Animals
All animal experiments were approved by the Southern Medical University Animal Care and Use Committee (SMUL2021014). 10-week-old male C57/BL6 mice (23–30g) were purchased from the Laboratory Animal Centre of Southern Medical University (No.44002100018098).
Mouse model
10-week-old male C57/BL6 mice (n=48) were subjected to intra-articular injection collagenase to induce CIOA as previously described.[29] Briefly, 1 U of collagenase (C0773; Sigma-Aldrich, St. Louis, MO, USA) was injected into the right knee joint twice on alternate days. Only skin of the right knee joint was resected in the sham-operated group. Some mice from the CIOA group were treated with fargesin (5, 10, or 20 mg/kg, 10ul, n=36; PHL82537, Sigma, USA), while others were treated with vehicle (20% DMSO dissolved in saline) by intra-articular injection twice a week for 1, 3, or 6 weeks (10ul, n=12). 1, 3, or 6 weeks after operation, mice from each group were sacrificed for collection of the right knee joint. (Additional file 1: figure S1A) Articular cartilage degeneration was quantified using the Osteoarthritis Research Society International (OARSI) scoring system. H&E were used to evaluate synovial activation by scoring synovial lining cell thickness (0-3), as previously described.[29]. Then the sum of medial and lateral compartments of the joint is presented (0-6).
Cells
Raw264.7 macrophages from the American Type Culture Collection (ATCC, USA) were grown in Dulbecco’s Modified Eagle’s Medium (DMEM) with high glucose (4.5 g/L; Gibco, USA), containing 100 U/mL penicillin, 100 mg/mL streptomycin sulfate (Life Technologies, USA), and 10% FBS (Gibco, USA). The pre-chondrocyte cell line ATDC5 (Tsukuba, Japan) was maintained in DMEM/F12 (Gibco, USA), containing 100 U/mL penicillin, 100 mg/mL streptomycin sulfate, 5% FBS and 1x ITS universal culture supplement premix reagent (BD Biosciences).
Drug treatment
Raw264.7 cells were treated with lipopolysaccharides (LPS) (100 ng/mL; Peprotech, USA) for 24 h to induce M1-like macrophages and IL-4 (20 ng/mL; Peprotech, USA) for 24 h to induce M2-like macrophages. Fargesin (10, 20, and 40 µM) was administered in Raw264.7 cells for 24 h. Cells were administered with 0.1% DMSO served as the control in vitro. Media samples were then collected and kept at -20°C until further analysis for cytokine determination.
Primary chondrocyte culture
We dissected rib cartilage from newborn mice (24–72 h) under a stereo light microscope to harvest primary chondrocytes. After trypsin digestion for 30 min, primary chondrocytes were separated and purified, and digested in 0.1% collagenase type II (Sigma, USA) with 10% FBS, 100 U/ml penicillin, and 100 mg/ml streptomycin sulfate at 37°C for 4–6 h. Primary chondrocytes were resuspended and seeded in a 24-well plate and cultured in DMEM/F12 with 10% FBS, 100 U/ml penicillin, and 100 mg/ml streptomycin sulfate at 37°C with 5% CO2.
Co-culture
Raw264.7 cells were treated with LPS (100 ng/mL) for 24 h to induce M1-like macrophages. Vehicle or Fargesin (20µM) was administered in Raw264.7 cells induced LPS for 24 h. Surperanants were collected, then co-cultured with ATDC5 for 24h in a 24-well plate. Moreover, ATDC5 cells were treated with IL-1β (100 ng/mL) for 24 h to induce OA-like cells. Vehicle or Fargesin (20µM) was administered in ATDC5 cells induced IL-1βfor 24 h. Surperanants were collected, then co-cultured with Raw264.7 for 24h in a 24-well plate.
ELISA
Serum and cell supernatants were analyzed using mouse IL-6, IL-10, and IL-1β ELISA kit (#E-EL-M0044c, #E-EL-M0046c, #E-EL-M0037c; Elabscience Biotechnology). ELISA analysis was performed according to the manufacturers’ instructions.
