Anti-invasive effects of Celastrus Orbiculatus extract on interleukin-1 beta and tumour necrosis factor-alpha combination-stimulated fibroblast-like synoviocytes

Abstract

Background: Invasion of fibroblast-like synoviocytes (FLSs) is critical in the pathogenesis of rheumatoid arthritis (RA). The metalloproteinases (MMPs) and activator of nuclear factor-kappa B (NF-κB) pathway play a critical role in RA-FLS invasion induced by interleukin-1 beta (IL-1β) and tumour necrosis factor-alpha (TNF-α). The present study aimed to explore the anti-invasive activity and mechanism of Celastrus orbiculatus extract (COE) on IL-1β and TNF-α combination-stimulated human RA-FLSs.

Methods: We investigated the effect of COE on IL-1β and TNF-α combination-induced FLS invasion as well as MMP expression and explored upstream signal transduction.

Results: COE suppressed IL-1β and TNF-α combination-stimulated FLSs invasion by inhibiting MMP-9 expression and activity. Furthermore, our results revealed that COE inhibited the transcriptional activity of MMP-9 by suppression of the binding activity of NF-κB in the MMP-9 promoter, and inhibited IκBα phosphorylation and nuclear translocation of NF-κB.

Conclusions: COE inhibits IL-1β and TNF-α combination-induced FLSs invasion by suppressing NF-κB-mediated MMP-9 expression.

Figure 1

Effect of COE on IL-1β and TNF-α-induced FLSs migration and invasion. (A) FLSs were incubated with the indicated concentrations of COE in serum containing medium for 24 h, and cell viability was measured by MTT assay. ^*P < 0.05, ^**P < 0.01 versus normal control group. (B) FLSs were incubated with the indicated concentrations of COE for 24 h. Cells were harvested and the cell cycle distribution in the sub-G1 phase was determined by FCM analysis. ^*P < 0.05 versus normal control group. The migration (C) and invasion (D) abilities of FLSs were determined by cell migration and invasion assays. FLSs were pretreated with the indicated concentrations of COE for 1 h. Then, FLSs were allowed to migrate with or without IL-1β (10 ng/ml), TNF-α (10 ng/ml), or IL-1β (10 ng/ml) and TNF-α (10 ng/ml) for 48 h, respectively. The number of migrating and invasive cells in each chamber was plotted as the mean ± SD in three independent experiments. The results were analysed by ANOVA. ^*P < 0.05, ^**P < 0.01 versus normal control group, ^◆P < 0.05, ^◆◆P < 0.01 versus IL-1β and TNF-α combination-treated group.

Figure 2

Effect of COE on mRNA expression of MMPs in IL-1β and TNF-α-induced FLSs. FLSs were incubated with the indicated concentrations of COE for 1 h followed by IL-1β (10 ng/ml) and TNF-α (10 ng/ml) stimulation. After 48 h, the mRNA levels of endogenous MMP-1(A), MMP-2 (B), MMP-3 (C) and MMP-9 (D) were measured by qRT-PCR. GAPDH and β-actin were used as internal controls, respectively. The histogram shows the mRNA levels from three independent experiments. ^*P < 0.05, ^**P < 0.01 versus normal control group, ^◆P < 0.05, ^◆◆P < 0.01 versus IL-1β and TNF-α combination-treated group.

Figure 3

Effect of COE on MMP-2 and MMP-9 activity in IL-1β and TNF-α-induced FLSs. FLSs were pretreated with the indicated concentrations of COE for 1 h followed by IL-1β (10 ng/ml) and TNF-α (10 ng/ml) stimulation. After 48 h, the supernatants were collected and assayed for the amount and activity of secreted MMP-2 and MMP-9, by ELISA (A and B), western blot (C and D) and gelatin zymography (E) respectively. The histogram shows the ELISA and western blot results from three independent experiments. ^*P < 0.05, ^**P < 0.01 versus normal control group, ^◆P < 0.05, ^◆◆P < 0.01 versus IL-1β and TNF-α combination-treated group.

