A novel function of artesunate on inhibiting migration and invasion of fibroblast-like synoviocytes from rheumatoid arthritis patients

Abstract

Introduction: Anti-malarial drug artesunate can suppress inflammation and prevent cartilage and bone destruction in collagen-induced arthritis model in rats-suggesting it may be a potent drug for rheumatoid arthritis (RA) therapy. We aimed to investigate its effect on the invasive property of fibroblast-like synoviocytes (FLS) from patients with RA.

Methods: Synovial tissues were obtained by closed needle biopsy from active RA patients, and FLS were isolated and cultured in vitro. RA-FLS were treated with artesunate at various concentrations, while methotrexate or hydroxychloroquine was employed as comparator drugs. Cell viability, proliferation, cell cycle, apoptosis, migration, invasion, and pseudopodium formation of RA-FLS were assessed by CCK-8 assays, EdU staining, Annexin V-FITC/PI staining, transwell assays, or F-actin staining, respectively. Further, relative changes of expressed proteases were analyzed by Proteome profiler human protease array and verified by quantitative real-time PCR (qPCR), Western blot, and ELISA. The expression of signaling molecules of MAPK, NF-κB, AP-1, and PI3K/Akt pathways were measured by qPCR and Western blot. PDK-1 knockdown by specific inhibitor AR-12 or siRNA transfection was used to verify the pharmacological mechanism of artesunate on RA-FLS.

Results: Artesunate significantly inhibited the migration and invasion of RA-FLS in a dose-dependent manner with or without TNF-α stimulation. The effect was mediated through artesunate inhibition of MMP-2 and MMP-9 production, and pre-treatment with exogenous MMP-9 reversed the inhibitory effect of artesunate on RA-FLS invasion. Artesunate had a stronger inhibitory effect on migration and invasion of RA-FLS as well as greater anti-inflammatory effect than those of hydroxychloroquine. Similar inhibitory effect was detected between artesunate and methotrexate, and synergy was observed when combined. Mechanistically, artesunate significantly inhibited PDK-1 expression as well as Akt and RSK2 phosphorylation-in a similar manner to PDK-1-specific inhibitor AR-12 or PDK-1 knockdown by siRNA transfection. This inhibition results in suppression of RA-FLS migration and invasion as well as decreased MMP-2 and MMP-9 expression.

Conclusions: Our study demonstrates artesunate is capable of inhibiting migration and invasion of RA-FLS through suppression of PDK1-induced activation of Akt and RSK2 phosphorylation-suggesting that artesunate may be a potential disease-modifying anti-rheumatic drug for RA.

Keywords: Artesunate; Fibroblast-like synoviocytes; Invasion; Migration; Rheumatoid arthritis.

Fig. 1

Effects of artesunate on proliferation, cell cycle, apoptosis and cytokine production of primary RA-FLS. RA-FLS were treated with different concentrations of artesunate, MTX (10 nM), or HCQ (20 μM) for 24 h. a EdU assays were performed to evaluate the proliferation of RA-FLS. Representative images showed proliferation of RA-FLS labeled with EdU (green) and nuclei stained with DAPI (blue, original magnification, × 100). b Cell cycle was detected by flow cytometry with PI staining. c Apoptosis was detected by flow cytometry with annexin V-FITC/PI double staining. d Bars were representative as effects of artesunate (60 μM), MTX (10 nM), or HCQ (20 μM) on proliferation, cell cycle, and apoptosis of primary RA-FLS measured by EdU, PI, and annexin V-FITC/PI assays, respectively. e The levels of IL-1β, IL-6, and IL-8 in culture supernatant of primary RA-FLS with or without 100 pg/mL TNF-α stimulation were measured by ELISA. Data were representative as means ± SD from 6 RA patients. *P < 0.05, **P < 0.01, compared with RA-FLS without TNF-α or artesunate treatment. ^△P < 0.05, ^△△P < 0.01, compared with RA-FLS treated with TNF-α alone

