Mmp 13

The joints were fixed in 4% paraformaldehyde and subsequently decalcified with a 14% ethylenediaminetetraacetic acid (EDTA) solution for ten days. Next the joints were embedded in paraffin and cut into 5 μm thick slices by sharp blade. HE (Solarbio, China) and safranin O-fast green (Solarbio, China) staining were performed for histomorphological analysis. The severities of OA were graded by three independent observers by using the Osteoarthritis Research Society International (OARSI) score (scale of 0–24) [47 ]. Furthermore, Immunohistochemical staining for MMP13 (1:200, Boster) was performed to evaluate the anti-inflammatory effects of the probes.

Proteins were extracted from synoviocytes and protein concentrations were adjusted using the bicinchoninic acid protein assay kit (Fermentas, Thermo Fisher Scientific, Waltham, MA, USA). Every well in the 10% separating SDS-PAGE was loaded with 20 μL protein. SDS-PAGE was used to separate equal quantities of protein and separated proteins were transferred to nitrocellulose membranes (Fermentas, Thermo Fisher Scientific, Waltham, MA USA). The membranes were treated with PBS containing 5% nonfat dry milk, and incubated with primary antibodies [MMP-3 (1:1,000, Boster, Wuhan, China), MMP-13 (1:1,000, Boster, Wuhan, China), COX-2 (1:1,000, Boster, Wuhan, China) and iNOS (1:1,000, Boster, Wuhan, China)] overnight at 4 °C. After washing, the membranes were treated with horseradish peroxidase-conjugated secondary antibodies, followed by visualization using an enhanced chemiluminescence kit (Fermentas, Thermo Fisher Scientific, Waltham, MA, USA). Blots were scanned using a gel imaging system (GelDoc-It 310, UVP Co., Upland, CA, USA) and densitometric analyses were performed using Image Lab 4.1 software (Bio-Rad Laboratories, Hercules, CA, USA).

Vindoline (Vin), 99.33% purity, was purchased from MedChem Express (Monmouth, NJ, USA) and the molecular structure is presented in Figure 1A. Dimethyl sulfoxide was used to dissolve Vin to yield a stock solution (50 mM), which was stored at −80°C. Dulbecco’s modified Eagle’s medium (DMEM)-high glucose was bought from Solarbio (Beijing, China). Fetal bovine serum (FBS) and DMEM/F12 were from Gibco (Grand Island, NY, United States). Cell Counting Kit-8 (CCK-8) and recombinant mouse RANKL were from BestBio (Shanghai, China) and R&D Systems (Minneapolis, MN, United States), respectively. Recombinant human macrophage colony-stimulating factor (M-CSF) and recombinant murine IL-1β were obtained from PeproTech (Rocky Hill, NJ, USA). The staining kit for tartrate-resistant acid phosphatase (TRAP) was from Sigma-Aldrich (St. Louis, MO, United States). Toluidine blue solution was purchased from Solarbio (Beijing, China). Primary antibodies against extracellular signal–regulated kinase (ERK), inhibitor of NF-κB (IκBα), p-65, phosphorylated-ERK (p-ERK; Thr202/Tyr204), p-p65 (Ser536), nuclear factor of activated T cells 1 (NFATc1), c-fos and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were from Cell Signaling Technology (Danvers, MA, United States). Primary antibodies against COL2a1, aggrecan and MMP13 were obtained from Boster Biological Technology (Wuhan, China).

A qualitative commercial enzyme-linked immunosorbent assay test (Multi-analyte ELISArray kit (SABiosciences - QIAGEN, Maryland, USA) has been used to simultaneously profile the level of multiple cytokines (IL-1α, IL-1ß, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-17A, IFNγ, TNF-α, GM-CSF) in cell culture supernatants.
A cutoff of twice the absorbance value (read at 450 nm) of the negative control for every cytokine (A₄₅₀) was used and the results were reported as positive (A₄₅₀ ≥ specific cutoff) or negative (A₄₅₀ < specific cutoff).
Protein concentration of detectable cytokines IL-6, IL-8, MMP-3, MMP-13 (Boster Biological Technology, Fremont, CA, USA), TIMP-3 and TIMP-4 (R&D Systems, Minneapolis, MN, USA) was then determined in cell-free supernatants using quantitative commercially available ELISA kits.