Figure 4

COE inhibits the transcriptional activity of MMP-9 by suppression of NF-κB activity. (A) RA-FLSs were transfected with pGL2-MMP-9WT and Renilla luciferase reporter vector plasmids. The transfected cells were pretreated with the indicated concentrations of COE for 1 h, followed by IL-1β (10 ng/ml) and TNF-α (10 ng/ml) stimulation for 48 h. The relative luciferase activity in the cell extract was normalized by Renilla luciferase activity. Each value represents the mean ± SD of triplicate experiments and is expressed relative to the control. FLSs were transfected with pGL2-MMP-9 mNF-κB (B), pGL2-MMP-9 mAP-1-U (C) and pGL2-MMP-9 mAP-1-P (D). The relative luciferase activity is normalized to Renilla luciferase activity and is expressed relative to the controls. ^*P < 0.05, ^**P < 0.01 versus normal control group, ^◆P < 0.05, ^◆◆P < 0.01 versus IL-1β and TNF-α combination-treated group.

Figure 5

NF-κB mediates IL-1β and TNF-α-induced MMP-9 expression. (A) FLSs were pretreated with BAY (5, 10 μM) for 1 h and then stimulated with IL-1β (10 ng/ml) and TNF-α (10 ng/ml) for 48 h. MMP-9 mRNA expression in cells was analysed by qRT-PCR. GAPDH was used as an internal control. The histogram shows the mRNA levels from three independent experiments. (B) and (C) FLSs were pretreated with BAY (5, 10 μM) for 1 h and then stimulated with IL-1β (10 ng/ml) and TNF-α (10 ng/ml). After 48 h, conditioned media was collected and gelatin zymography, western blotting or ELISA was performed. (D) FLSs were transfected with pGL2-MMP-9WT reporter plasmids and then cultured with BAY (5, 10 μM) with or without IL-1β (10 ng/ml) and TNF-α (10 ng/ml) for 48 h. Luciferase activity in the cell extracts was determined. The data are presented as mean ± SD of triplicate experiments. ^**P < 0.01 versus normal control group, ^◆P < 0.05, ^◆◆P < 0.01 versus IL-1β and TNF-α combination-treated group.

Figure 6

COE inhibits the binding activity of NF-κB in the MMP-9 promoter. FLSs were pretreated with the indicated amounts of COE for 1 h, and stimulated with IL-1β (10 ng/ml) and TNF-α (10 ng/ml) for 48 h. (A) The DNA binding activity of the nuclear extracts was examined by EMSA using a probe containing the NF-κB motif in the MMP-9 promoter. NF-κB DNA binding activity was analysed by EMSA and shown is a representative blot from three independent experiments. (B) The cross-linked chromatin was prepared and immunoprecipitated with antibody and IgG to NF-κB p65 before amplification of the MMP-9 gene region containing the NF-κB site. Immunoprecipitates were analysed by PCR for the presence of the MMP-9 gene promoter sequence using the primer pair described in material and methods. DNA purified from the sonicated chromatin was directly analysed by PCR using the same primer set, which served as an input control (Input). Similar results were obtained in three independent experiments. (C) FLSs were harvested and whole cell extracts were subjected to western blot analysis for the indicated proteins (IκBα, p-IκBα, p65 and p-p65). A representative protein blot of three independent experiments is shown. β-actin served as a control. The histogram shows the protein relative expression changes from three independent experiments. ^*P < 0.05, ^**P < 0.01 versus normal control group, ^◆P < 0.05, ^◆◆P < 0.01 versus IL-1β and TNF-α combination-treated group.

Figure 7

Schematic diagram. COE inhibits the IL-1β and TNF-α-induced migration and invasion signal pathway in FLSs. COE blocks IL-1β and TNF-α-induced phosphorylation and degradation of IκBα, which inhibits the nuclear translocation of p65. This further suppresses the activation of NF-κB, and, thus, decreases MMP-9 expression and the subsequent migration and invasion of FLSs.