Metabolomic Elucidation of the Effects of Curcumin on Fibroblast-Like Synoviocytes in Rheumatoid Arthritis

Abstract

Rheumatoid arthritis (RA) is a chronic systemic inflammatory disease characterized by synovial inflammation and joint disability. Curcumin is known to be effective in ameliorating joint inflammation in RA. To obtain new insights into the effect of curcumin on primary fibroblast-like synoviocytes (FLS, N = 3), which are key effector cells in RA, we employed gas chromatography/time-of-flight mass spectrometry (GC/TOF-MS)-based metabolomics. Metabolomic profiling of tumor necrosis factor (TNF)-α-stimulated and curcumin-treated FLS was performed using GC/TOF-MS in conjunction with univariate and multivariate statistical analyses. A total of 119 metabolites were identified. Metabolomic analysis revealed that metabolite profiles were clearly distinct between TNF-α-stimulated vs. the control group (not stimulated by TNF-α or curcumin). Treatment of FLS with curcumin showed that the metabolic perturbation by TNF-α could be reversed to that of the control group to a considerable extent. Curcumin-treated FLS had higher restoration of amino acid and fatty acid metabolism, as indicated by the prominent metabolic restoration of intermediates of amino acid and fatty acid metabolism, compared with that observed in TNF-α-stimulated FLS. In particular, the abundance of glycine, citrulline, arachidonic acid, and saturated fatty acids in TNF-α-stimulated FLS was restored to the control level after treatment with curcumin, suggesting that the effect of curcumin on preventing joint inflammation may be elucidated with the levels of these metabolites. Our results suggest that GC/TOF-MS-based metabolomic investigation using FLS has the potential for discovering the mechanism of action of curcumin and new targets for therapeutic drugs in RA.

Fig 1. PCA score (A) and loading plots (B) of RA fibroblast-like synoviocytes (FLS), which were not stimulated (Control), stimulated with TNF-α (TNF), and treated with curcumin (Curcumin).

(A) Principal component (PC)1 explained the significant separation of metabolite profiles between the TNF-α-stimulated group on the negative region of the PC1, and the control and curcumin-treated groups on the positive region of the PC1. Further, the control group was clearly separated from the curcumin-treated group on PC2. (B) PC1 was explained by 84 metabolites that correlated positively with the axis, and 35 metabolites that correlated negatively.

Fig 2. Hierarchical clustering analysis of 119 identified metabolites from RA FLS.

The results of heat mapping generated through metabolomic analysis and the relevant changes discovered. A heat map showed that the metabolite profiles of controls were similar to those of the curcumin-treated group. Red color reflects an increase, and blue color a decrease.

Fig 3. Inhibition of TNF-α-induced production of (A) IL-6, (B) IL-8, (C) MMP-1, and (D) MMP-3 by curcumin in RA FLS.

RA FLS (N = 3) were incubated with DMSO-containing vehicle or curcumin for 1 h, followed by stimulation with TNF-α (100 ng/mL) for 24 h. Production of IL-6, IL-8, MMP-1, and MMP-3 in the culture supernatants was measured using a commercially available ELISA kit. Values are mean ± standard deviation. * = P < 0.05, by the Kruskal-Wallis test.

Fig 4. The intensities of metabolites related to amino acid metabolism (A) and fatty acid metabolism (B).