Reagents and materials
Paroxetine was purchased from Apexbio (Suzhou, China). Alpha-MEM and DMEM were procured from Biosharp (Anhui, China). The IL-1β and Cell Counting Kit-8 (CCK-8) were purchased from Med Chem Express (New Jersey, USA). RANKL and M-CSF were procured from LifeTein (Beijing, China). Primary antibodies against Nlrp3 (ab263899), IL-1β (ab254360), caspase1 (ab179515) and Adamts5 (ab185722) were purchased from Abcam (Cambridge, UK). The aggrecan neo polyclonal antibody (PA1-1746) was procured from Thermo Fisher Scientific (Waltham, MA, USA), and MMP3 (ET1705-98) and SOX9 (ET1611-56) antibodies were purchased from Huaan Biotechnology (Hangzhou, China). Primary antibodies against P65 (8242), p-p65 (3033), iκBα (4812), p-iκBα (2859), and β-actin (4970) were procured from Cell Signaling Technology (Danvers, MA, USA).
Cell viability and cytotoxicity assays
Six-week-old C57 mice were used for the isolation of bone marrow-derived macrophages (BMMs). The bone marrow was washed with MEM containing 10% serum and 30 ng/ml M-CSF and cultured in an incubator under conditions of 37°C and 5% CO2. The medium was changed every other day and the adherent cells were considered to be BMMs. The ATDC5 cell line was purchased from the European Collection of Authenticated Cell Cultures and cultured in DMEM containing 5% serum. To investigate the cytotoxic effects of paroxetine, ATDC5 cells were treated with different concentrations of paroxetine for 24 and 48 h and the cell counting kit-8 assay was performed according to the manufacturer’s instructions. The absorbance at 450 nm of each well was measured with the Multiskan FC microplate photometer (Thermo Fisher Scientific). In another experiment, BMMs were treated with different concentrations of paroxetine for 48 and 96 h and assayed.
Assay of extracellular matrix components
To observe the effects of paroxetine on cartilage production, ATDC5 cells were cultured at high density. After the cells had adhered to the multi-well plates, MEM containing IL-1β and different concentrations of paroxetine was added. The cells were stained with toluidine blue after 7 days of treatment and the multi-well plates were scanned with an Epson V600 Photo Scanner (Japan). In another experiment, ATDC5 cells were treated with IL-1β and different concentrations of paroxetine for 48 h for the extraction of RNA and protein to examine the synthesis and catabolism of extracellular matrix components.
Osteoclast differentiation and osteoclast-related gene expression
The effects of paroxetine on osteoclast differentiation in vitro were examined. BMMs were treated with 50 ng/ml RANKL and different concentrations of paroxetine, followed by TRAP staining and filamentous actin (F-actin) ring formation assay. The number and area of osteoclasts were determined for statistical analysis. To observe the time-dependent effects of paroxetine, BMMs at different stages of osteoblast differentiation (1–3 days, 3–5 days, and 5–7 days) were treated with the compound as indicated above.
To investigate the effects of paroxetine on osteoclast marker genes, BMMs were treated with RANKL and different concentrations of paroxetine. After significant osteoclast formation was observed microscopically, total RNA was extracted for reverse transcription and quantitative polymerase chain reaction (qPCR). The expression of osteoclast-related genes (c-Fos, NFATC1, ACP5, V-ATPase D2, DC-STAMP, and CTSK) was examined by qPCR.
Quantitative polymerase chain reaction
Total RNA was extracted using TRIzol reagent (Gibco, Grand Island, NY, USA), and complementary DNA (cDNA) was synthesized by reverse transcription using the HiFiScript cDNA synthesis kit (CWBiotech, China). Quantitative polymerase chain reactions were performed on an ABI 7300 plus real-time PCR system (Applied Biosystems, Foster City, CA, USA) using the ChamQ Universal SYBR qPCR Master Mix (Vazyme, China) and corresponding primers. GAPDH was used as an internal reference for the normalization of gene expression. The 2−ΔΔCT method was used to calculate the relative expression of each gene. Primer sequences for the detection of target genes are shown in Table 1.
Western blotting
Total proteins were separated with radioimmunoprecipitation assay buffer (Biosharp, Hefei, China) containing 1 mM phenylmethylsulfonyl fluoride and a phosphatase inhibitor cocktail (MedChemExpress, NJ, USA). After centrifugation at 12,000 rpm for 15 min at 4°C, the concentration of proteins was determined with the BCA protein assay kit (AMEKO, Shanghai, China). The proteins (30 μg) were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis using 10% gels (Epizyme, Shanghai, China) and transferred to polyvinylidene difluoride membranes. The membranes were blocked with blocking buffer (Biosharp) for 1–4 h and incubated with primary antibody for 12 h at 4°C. Next, the membranes were incubated with secondary antibody for 1 h at room temperature, followed by the detection of target proteins with an ultra-sensitive ECL chemiluminescence kit (NCM Biotech, Suzhou, China) and the ImageQuant LAS 500 system (GE Healthcare, Fairfield, CT, USA). The intensity of each band was quantified by ImageJ software and normalized using β-actin.
Immunofluorescence
ATDC5 cells were treated with IL-1β and paroxetine for 24 h and fixed in 4% paraformaldehyde for 15 min. The fixed cells were permeabilized with 0.25% Triton X-100 for 20 min and blocked at room temperature for 30 min, followed by the incubation of cells in primary antibody at 4°C overnight and secondary antibody at room temperature for 1 h. The nuclei were stained with DAPI, and the cells were viewed by confocal microscopy.
Animal model
Ten-week-old male C57BL/6J mice were randomly divided as follows: sham-operated group, OA group, OA + 5 mg/kg paroxetine group, and OA + 20 mg/kg paroxetine group (n = 6/group). After the mice were anesthetized with an intraperitoneal injection of chloral hydrate, the medial meniscus-tibial ligament (MML) was exposed by making an incision next to the medial knee ligament. For OA and OA + paroxetine treatment groups, the MML was excised to destabilize of the medial meniscus. The MML was intact in the sham-operated group. After surgery, the mice were treated intraperitoneally with different drugs for 4 weeks, while the sham-operated and OA groups were treated with phosphate buffered saline on alternate days.
Histological observations
The knee joints (n = 6/group) were fixed in 4% paraformaldehyde for 24 h and decalcified with 10% EDTA solution for 4 weeks. Paraffin-embedded tissues were cut into 5-μm-thick sections and stained with hematoxylin–eosin (HE), saffron O–sturdy green (SO), or TRAP. The degree of cartilage degeneration was assessed according to the Osteoarthritis Research Society International (OARSI) scoring system. The TRAP-positive cell number and Oc.S/BS tissue ratio were recorded.
Statistical analysis
GraphPad Prism 5.0 software (GraphPad Software, La Jolla, CA, USA) was used for data processing. One-way analysis of variance (ANOVA) was used for comparisons of means among three or more groups, and Student's t-test was used for comparisons of means between two groups. All experiments were performed at least three independent times, and p-values < 0.05 were considered statistically significant.