Multiple-Factors-Induced Rheumatoid Arthritis Synoviocyte Activation Is Attenuated by the α2-Adrenergic Receptor Agonist Dexmedetomidine

Abstract

Dexmedetomidine (Dex) has analgesic and sedative properties and anti-inflammatory functions. Although the effects of Dex on arthritis have been revealed, the physiological mechanism underlying the interaction between Dex and rheumatoid arthritis (RA)-mediated inflammatory cytokines has not been fully studied. Inflamed and migrated fibroblast-like synoviocytes (FLSs) are involved in RA severity. Thus, we aimed to determine the effects of Dex on RA-FLSs treated with inflammatory cytokines and a growth factor as multiple stimulating inputs. TNF-α, IL-6, and EGF as multiple stimulating inputs increased the cAMP concentration of RA-FLSs, while Dex treatment reduced cAMP concentration. Dex reduced electroneutral sodium-bicarbonate cotransporter 1 (NBCn1) expression, NBC activity, and subsequent RA-FLS migration. The mRNA expression levels of RA-related factors, such as inflammatory cytokines and osteoclastogenesis factors, were enhanced by multiple-input treatment. Notably, Dex effectively reduced these expression levels in RA-FLSs. These results indicate that multiple inflammatory or stimulating inputs enhance RA-FLS migration, and treatment with Dex relieves activated RA-FLSs, suggesting that Dex is a potential therapeutic drug for RA.

Keywords: EGF; FLS; IL-6; TNF-α; dexmedetomidine; rheumatoid arthritis.

Figure 1

Dex inhibited NBC activities stimulated by multiple inflammatory or proliferative inputs in RA-FLSs. (a) mRNA expression of α2-AR in human RA-FLS cells with or without Dex treatment. (b) Dot plots represent the cellular viability of RA-FLSs following dose-dependent treatment with Dex (n = 3, ** p < 0.01). (c) Dot plots represent the cellular viability of RA-FLSs following time-dependent treatment with Dex (100 ng/mL, n = 3, * p < 0.05). (d) Protein expression of NBCn1 and IRBIT in human RA-FLS cells with or without Dex. (e) Bars represent the mean ± SEM (n = 3). (f) Bars represent the mean ± SEM of cAMP concentration in RA-FLSs with or without Dex treatment (n = 4, * p < 0.05, ** p < 0.01). (g–l) NBC activity was assessed by measuring the changes in pH_i with 10 ng/mL TNF-α (g), 50 ng/mL IL-6 (i), and 10 ng/mL EGF (k) with or without 100 ng/mL Dex in RA-FLSs for 6 h using transwell assay. Bars represent the mean ± SEM of TNF-α (h), IL-6 (j), and EGF (l) (n = 5, ** p < 0.01 and *** p < 0.001).

Figure 2

Inhibitory role of Dex in the intracellular signaling pathway in RA-FLSs. (a) Protein expression of Akt, Erk, and NF-κB (total and phosphorylated forms) in human RA-FLS cells with or without 10 ng/mL TNF-α and 100 ng/mL Dex. (b–d) Bars represent the mean ± SEM of p-Akt (b), p-Erk (c), and p-NF-κB (d) protein expression (n = 3, * p < 0.05, ** p < 0.01). (e) Protein expression of Akt, Erk, and NF-κB (total and phosphorylated forms) in human RA-FLS cells with or without 50 ng/mL IL-6 and 100 ng/mL Dex. (f–h) Bars represent the mean ± SEM of p-Akt (f), p-Erk (g), and p-NF-κB (h) protein expression. (i) Protein expression of Akt, Erk, and NF-κB (total and phosphorylated forms) in human RA-FLS cells with or without 10 ng/mL EGF and 100 ng/mL Dex. (j–l) Bars represent the mean ± SEM of p-Akt (j), p-Erk (k), and p-NF-κB (l) protein expression (n = 3, ** p < 0.01).

Figure 3

Dex treatment attenuated the migration of RA-FLSs stimulated by multiple inputs. RA-FLS migration was measured following stimulation with 10 ng/mL TNF-α (a), 50 ng/mL IL-6 (c), and 10 ng/mL EGF (e) in the transwell culture system with or without 100 ng/mL Dex. Analysis of the total DAPI intensity for RA-FLS migration with TNF-α (b), IL-6 (d), and EGF (f). Bars represent the mean ± SEM (n = 5, * p < 0.05 and ** p < 0.01).

Figure 4

Directional stimulation of TNF-α- and EGF-mediated membranous NBCn1 expression in RA-FLSs was attenuated by Dex. (a) Immunofluorescence staining of NBCn1 (red) using 2D culture on coverslips or the transwell membrane. Left images (−) indicate the negative control (absence of NBCn1 antibodies). Scale bar represents 20 μm. (b) Immunofluorescence staining of NBCn1 (red) using transwell membrane after treatment with 10 ng/mL TNF-α, 50 ng/mL IL-6, and 10 ng/mL EGF on the bottom plate with or without 100 ng/mL Dex in the upper chamber for 6 h. Scale bar represents 20 μm. (c) Bars represent the mean ± SEM of the normalized band intensity of NBCn1 in RA-FLSs (n = 5, ** p < 0.01 and *** p < 0.001).

Figure 5

TNF-α- and EGF-mediated NBCn1 expression was attenuated by Dex with no change in IRBIT in RA-FLSs. (a–f) Protein expression levels of NBCn1 and IRBIT following treatment with 10 ng/mL TNF-α (a), 50 ng/mL IL-6 (c), and 10 ng/mL EGF (e) with or without Dex in RA-FLSs for 6 h using a transwell membrane. β-actin was used as a loading control. Bars represent the mean  ±  SEM of the normalized band intensity of NBCn1 and IRBIT following treatment with 10 ng/mL TNF-α (b), 50 ng/mL IL-6 (d), and 10 ng/mL EGF (f) with or without Dex in RA-FLSs (n = 3, * p < 0.05). (g) Immunofluorescence staining of IRBIT (red) using a transwell membrane after treatment with 10 ng/mL TNF-α, 50 ng/mL IL-6, and 10 ng/mL EGF on the bottom plate with or without 100 ng/mL Dex in the upper chamber for 6 h. Scale bar represents 20 μm.

Figure 6

Dex inhibited multiple-input-associated RA factors in RA-FLSs. The relative mRNA levels of IL-6, IL-8, IL-1β, IL-27, COX-2, DKK-1, and RANKL stimulated with 10 ng/mL TNF-α (a), 50 ng/mL IL-6 (b), and 10 ng/mL EGF (c) with or without 100 ng/mL Dex for 48 h in transwell-cultured RA-FLSs. Bars represent mean ± SEM (n = 4, * p < 0.05, ** p < 0.01, and *** p < 0.001).

Figure 7

Schematic illustration of RA-FLS activation by multiple inputs and the inhibitory role of Dex. Multiple-input-mediated cAMP stimulation increased NBCn1 expression and activity by upregulating RA factors, such as IL-6, IL-8, IL-1β, IL-27, COX-2, DKK-1, and RANKL, which were attenuated by Dex treatment. Dex: dexmedetomidine, AR: adrenergic receptor, IL: interleukin, COX-2: cyclooxygenase-2, DKK-1: Dickkopf-1, RANKL: receptor activator of nuclear factors κB ligand, IRBIT: the IP₃ receptor-binding protein released with IP₃, NBCn1: electroneutral sodium-bicarbonate cotransporter 1.