (Abcam, ab9722, ab9324, ab9739, ab39012, ab34712, ab39012, ab178870, ab32518, and ab16502). Briefly, the sections were dewaxed for 1 h with xylene and then subjected to gradient hydration, antigen retrieval with hyaluronidase (1 h, 37°C), and pepsin (25 min, RT). Next, the sample sections were blocked with bovine serum albumin (BSA, 1 h, room temperature (RT)), followed by incubation with correspondent primary antibodies (12 h, 4°C). After washing with PBS, sections were incubated with secondary antibodies (30 min, RT). The chromogenic reaction was induced by a DAB horseradish peroxidase color development kit (Beyotime, Shanghai, China). In the semi-quantitative analysis, positive cell counts were randomly selected under the microscope to count positive cells and non-positive cells, and the proportion of positive cells was obtained. The average number was used as the proportion of positive cells in the section. The average optical density analysis was completed with Image Pro-Plus 6.0 software, and uniform white balance and exposure time settings used in the screenshot were adopted. Because col II is only concentrated on the surface of the cartilage, the only semi-quantitative analysis was performed on the surface of the cartilage.
2.7 Cell culture
Rat primary articular chondrocytes (Cat NO.: CP-R092) was purchased from Procell (Wuhan, China). After digestion by 0.25% trypsin, chondrocytes were cultured in F-12 medium mixed with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin in a regular incubator (37°C, 5% CO2). The medium was changed every 3 days. Cells subculturing was performed when the cells reached 80-90% confluency after washing and digestion. Cells were then treated with AKBA in low (2 µM) or high (8 µM) concentrations.
2.8 Cell viability assay
Chondrocytes were seeded (5 × 103/well in 0.5 ml of F-12 medium) in 96-well plates and cultured with a range of concentrations of AKBA (0, 2, 4, 8, 16, 32, 64, and 128 µM) for 24, 48, and 72 h after adherence. After reaching the corresponding time point, the PBS-washed cells were incubated with a cell counting kit-8 (CCK-8) (Dojindo, Shanghai, China) (3h, 37°C) and the optical density (OD) of chondrocytes at 450 nm was determined. The cell viability to control was calculated using the equation: Cell viability to control (%) = OD drug-treated group/OD control group.
2.9 Western blot analysis
After treatment, cell proteins were extracted using radioimmunoprecipitation assay (RIPA) buffer with protease inhibitors and phosphatase inhibitors. The protein concentration was determined by a nucleic acid-protein quantometer (Thermo Scientific, Shanghai, China), and then separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The resolved proteins were transferred to a polyvinylidene difluoride (PVDF) membrane and blocked with blocking solution (1 h, RT). Next, PVDF membranes were incubated with corresponding primary antibodies (overnight, 4°C), followed by incubation with the secondary antibody (1 h, 4°C). The reactive protein bands were detected by the Western blot detection kit. Image analysis was performed using Image J software.
2.10 Quantitative real-time (qRT)-PCR
After treatment, total RNA was extracted using Beyozol reagent (Beyotime, Shanghai, China). The quality of RNA was confirmed by measuring the absorbance of the A260/A280 and A260/A230 ratio in the range of 1.8-2.0, respectively. cDNA was synthesized with MonScript™ RTIII Super Mix with dsDNase (Monad, Wuhan, China) following the manufacturer’s protocols. Subsequently, MonAmp™ ChemoHS qPCR Mix (Monad, Wuhan, China) was used to perform qRT-PCR. The parameters were as follows: 95.0°C for 10 min, 40 cycles of 95.0°C for 10 s, 60.0°C for 10 s, and 72.0°C for 10 s. All samples were analyzed in triplicate and three independent experiments. The comparative 2−ΔΔCq method was used for analysis. GraphPad Prism 8 software was used to perform statistical analysis and draw graphs. The primer sequences are shown in Table 1.
2.11 Immunofluorescence (IF) assay
Chondrocytes climbing slices were incubated with or without LPS (1 µg/ml) for 24 h, and then co-incubated with or without AKBA (1 µM) for 24 h. PBS-washed slices were fixed in 4% paraformaldehyde solution (15 min, RT), washed thrice with PBS, and then treated with 0.1% TritonX-100 (5 min, RT). Climbing slices were then shifted into a wet box, blocked with blocking solution (1 h, 37°C), and then incubated with primary antibodies (MMP13 (1:200) and p-p65 (1: 200)) diluted with their dilutions for 12 h at 4°C. After incubating with iFluor™ 488 goat anti-rabbit IgG antibody (1:1000, AAT Bioquest, Calif, USA) in darkness for 1 h, DAPI (4′,6-diamidino-2-phenylindole) was used to stain the cell nuclei. The slices were dried and sealed by an anti-fluorescence quenching agent and imaged using confocal microscopy (Carl Zeiss AG, Jena, Germany) after aspirating the liquid from the climbing slices.
2.12 Statistical analysis
The quantitative data were expressed as the means ± standard deviation (SD). GraphPad Prism 8 (GraphPad Software Inc, USA) was utilized for data analysis and statistical mapping. The two samples were compared using an independent sample t-test. A P-value <0.05 was considered statistically significant.