Fig. 2

Effects of artesunate on migration and invasion of primary RA-FLS. a–c Concentrations of artesunate, MTX (10 nM), or HCQ (20 μM) without TNF-α stimulation; wound healing and transwell assays showed the capacity of horizontal migration (a), vertical migration (b), and invasion (c) of primary RA-FLS. d F-actin was stained with phalloidin. Representative images were shown (original magnification, × 400 above and × 1000 below). The green arrow indicates lamellipodia formation, and the yellow arrow indicates filopodia formation. e Bars were representative as effects indicated concentrations of artesunate, MTX (10 nM), or HCQ (20 μM) on primary RA-FLS migration and invasion measured by wound healing and transwell assays of migration and invasion. Data were representative as means ± SD from 12 RA patients. *P < 0.05, **P < 0.01, compared with RA-FLS without TNF-α or artesunate treatment. ^△P < 0.05, ^△△P < 0.01, ^△△△P < 0.001, compared with RA-FLS treated with TNF-α alone

Fig. 3

Effects of artesunate on MMP and TIMP expression in primary RA-FLS. a–c After artesunate, MTX, or HCQ treatment for 24 h, expression of MMP-2, MMP-9, TIMP-1, and TIMP-2 of primary RA-FLS were analyzed by Western blot (a), qPCR (b), and ELISA (c). a–c shared the same figure key. Each bar represents mean ± SD from 6 RA patients. *P < 0.05, **P < 0.01, compared with RA-FLS without treatment. d After treatment of MMP-2 (6 ng/mL) and/or MMP-9 (12 ng/mL) in combination with artesunate (60 μM) for 24 h, wound healing and transwell assays showed the capacity of horizontal migration, vertical migration, and invasion of primary RA-FLS. Bars are representative as means ± SD from 6 RA patients. **P < 0.01, compared with RA-FLS without artesunate treatment. ^△P < 0.05, ^△△P < 0.01 compared with RA-FLS treated with artesunate alone

Fig. 4

Effects of combined treatment with artesunate and MTX on primary RA-FLS. a–c After combined treatment with artesunate and MTX for 24 h, wound healing and transwell assays showed the capacity of horizontal migration (a), vertical migration (b), and invasion (c) of primary RA-FLS. d–f Effects of combined treatment with artesunate and MTX on the expression of MMP-2, MMP-9, TIMP-1, and TIMP-2 in primary RA-FLS were measured by Western blot (d), qPCR (e), and ELISA (f). d–f shared the same figure key. Each bar represents mean ± SD from 6 RA patients. *P < 0.05, **P < 0.01, ***P < 0.001, compared with RA-FLS without artesunate treatment. ^△P < 0.05, ^△△P < 0.01, compared with RA-FLS treatment with MTX (10 nM)

Fig. 5

Effects of artesunate on PI3K/Akt/RSK2 pathway in primary RA-FLS. a Effects of artesunate (0–60 μM) on the expression of PIP2, PIP3, PI3K, p-PI3K, PDK-1, Akt, p-Akt, RSK2, and p-RSK2 were measured by Western blot. b Effects of AR-12 (5 μM) or si-PDK-1 on the expression of PDK-1, Akt, p-Akt, RSK, and p-RSK in primary RA-FLS were measured by Western blot. c Effects of AR-12 (5 μM) or si-PDK-1 on the expression of MMP-2, MMP-9, TIMP-1, and TIMP-2 in primary RA-FLS were measured by Western blot (c), qPCR (d), and ELISA (e). f–h After treatment with AR-12 (5 μM) or si-PDK-1, wound healing and transwell assays showed the capacity of horizontal migration (f), vertical migration (g), and invasion (h) of primary RA-FLS. Each bar represents mean ± SD from 6 RA patients. *P < 0.05, **P < 0.01, ***P < 0.001, compared with RA-FLS without treatment or si-NC group

Fig. 6

Proposed mechanism of artesunate on inhibition of RA-FLS migration and invasion