Apremilast prevents IL‑17‑induced cellular senescence in ATDC5 chondrocytes mediated by SIRT1

Abstract

Osteoarthritis is the most prevalent joint degenerative disease and has been considered a major cause of severe joint pain and physical disability in the elderly. The chondrocyte is the only cell type found in articular cartilage and chondrocyte senescence plays a pivotal role in the pathogenesis of osteoarthritis. Apremilast is an oral PDE4 inhibitor and has been used for the treatment of patients with active psoriatic arthritis. In the present study, the biological function of apremilast was examined in an interleukin (IL)‑17‑treated chondrocyte model. Expression levels of target genes and proteins were measured using reverse transcription‑quantitative PCR, ELISA, and western blotting, respectively. ROS levels in chondrocytes were examined using the fluorescent dye DCFH‑DA. Cellular senescence was determined using senescence-associated-β-galactosidase staining. The profile of cell cycle phases was analyzed via flow cytometry. It was revealed that treatment with apremilast reduced the expression of IL‑1β, MCP‑1, and the production of ROS. SA‑β‑gal staining results indicated that the presence of apremilast suppressed IL‑17‑induced cellular senescence. Furthermore, apremilast prevented IL‑17‑induced G0/G1 phase cell cycle arrest. In addition, it was demonstrated that apremilast suppressed IL‑17‑induced expression of p21 and PAI‑1. Notably, the silencing of sirtuin 1 (SIRT1) abolished the protective effect of apremilast against IL‑17‑induced cellular senescence, suggesting that the action of apremilast in chondrocytes is dependent on SIRT1. In conclusion, the present results revealed that apremilast exerted a beneficial effect, thereby protecting chondrocytes from senescence induced by IL‑17.

Keywords: osteoarthritis; chondrocyte senescence; apremilast; plasminogen activator inhibitor‑1; sirtuin 1.

Figure 1

Apremilast prevents IL-17-induced expression and secretions of pro-inflammatory cytokines in mouse ATDC5 chondrocytes. Cells were treated with IL-17 (10 ng/ml) in the presence or absence of apremilast (0.5 and 1 µM) for 24 h. (A) Molecular structure of apremilast. (B) mRNA levels of IL-1β and MCP-1. (C) Secretions of IL-1β and MCP-1. ^####P<0.0001 vs. the vehicle control group; ^**P<0.01 and ^***P<0.001 vs. the IL-17 group. IL, interleukin; MCP-1, monocyte chemoattractant protein-1.

Figure 2

Apremilast prevents IL-17-induced production of ROS in mouse ATDC5 chondrocytes. Cells were treated with IL-17 (10 ng/ml) in the presence or absence of apremilast (0.5 and 1 µM) for 24 h. Production of ROS was measured using DCFH-DA staining (magnification, ×10). ^####P<0.0001 vs. the vehicle control group; ^**P<0.01 and ^***P<0.001 vs. the IL-17 group. ROS, reactive oxygen species; IL, interleukin; DCFH-DA, 2,7-dichlorodihy-drofluorescein diacetate.

Figure 3

Apremilast prevents IL-17-induced cellular senescence in mouse ATDC5 chondrocytes. Cells were treated with IL-17 (10 ng/ml) in the presence or absence of apremilast (1 µM) for 7 days. Cellular senescence was measured using SA-β-gal staining. ^##P<0.01 vs. the vehicle control group; ^**P<0.01 vs. the IL-17 group. IL, interleukin; SA-β-gal, senescence-associa ted-β-galactosidase.

Figure 4

Apremilast prevents IL-17-induced cell cycle arrest in the G0/G1 phase in mouse ATDC5 chondrocytes. Cells were treated with IL-17 (10 ng/ml) in the presence or absence of apremilast (1 µM) for 7 days. The cell cycle was assayed using flow cytometry. G0/G1 phase, G2/M phase, and S phase of cells were measured. ^##P<0.01 vs. the vehicle control group; ^**P<0.01 vs. the IL-17 group. IL, interleukin.

Figure 5

Apremilast prevents IL-17-induced expression of p21 and PAI-1 in mouse ATDC5 chondrocytes. Cells were treated with IL-17 (10 ng/ml) in the presence or absence of apremilast (1 µM) for 24 h. (A) mRNA levels of p21 and PAI-1. (B) Protein levels of p21 and PAI-1 as measured by western blotting. ^####P<0.0001 vs. the vehicle control group; ^***P<0.001 vs. the IL-17 group. IL, interleukin; PAI-1, plasminogen activator inhibitor-1.

Figure 6

Apremilast prevents IL-17-induced reduction of SIRT1 in mouse ATDC5 chondrocytes. Cells were treated with IL-17 (10 ng/ml) in the presence or absence of a Apremilast (1 µM) for 24 h. (A) mRNA of SIRT1. (B) Protein level of SIRT1. ^####P<0.0001 vs. the vehicle control group; ^***P<0.001 vs. the IL-17 group. IL, interleukin; SIRT1, sirtuin 1.

Figure 7

Knockdown of SIRT1 abolishes the protective effects of apremilast against IL-17-induced cellular senescence and the expression levels of p21 and PAI-1. Cells were transfected with SIRT1 siRNA, followed by stimulation with IL-17 (10 ng/ml) in the presence or absence of apremilast (1 µM) for 24 h. (A) Cellular senescence. (B) mRNA levels of p21 and PAI-1. (C) Protein levels of p21 and PAI-1 as measured by western blotting. ^####P<0.0001 vs. the vehicle control group; ****P<0.0001 vs. the IL-17 group; ^$$$$P<0.0001 vs. the IL-17 + apremilast group. SIRT1, sirtuin 1; IL, interleukin; PAI-1, plasminogen activator inhibitor-1.

Figure 8

Graphical representation of the underlying mechanism, whereby apremilast prevents IL-17-induced cellular senescence in ATDC5 chondrocytes. IL, interleukin; ROS, reactive oxygen species; SIRT1, sirtuin 1; MCP-1, monocyte chemoattractant protein-1; PAI-1, plasminogen activator inhibitor-1.