Quantitative reverse transcription-polymerase chain reaction
Total RNA was extracted from tissues or cultured cells using TRIzol reagent (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA), as previously described[24]. cDNA was reverse transcribed using TaKaRa reverse transcription reagents (TaKaRa Bio Inc., Shiga, Japan) and PCR was performed using Real-Time PCR Mix (TaKaRa) on a light cycler (Roche, Basel, Switzerland) with the following primers: ColX (forward primer 5’-AAA GCT TAC CCA GCA GTA GG-3’ and reverse primer 5’-ACG TAC TCA GAG GAG TAG AG-3’), MMP13 (forward primer 5’- CTT CTT CTT GTT GAG CTG GA CTC-3’ and reverse primer 5’- CTG TGG AGG TCA CTG TAG ACT-3’), Runx2 (forward primer 5’-TCC CCG GGA ACC AAG AAG GCA-3’ and reverse primer 5’-AGG GAG GGC CGT GGG TTC TG-3’), GAPDH (forward primer 5’- AGG TCG GTG TGA ACG GAT TTG-3’ and reverse primer 5’- TGT AGA CCA TGT AGT TGA GGT CA-3’). The glyceraldehyde 3-phosphate dehydrogenase gene was used as an endogenous control to normalize for differences in the amount of total RNA.
Preparation of decalcified sections, histochemistry, immunostaining, and immunohistochemistry
Freshly dissected mouse knee joints were fixed in 4% paraformaldehyde for 24 h at 4°C and decalcified in 14% EDTA (pH 7.4) for 30 days at 25°C. Tissues were embedded in paraffin and sectioned continuously (3-μm thick). Safranin-O/Fast Green staining was performed as previously described.[24] For immunohistochemistry and immunofluorescence, sections were soaked in citrate buffer (10 mM citric acid, pH 6.0) for 16 h at 62°C or treated with 0.1 mg/mL proteinase K (Sigma-Aldrich) for 15 min at 37°C to unmask antigens after deparaffinization and rehydration. For immunohistochemistry, 3% hydrogen peroxide solution was added for 15 min. Sections were blocked with 10% sheep serum at 37°C for 1–2 h and incubated with primary antibodies (in 1% BSA, 0.1% Triton X-100) at 4°C overnight. Sections were then incubated with secondary antibodies at 37°C for 1 h. Furthermore, 3,3'-diaminobenzidine was used to observe chromogen and hematoxylin during counterstaining. For immunofluorescence, species-matched antibodies labeled with Alexa Fluor 488 and 594 or horseradish peroxidase (HRP) were used (1:100 in 1% BSA) as previously described.(24) Nuclei were labeled with 4',6-diamidino-2-phenylindole (Thermo) before imaging.
Western blotting
Lysis buffer was prepared with 10% glycerol, 2% sodium dodecyl sulfate, 10 mM dithiothreitol, 10 mM Tris–HCl (pH 6.8), 1 mM phenylmethylsulfonyl fluoride, and 10% β-mercaptoethanol. Tissues and cells were lysed at 98°C for 10 min. Samples were separated with SDS-PAGE for 90 min, blotted onto nitrocellulose membranes for 1 h, and blocked with 5% milk at 37°C for 1–2 h. Then, membranes were incubated with primary antibodies (in 5% BSA, 0.2% NaN3) at 4°C overnight. Samples were incubated with secondary antibodies at 37°C for 1 h.
Antibodies
The following antibodies were used in this study: rabbit anti-Col X [1:100 for immunohistochemistry (IHC); Abcam, USA; ab58632], rabbit anti-MMP-13 (1:100 for IHC; Abcam, USA; ab39012), rabbit anti-RUNX2 (1:100 for IHC; Abclone, Australia; A2851) , rabbit anti-Mannose Receptor (1:100 for IF; Acam, USA; ab64693), rabbit anti-iNOS (1:100 for IF; Abclone, Australia; A3200), anti-rabbit IgG light chain (1:100 for IHC; Abbkine, USA; A25022), HRP-labeled goat anti-rabbit IgG H&L (1:1,000 for western blots, 1:100 for IHC; Jackson Immuno Research, USA; 111-035-003), HRP-labeled goat anti-mouse IgG H&L (1:3,000 for western blots; Jackson Immuno Research; 115-035-003), Alexa Fluor 594-labeled goat anti-mouse IgG H&L [1:500 for immunofluorescent labeling (I); Abcam; ab150120], and Alexa Fluor 488-labeled goat anti-rabbit IgG H&L (1:500 for IF; Abcam; ab150077).
Materials
Dimethyl-sulphoxide (DMSO) were obtained from Sigma-Aldrich (St Louis, MO, USA). Cell Counting Kit-8 (CCK-8) was purchased from Keygen Biotech (Nanjing, China). Fargesin (purity > 98%) was dissolved in 100% DMSO (400 mg/mL). Then, fargesin storage solution dissolved in saline.
Statistical analysis
All experiments were performed three times. Data were represented as mean ± SD using SPSS version 19.0 software (SPSS, USA). Curve analysis was performed using GraphPad Prism 5.0 (USA). Data in each group were analyzed using unpaired, two-tailed Student’s t-test. The level of significance was set at p < 0.05.