Enhanced expression of inflammatory cytokines and nuclear factor-κB in microglia by overdose fluoride

Abstract

Objective To investigate fluoride-induced inflammation and nuclear factor-κB (NF-κB) signaling pathway in cultured human acute monocytic leukemia cells (THP-1). Methods In vitro cultured THP-1 cells were used as a model of microglia. After cultured with different concentrations of [0 (negative control group), 10, 50, 100, 500, 1 000 and 5 000 μmol/L] sodium fluoride (NaF) for 48 h, the survival of cells was detected by CCK8. THP-1 cells were divided into 3 groups: control group, low dose and high dose fluoride groups according to the results of CCK8 assay, and then treated with different concentrations of sodium fluoride (0, 500, 5 000 μmol/L) for 48 h, concentrations of inflammatory cytokines, such as Interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) were measured by enzyme linked immunosorbent assay (ELISA) in THP-1 mononuclear cell culture medium. The protein levels of IκBα, phospho-NF-κB p65 and phospho-IκB-α were detected by Western blotting. Results THP-1 cells were treated with different concentrations of sodium fluoride (500, 1 000, 5 000 μmol/L) for 48 h. Fluoride group THP-1 cell survival rate [(73.21 ± 3.67)%, (31.40 ± 4.56)%, (0.40 ± 0.24)%] was lower than that of the negative control group [(100.00 ± 0.00)%, all P < 0.01]. Compared to the control groups [(0.36 ± 0.07), (31.07 ± 0.81)ng/L], significant increases of the inflammatory cytokines IL-1β [(1.42 ± 0.79), (19.47 ± 2.90)ng/L] and TNF-α [(61.06 ± 2.20), (172.72 ± 2.29)ng/L] were detected in culture medium in low-fluoride and high fluoride groups, respectively. Interestingly, compared to the control groups [(100.00 ± 5.48)%, (100.00 ± 14.82)%], significant increases of phospho-NF-κB p65 [(113.71 ± 8.99)%, (134.74 ± 1.93)%] and phospho-IκB-α [(152.61 ± 14.16)%, (176.91 ± 7.95)%] were observed in both low-fluoride and high fluoride groups. Meanwhile, the protein level of IκBα in high fluoride group [(63.53 ± 9.67)%] was significantly lower than that of the control group [(100.00 ± 10.99)%, P < 0.01]. Furthermore, significant positive correlation was detected between increased IL-1β, TNF-α and phospho-NF-κB p65 (r = 0.74, 0.72, all P < 0.05). Conclusions Excessive fluoride can induce microglial cells to release inflammatory cytokines and activate nuclear factor-κB signaling pathway. The release of inflammatory cytokines and activation of the signaling pathway may be one of the mechanisms of the damage of the central nervous system caused by sodium fluoride. Key words: Fluoride poisoning; Microglial; Nuclear factor κB