Acacetin inhibits expression of matrix metalloproteinases via a MAPK-dependent mechanism in fibroblast-like synoviocytes

Abstract

It is well known that rheumatoid arthritis (RA) is an autoimmune joint disease in which fibroblast-like synoviocytes (FLSs) play a pivotal role. In this study, we investigated the anti-arthritic properties of acacetin in FLSs. The expression of matrix metalloproteinase (MMP)-1, MMP-3 and MMP-13 were investigated by quantitative RT-PCR and western blot at gene and protein levels. At the same time, the phosphorylation of mitogen-activated protein kinases (MAPK) was investigated. The DNA-binding activity of NF-κB was investigated by electrophoretic mobility shift assay. We found that acacetin inhibits p38 and JNK phosphorylation and reduces MMP-1, MMP-3 and MMP-13 expression in interleukin-1β-induced FLSs. Our results suggest that acacetin has antiarthritic effects in FLSs. Thus, acacetin should be further studied for the treatment of arthritis.

Keywords: acacetin; fibroblast-like synoviocytes; interleukin-1β; matrix metalloproteinase; rheumatoid arthritis.

Figure 1

Effect of acacetin on cell viability. Cells were seeded in 96-well plates at 6 × 10³/well and cultured with different concentrations of acacetin for 24 hrs, followed by MTT assay analysis. Data are expressed as means ± SD. *P < 0.05 compared with cells treated with culture medium only.

Figure 2

Effect of acacetin on IL-6 production. Cells (1 × 10⁵/well in six-well plates) were treated with acacetin for 1 hr prior to treatment with IL-1β (10 ng/ml) and were collected after 24 hrs. Conditioned media was collected for IL-6 measurement with an ELISA kit. Data are expressed as means ± SD. *P < 0.05 compared with cells stimulated with IL-1β in the absence of acacetin.

Figure 3

Effect of acacetin on MMP-1, MMP-3 and MMP-13 expression in FLSs. Cells (1 × 10⁵/well in six-well plates) were treated with acacetin for 1 hr prior to treatment with IL-1β (10 ng/ml) and were collected after 24 hrs. (A) Quantitative real-time PCR and (B) Western blot analyses were performed to analyse MMP-1, MMP-3 and MMP-13 mRNA and protein expression respectively. *P < 0.05 compared with cells stimulated with IL-1β in the absence of acacetin.

Figure 4

Effect of acacetin on IL-1β-induced phosphorylation of MAPKs in FLSs. Western blotting was performed to detect the phosphorylation levels of JNK, ERK1/2 and p38 in cells pre-treated with acacetin for 2 hrs before stimulation with IL-1β for 30 min. (A). The ratio between the intensity of the phosphorylated and non-phosphorylated band are shown in B. Cells were treated with a p38 inhibitor or a JNK inhibitor for 2 hrs before stimulation with IL-1β for 30 min., and MMP-1, MMP-3 and MMP-13 expression were assessed (C). *P < 0.05 compared with cells stimulated with IL-1β in the absence of acacetin.

Figure 5

Effect of acacetin on DNA-binding activity of NF-κB. Cells were pre-treated with various concentration of acacetin for 30 min. before stimulation with IL-1β (10 ng/ml) for 90 min. Nuclear extracts were prepared and analysed by electrophoretic mobility shift assay. Lane 1: nuclear extracts incubated with unlabelled consensus oligonucleotide (Positive) to confirm the specificity of binding. Lane 2–6 biotin-NF-κB oligonucleotide plus IL-1β and various concentrations of acacetin. Lane 7 biotin-NF-κB oligonucleotide (Negative), no nuclear extracts for probe to bind. IL-1β stimulation increased nuclear levels of NF-κB, and acacetin did not inhibit the DNA-binding activity of NF-κB. NSB: Non-Specific Binding; P: Free Biotin-labelled Probe.