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Fig. 6 | Arthritis Research & Therapy

Fig. 6

From: EC-18 prevents autoimmune arthritis by suppressing inflammatory cytokines and osteoclastogenesis

Fig. 6

EC-18 suppressed the production of inflammatory cytokines in human cells by reducing signal transducer and activator of transcription (STAT) 3 phosphorylation. A Peripheral blood mononuclear cells (PBMCs) (1 × 106 cells/1 ml) from healthy controls (n = 3, white bar) or patients with RA (n = 4, black bar) were cultured with EC-18 (10 or 100 μg/ml) or vehicle for 2 h and then untreated (Nil) or treated with anti-CD3 Ab for 3 days. Levels of IL-6, TNF-α, and IL-17 in the culture supernatant were determined using ELISA. B PBMCs (1 × 106 cells/1 ml) from healthy controls were cultured with EC-18 (10 or 100 μg/ml) or vehicle for 2 h and then untreated (Nil) or treated with anti-CD3 Ab for 3 days. To detect intracellular IL-17, the cells were re-stimulated with PMA and ionomycin in the presence of GolgiStop for 4 h. The percentages of IL-17+ or CD25+Foxp3+ cells among CD4+ cells were analyzed using flow cytometry. Values are percentages of positive cells. C PBMCs (5 × 106 cells/5 ml) from healthy controls were cultured with EC-18 (10 or 100 μg/ml) or vehicle for 2 h and then untreated (Nil) or treated with anti-CD3 Ab for 12 h. p-STAT3 (Tyr705) and STAT3 levels were determined via western blot analysis. Data are representative of three independent experiments. D PBMCs (1 × 106 cells/1 ml) from healthy controls were cultured with EC-18 (1 μg/ml) or vehicle for 2 h and then with or without anti-CD3 Ab for 48 h. RORc and Foxp3 mRNA expression was determined using real-time polymerase chain reaction (PCR). Data are presented as the means ± SEM of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 vs. vehicle-treated control

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