Cell and Materials
Normal human chondrocyte were purchased from iCell Bioscience Inc(Shanghai, CHINA). AGEs were purchased from BioVision Inc.(Milpitas CA, USA ).Curcumin, the selective AMPK activator AICAR and AMPK inhibitor Compound C were purchased from Sigma (St Louis, MO, USA). Recombinant human MMP-3, MMP-13 quantikine enzyme-linked immunosorbent assay (ELISA) kits were purchased from R&D Systems, Inc. (Minneapolis, MN). Human small interfering RNAs (siRNAs) for AMPKa, PGC-1α and the control siRNA were from Invitrogen. JC-1, ATP production assay kit and NO release assay kit were from Beyotime Institute of Biotechnology, China. All other reagents were of highest purity available.
Rabbit cartilage explants preparation
Rabbit articular cartilage from the metacarpophalangeal joints of 5-week-old male rabbits was dissected into 25 cm3 discs. The cartilage was then incubated in DMEM(supplemented with 15% fetal bovine serum and 1% penicillin-streptomycin) for 30 min at 37°C and 5% CO2 in a petri dish. After that, the cartilage was incubated in refreshed medium for 24h to ensure sterility. Then 30-35 mg portions of cartilage were placed into wells of 24 well-plates and cultured at 37°C and 5% CO2 in DMEM. The study was approved by the Ethics Committee of Gan Su Province Hospital(No.2020092).
Hematoxylin and eosin (H&E)
Cartilage samples were fxed in 4% paraformaldehyde and embedded in wax, then were then cut into 5 mm thick sections perpendicular to the articular cartilage surface. The sections were evaluated for tissue morphology by staining with Hematoxylin and eosin (H&E).
ATP Bioluminescence Assay
The ATP levels were evaluated using an ATP bioluminescence assay kit. This technique is well established and uses the ATP dependence of the light omitting luciferase-catalyzed oxidation of luciferin for the measurement of extremely low concentrations of ATP.
Determination of mitochondrial membrane potential
Mitochondrial membrane potential was assessed by using JC-1. The decline in mitochondrial membrane potential will lead to leakage of JC-1 from mitochondria. JC-1was added to cell cultures for 30 min at room temperature, and then the cells were washed twice with phosphate-buffered saline. The ﬂuorescence of JC-1 was observed using a confocal laser scanning microscope (Leica TCS SP2, German) with excitation at 488nm and emission at 510nm.
Measurement of the release of NO, MMP-3，MMP-13 and ROS production
Levels of NO and MMP-3 and MMP-13 in conditioned media were assayed using the Griess reaction method and ELISA, respectively. The determination of ROS was based on the oxidation of 2’, 7’-dichlorofluorescein diacetate (DCFH-DA) by peroxide. In brief, cells were washed and incubated with DCFH-DA for 20 min at 37 °C in the dark. Cells were then washed twice and harvested in PBS. The fluorescence of DCFH was detected with a flow cytometer (FAC-SCalibur; BD Biosciences, San Jose, CA, USA) with excitation at 488 nm and emission at 530 nm.
SiRNA of PGC-1α and AMPKα in chondrocytes
Small interfering RNA (siRNA) construction and transfection PGC-1α and AMPKα siRNA assays were performed using Silencer Select Predesigned siRNA. Chondrocytes were grown to 30% confluence before transfection, according to the manufacturer’s protocol. Transfection complexes were prepared in Opti-MEM serum-free medium by mixing 1.5 mL of Oligofectamine and 2 mM of siRNA. Forty-eight hours after siRNA transfection, cells were subjected to AGEs or curcumin, and subsequently analyzed for the expression of the related indexes. Level of expression of PGC-1α or AMPKα was examined by western blot analysis. Densitometry was performed using the Image J program (National Institutes of Health).
Western Blot Analysis
After the indicated treatments, cell extracts were prepared in phosphate-buffered saline that contained 25 μl of protease inhibitor cocktail. Aliquots of the cell extract were separated by sodium dodecyl sulfate polyacrylamide gel electro-phoresis, and western blot analyses were carried out using the indicated antibodies. Antibody binding was detected by enhanced chemiluminescence. The bands were scanned and densitometrically analyzed using an automatic image analysis system (Alpha Innotech Corp., San Leandro, Calif., USA). These quantitative analyses were normalized to GAPDH.
All values are expressed as mean ± SD. Data were analyzed using a one-way or two-way ANOVA followed by Newman-Student’s t-test. P<0.05 was considered significant.