RA human fibroblast-like synoviocytes (HFLS-RAs) (ATCC, USA) were cultured in RPMI-1640 (Thermo Fisher, Waltham, Massachusetts, USA) containing 2% fetal bovine serum (FBS, Thermo Fisher). The medium was replaced every 2 days. HFLS-RAs with a confluence above 80% at passages 4 to 10 were used in this study.
To probe into the effects of plumbagin on the HFLS-RAs, HFLS were treated with plumbagin (Sigma, USA) at different concentrations (0.125, 0.25, 0.5, 1, 1.5, 2, 2.5, 3, 3.5 μM). Since methotrexate is a folate antagonist and a first-line drug for the treatment of RA [14 (link),15 (link)], it was used to treat HFLS-RAs at 2.2 μM as the positive control. 24, 48 and 72 hours (h) after the initiation of culture, cell viability was examined by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.
Additionally, to explore the effect of plumbagin on the IL-1β-induced HFLS-RAs, IL-1β (10 ng/mL) was used to establish an inflammatory model in these HFLS-RAs [16 ], HFLS-RAs were treated with 10 ng/mL IL-1β for 24 h, and then treated with plumbagin (0.125, 0.25, 0.5 μM) for 48 h and then the cells were collected for following experiment.
To probe into the effects of plumbagin on the HFLS-RAs, HFLS were treated with plumbagin (Sigma, USA) at different concentrations (0.125, 0.25, 0.5, 1, 1.5, 2, 2.5, 3, 3.5 μM). Since methotrexate is a folate antagonist and a first-line drug for the treatment of RA [14 (link),15 (link)], it was used to treat HFLS-RAs at 2.2 μM as the positive control. 24, 48 and 72 hours (h) after the initiation of culture, cell viability was examined by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.
Additionally, to explore the effect of plumbagin on the IL-1β-induced HFLS-RAs, IL-1β (10 ng/mL) was used to establish an inflammatory model in these HFLS-RAs [16 ], HFLS-RAs were treated with 10 ng/mL IL-1β for 24 h, and then treated with plumbagin (0.125, 0.25, 0.5 μM) for 48 h and then the cells were collected for following experiment.
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