Paroxetine Attenuates Chondrocyte Pyroptosis and Inhibits Osteoclast Formation by Inhibiting NF-κB Pathway Activation to Delay Osteoarthritis Progression

Abstract

Background: Osteoarthritis (OA), a common chronic joint disease, is characterized by cartilage degeneration and subchondral bone reconstruction. NF-κB signaling pathway-activated inflammation and NLRP3-induced pyroptosis play essential roles in the development of OA. In this study, we examine whether paroxetine can inhibit pyroptosis and reduce osteoclast formation, thereby delaying the destruction of knee joints.

Methods: We employed high-density cultures, along with quantitative polymerase chain reactions and Western blotting techniques, to investigate the effects of paroxetine on extracellular matrix synthesis and degradation. The expression levels of NF-κB and pyroptosis-related signaling pathway proteins were examined by Western blotting and immunofluorescence. Furthermore, the impact of paroxetine on RANKL-induced osteoclast formation was evaluated through TRAP staining and F-actin ring fluorescence detection. To investigate the role of paroxetine in vivo, we constructed a mouse model with destabilization of the medial meniscus (DMM) surgery. Safranin O-Fast Green staining, Hematoxylin-Eosin staining, and immunohistochemistry were conducted to observe the extent of knee joint cartilage deformation. In addition, TRAP staining was used to observe the formation of osteoclasts in the subchondral bone.

Results: In the in vitro experiments with ATDC5, paroxetine treatment attenuated IL-1β-induced activation of the pyroptosis-related pathway and suppressed extracellular matrix catabolism by inhibiting the NF-kB signaling pathway. In addition, paroxetine treatment decreased the expression of RANKL-induced osteoclast marker genes and reduced osteoclast formation. In animal experiments conducted in vivo, mice treated with paroxetine exhibited thicker knee cartilage with a smoother surface compared to the DMM group. Additionally, the formation of osteoclasts in the subchondral bone was reduced in the paroxetine-treated mice. Further analysis revealed that paroxetine treatment played a role in preserving the balance of the extracellular matrix and delaying knee joint degeneration.

Conclusion: Paroxetine can inhibit pyroptosis and reduce osteoclast formation via inhibiting the NF-κB signaling pathway, suggesting that it may have therapeutic effects in patients with OA.

Keywords: inflammation; osteoarthritis; osteoclasts; paroxetine; pyroptosis.

Figure 1

Paroxetine promotes extracellular matrix synthesis and inhibits catabolism. (A) Chemical structure of paroxetine. (B) Cytotoxic effects of paroxetine in ATDC5 cells after treatment for 24 and 48 h. (C) Toluidine blue staining of high-density-cultured ATDC5 cells after treatment with different concentrations of paroxetine and IL-1β. (D) Toluidine blue (+) area (%/area of interest) analysis as quantified by ImageJ. (E–J) Western blotting results of extracellular matrix anabolic- and catabolic-related proteins (SOX9, Aggrecan, MMP3, ADAMTS5) in ATDC5 cells as quantified by ImageJ. (K) The expression levels of genes related to extracellular matrix synthesis and catabolism were quantified by RT-PCR. Data are presented as mean ± S.D. (n = 3/group). Significant differences between groups are indicated as ***p < 0.001, **p < 0.01.

Figure 2

Paroxetine alleviates IL-1β-induced pyroptosis in ATDC5. (A–C) Western blotting results of pyroptosis-related proteins (Nlrp3, IL-1β and Caspase1 [CASP1]). (D–H) Protein levels were quantified by ImageJ. (I) Nlrp3 was assessed by immunofluorescence staining combined with DAPI staining. Values are mean ± SD (n = 3/group). Significant differences between groups are indicated as ***p < 0.001.

Figure 3

Paroxetine inhibits NF-κB signaling pathway activation in ATDC5. (A–E) Western blotting results of NF-κB-related signaling pathway proteins (p-P65, P65, p-IκB and IκB). (F) P65 localization was assessed by immunofluorescence. Values are mean ± SD (n = 3/group). Significant differences between groups are indicated as ***p < 0.001.

Figure 4

Paroxetine inhibits RANKL-induced osteoclast differentiation in vitro. (A) Cytotoxic effects of paroxetine in BMMs after treatment for 48 and 96 h. (B–D) TRAP staining of BMMs to observe the effects of different concentrations of paroxetine on RANKL-induced osteoclastic differentiation. (E–G) BMMs were treated with paroxetine for different periods of time to counteract the effects of RANKL. (H–J) F-actin rings were observed in osteoclasts by confocal microscopy. Osteoclasts with F-actin rings are marked with white arrows. Values are mean ± SD (n = 3/group). Significant differences between groups are indicated as ***p < 0.001, ** p < 0.01, and * p < 0.05.

Figure 5

Paroxetine inhibits osteoclast formation by suppressing the NF-κB signaling pathway. (A) RT-PCR results of osteoclast-related genes. (B–H) Western blotting results of NFATc1 and NF-κB signaling pathway-related proteins (p-P65, P65, p-IκB and IκB). (I) P65 localization was assessed by immunofluorescence. Values are mean ± SD (n = 3/group). Significant differences between groups are indicated as ***p < 0.001 and ** p < 0.01.

Figure 6

Paroxetine delays the progression of osteoarthritis in murine DMM model. (A) Sections of knee joints were stained with HE, SO, and TRAP. (B) OARSI scores of cartilage specimens of different groups. (C) TRAP positive number were recorded. (D–G) Aggrecan, MMP3, and Nlrp3 expression levels in mouse cartilage were assessed by immunohistochemistry. The positive cell staining rates were statistically analyzed with ImageJ. Values are mean ± SD (n = 3/group). Significant differences between groups are indicated as ***p < 0.001 and ** p < 0.01.

Figure 7

Mechanism by which paroxetine delayed the progression of osteoarthritis. Paroxetine was capable of attenuating chondrocyte pyroptosis and reducing osteoclast formation via inhibiting NF-κB pathway.