Sch C, dimethylsulfoxide (DMSO) were obtained from Sigma-Aldrich (MO, USA). Sch C was dissolved in DMSO and stored at -20℃. Human IL-1β was purchased from R&D systems (MN, USA). Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were procured from Gibco (CA, USA). Antibodies against p65, p-p65, IκBα, p-IκBα, ERK1/2, p-ERK, p38, p-p38 were supplied by Wanlei Life Science (Shenyang, China). Antibodies against JNK, p-JNK were acquired from Wanlei Life Science (Shenyang, China).
Cell culture and treatment
The human chondrosarcoma cell line SW-1353 was purchased from the Shanghai Institute of Cell Biology (Shanghai, China), and it was cultured in 5% DMEM supplemented with 10% FBS, L-glutamine, and antibiotics (50 U/ml penicillin and 50 µg/ml streptomycin). Cells grown to 70–80% confluency were washed twice with phosphate buffered saline prior to the treatment. Cells were incubated with various concentrations of Sch C in the absence or presence of IL-1β (10 ng/ml, Sigma-Aldrich, MO, USA). Control group were added with vehicle DMSO in the experiment. Cell viability was detected with a cell counting kit-8 (CCK-8, Wanlei Life Science, China) after 24 h of incubation. Matrix metalloproteinase (MMP3) in cell supernatants was detected by ELISA kits. The absorbance at 450 nm was measured using a microplate reader (Bio-Rad, CA, USA).
NO and PGE2 Measurement
To detect the level of NO and PGE2, SW-1353 cell supernatants were harvested after 24 h treatment with IL-1β (10 ng/ml) with or without different concentrations of Sch C. Griess reaction was conducted to measure the NO level and PGE2 concentration was detected with an ELISA kit (Elabscience, China) following the manufacturer’s protocol.
The total protein was extracted from SW-1353 cells using ice-cold RIPA buffer containing protease inhibitor (Boster Biological Technology, China) and stored at − 80 °C. The protein concentrations were quantified using the BCA assay (Beyotime Institute of Biotechnology, Shanghai, China). Aliquots (40 µg) were separated on 10% SDS-PAGE and transferred to the equilibrated polyvinylidene difluoride (PVDF) membrane (Millipore, MA, USA), which were blocked in 5% fat-free milk or 5% BSA for 2 h at room temperature and incubated at 4 °C overnight using the primary antibodies. Subsequently, blots were washed three times and incubated with horse-radish peroxidase (HRP)-labeled secondary antibody for 2 h at room temperature. Finally, the immunoreactive bands were detected with the chemiluminescence (ECL) kits (Millipore, MA, USA) and an imaging system (Bio-Rad, CA, USA). β-actin was used as the loading control and representative bands were shown.
Adult male New Zealand white rabbits (6 months old and weighing 3 ± 0.5 kg) were obtained from the Jinan Jinfeng Laboratory Animal Co. Ltd. The animals were housed singly and allowed freely with water and food. All animals were allowed to acclimate to their environment for 7 days before the experiment. All procedures were performed in agreement with the provision of Chinese Experimental Animals Administration Legislation and approved by the Animal Ethics Committee of Drug Nonclinical Evaluation and Research Center.
The animals were randomly divided into sham group and model group. Then rabbit OA models were established by open surgery including anterior cruciate ligament transection (ACL-T) and partial medial meniscectomy . After 4 weeks, the animals that had undergone surgery were randomly divided into model group and Sch C group. After grouping, intra-articular injection with Sch C (50 µM) or vehicle (equal volume) was performed every 7 days. After 4 weeks, the animals were sacrificed, and the knee samples were fixed with 4% paraformaldehyde solution.
Measurement of inflammatory cytokines in the articular cavity flushing fluid
1 ml normal saline was injected into the articular cavity to flush, and the flushing solution was extracted, centrifuged at 2000 × g for 10 min, then the supernatant was absorbed and stored at -20 °C. The level of IL-1β and TNF-α in the articular cavity flushing fluid were measured according to the instructions of ELISA kits.
Articular cartilage histopathology
Animals were euthanized and operated distal femur condyles were harvested. The specimens were fixed with 10% formalin solution for 24 h and then decalcified for 24 h. All specimens were cut into sections of 4 µm thickness and stained with hematoxylin-eosin (HE) for morphological analysis. Histological evaluation of OA was performed by eight individuals using modified Mankin’s score .
Data analysis was performed using Graph Pad Prism 5.0. Data are presented as mean ± SD and statistically analyzed using one-way analysis of variance, followed by the Tukey’s test. P values of > 0.05 were considered statistically significant.