Feprazone Ameliorates TNF-α-Induced Loss of Aggrecan via Inhibition of the SOX-4/ADAMTS-5 Signaling Pathway

Abstract

Background: Arthritis is a cartilage degenerative disease that is mainly induced by the degradation of the cartilage extracellular matrix (ECM), which is found to be regulated by the expression level of a disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMT-5), an enzyme degrading Aggrecans in the ECM. Feprazone is a classic nonsteroidal anti-inflammatory drug with promising efficacy in arthritis. The present study aims to investigate the protective effect of Feprazone on the degraded Aggrecan in the human chondrocytes induced with tumor necrosis factor-α (TNF-α) and to clarify the underlying mechanism. Methods: To investigate the effect of Feprazone, the CHON-001 chondrocytes were stimulated with TNF-α (10 ng/mL) in the presence or absence of Feprazone (3, 6 μM) for 24 h. Mitochondrial membrane potential was evaluated using the Rhodamine 123 assay. The gene expressions of interleukin-1β (IL-1β), interleukin-8 (IL-8), monocyte chemotactic protein 1 (MCP-1), and ADAMTS-5 in the treated chondrocytes were detected using real-time quantitative polymerase chain reaction (qRT-PCR), and the protein levels of these targets were determined using enzyme-linked immunosorbent assay (ELISA). SOX-4 was knocked down by transfecting the siRNA into the chondrocytes. Western blot analysis was utilized to evaluate the expression levels of SOX-4, Aggrecan, and protein kinase C (PKCα). Results: First, the reduced mitochondrial membrane potential (ΔΨ_m) and secretion of proinflammatory factors (IL-1β, IL-8, and MCP-1) induced by TNF-α were significantly reversed by treatment with Feprazone. Second, the expression of Aggrecan was significantly decreased by stimulation with TNF-α via upregulation of ADAMTS-5 but was dramatically reversed by the introduction of Feprazone. Third, we found that TNF-α elevated the expression of ADAMTS-5 by upregulating SOX-4, which was observed to be related to the activation of PKCα. Lastly, the elevated expression of SOX-4 induced by TNF-α was significantly reversed by Feprazone. Conclusions: Feprazone might ameliorate TNF-α-induced loss of Aggrecan via the inhibition of the SOX-4/ADAMTS-5 signaling pathway.

Figure 1

Feprazone ameliorated TNF-α-induced reduction of mitochondrial membrane potential (ΔΨ_m) in human CHON-001 chondrocytes. Cells were stimulated with TNF-α (10 ng/mL) in the presence or absence of Feprazone (3, 6 μM) for 24 h. (A) Molecular structure of Feprazone. (B) Mitochondrial membrane potential (ΔΨm) was assayed by Rhodamine 123 (####, P < 0.0001 vs vehicle group; &&, &&&, P < 0.01, 0.001 vs TNF-α group, N = 6).

Figure 2

Feprazone inhibited TNF-α-induced expression and secretions of IL-1β, IL-8, and MCP-1 in human CHON-001 chondrocytes. Cells were stimulated with TNF-α (10 ng/mL) in the presence or absence of Feprazone (3, 6 μM) for 24 h. (A) mRNA of IL-1β, IL-8, and MCP-1. (B) Secretions of IL-1β, IL-8, and MCP-1 (####, P < 0.0001 vs vehicle group; &&, &&&, P < 0.01, 0.001 vs TNF-α group, N = 5).

Figure 3

Feprazone attenuated TNF-α-induced reduction of Aggrecan in human CHON-001 chondrocytes. Cells were stimulated with TNF-α (10 ng/mL) in the presence or absence of Feprazone (3, 6 μM) for 24 h. Expression of Aggrecan was measured (####, P < 0.0001 vs vehicle group; &&, &&&, P < 0.01, 0.001 vs TNF-α group, N = 6).

Figure 4

Feprazone suppressed TNF-α-induced expression of ADAMTS-5 in human CHON-001 chondrocytes. (A, B) Cells were stimulated with TNF-α (5 and 10 ng/mL) for 24 h. mRNA and protein levels of ADAMTS-5 as measured by real-time polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA). (C, D) Cells were stimulated with TNF-α (10 ng/mL) in the presence or absence of Feprazone (3, 6 μM) for 24 h. mRNA and protein levels of ADAMTS-5 as measured by real-time PCR and ELISA (###, ####, P < 0.001, 0.0001 vs vehicle group; &&, &&&, P < 0.01, 0.001 vs TNF-α group, N = 5).

Figure 5

TNF-α increased expression of SOX-4 in human CHON-001 chondrocytes. Cells were stimulated with TNF-α (5 and 10 ng/mL) for 24 h. (A) mRNA of SOX-4. (B). Protein of SOX-4 (###, ####, P < 0.001, 0.0001 vs vehicle group, N = 5).

Figure 6

Effects of TNF-α on the expression of ADAMTS-5 is mediated by SOX-4. Cells were transfected with SOX-4 RNA, followed by stimulation with TNF-α (10 ng/mL). (A) Western blot analysis revealed successful knockdown of SOX-4. (B) mRNA of ADAMTS-5. (C) Protein of ADAMTS-5 (####, P < 0.0001 vs vehicle group; &&&, P < 0.001 vs TNF-α group, N = 5).

Figure 7

Effects of TNF-α on SOX-4 expression are mediated by PKCα. (A) Cells were stimulated with TNF-α (5 and 10 ng/mL) for 24 h. Expression of PKCα was measured. (B, C) Cells were stimulated with TNF-α (10 ng/mL) or the PKCα inhibitor safingol (5 μM) for 24 h. mRNA and protein levels of SOX-4 were measured (####, P < 0.0001 vs vehicle group; &&&, P < 0.001 vs TNF-α group, N = 5).

Figure 8

Feprazone reduced the expression of SOX-4. Cells were stimulated with TNF-α (10 ng/mL) in the presence or absence of Feprazone (3, 6 μM) for 24 h. (A) mRNA of SOX-4. (B) Protein of SOX-4 (####, P < 0.0001 vs vehicle group; &&, &&&, P < 0.01, 0.001 vs TNF-α group, N = 5).