RESUMEN
Objective:To investigate whether the necroptosis pathway receptor interacting protein 1-receptor interacting protein 3-mixed lineage kinase domain-like protein (RIP1-RIP3-MLKL) is involved in fluoride-induced osteoblastic death.Methods:Human osteosarcoma cell line (Saos-2 cells) were cultured in vitro and divided into NC group, sodium fluoride (NaF) groups (5.0, 10.0, 20.0 and 40.0 mg/L NaF), necroptosis inhibitor Necrostatin-1 (Nec-1) group (50.0 μmol/L Nec-1) and NaF + Nec-1 groups (5.0, 10.0, 20.0 and 40.0 mg/L NaF + 50.0 μmol/L Nec-1). After cultured for 24 and 48 h, respectively, cell proliferation was determined via the CCK-8 method, and the activity of lactate dehydrogenase (LDH) was determined by chemical colorimetry. To further analyze the influence of NaF on RIP1-RIP3-MLKL pathway, Saos-2 cells were divided into NCⅡgroup and NaFⅡgroups (2.5, 5.0, 10.0, 20.0 and 40.0 mg/L NaF). After cultured for 24 and 48 h, respectively, the protein expression levels of RIP1, RIP3 and MLKL were determined by Western blotting. Results:The cell proliferation rates (%: 100.00 ± 0.59, 104.90 ± 0.44, 104.16 ± 0.41, 82.45 ± 1.91, 64.59 ± 1.83, 103.56 ± 0.41, 107.18 ± 0.87, 105.35 ± 1.28, 89.63 ± 1.20, 77.51 ± 1.30; 100.00 ± 0.33, 107.92 ± 0.44, 101.78 ± 1.06, 75.45 ± 0.39, 57.94 ± 1.17, 106.74 ± 0.21, 111.85 ± 0.21, 107.82 ± 0.68, 82.34 ± 0.56, 70.19 ± 0.99) among all groups were significantly different at both 24 and 48 h ( F = 77.13, 2 313.43, P < 0.05). Except the cell proliferation rate of the 10.0 mg/L NaF + Nec-1 group that was not significantly different with that of the 10.0 mg/L NaF group at 24 h ( P > 0.05), the cell proliferation rates of other NaF + Nec-1 groups were significantly higher than those of corresponding NaF groups at both 24 and 48 h ( P < 0.05). The proliferation rate was negatively correlated with fluoride concentration ( r24 h = - 0.976, r48 h = - 0.969, P < 0.001). The LDH activity in all concentrations of NaF groups was significantly higher than that in NC group and corresponding NaF + Nec-1 groups at both 24 and 48 h ( P < 0.05). The LDH activity was positively correlated with fluoride concentration ( r24 h = 0.985, r48 h = 0.988, P < 0.001). The protein expression levels of RIP1, RIP3 and MLKL of 5.0 mg/L NaFⅡ group at 24 h, RIP3 of 5.0 mg/L NaFⅡ group at 48 h, and RIP1, RIP3 and MLKL of 10.0, 20.0 and 40.0 mg/L NaFⅡ groups at both 24 and 48 h were higher than that in NCⅡ group ( P < 0.05). The protein expression levels of RIP1, RIP3 and MLKL were positively correlated with fluoride concentration ( r24 h-RIP1 = 0.881, r48 h-RIP1 = 0.952, r24 h-RIP3 = 0.867, r48 h-RIP3 = 0.938, r24 h-MLKL = 0.758, r48 h-MLKL = 0.907, P < 0.001). Conclusion:Fluoride can directly cause necroptosis of osteoblasts through the RIP1-RIP3-MLKL pathway, and the severity of cell damage is closely related to fluoride concentration, Nec-1 has partially reversed the effects of fluoride.
RESUMEN
Endemic fluorosis is a chronic systemic disease that seriously endangers human health. In high fluoride areas, people consume excessive fluoride for a long time through drinking water or food, which leads to chronic cumulative fluorosis in the body. Fluorosis can cause changes in the expression of some miRNA in cells, and the miRNA can participate in fluoride-induced osteoblast activation through various signal pathways. To observe the differential expression of apoptosis-related microRNA (miRNA) in mouse osteoblasts under the action of excessive fluoride. Primary cultured mouse osteoblasts, identified by osteocalcin (OC) and alkaline phosphatase (ALP) staining, were treated with 20 mg/L sodium fluoride and 40 mg/L sodium fluoride for 12/24 hr, respectively, to establish the fluoride staining model for comparing and analyzing the sequence of miRNA among groups by bioinformatics methods; four miRNA chains were verified by fluorescence quantitative PCR. After treatment with 20 mg/L sodium fluoride for 12 hr and 24 hr, 128 miRNA expressions were up-regulated while 36 miRNA expressions were down-regulated. In Group 40 mg/L, 130 miRNA expressions were up-regulated while 29 miRNA expressions were down-regulated after 12 hr and 24 hr; 72 miRNA were up-regulated and 2 miRNA were down-regulated at the two time points. 10 up-regulated miRNA and 2 down-regulated miRNA with higher scores in Bioinformatics software were analyzed the target genes. Fluorescence quantitative PCR verified that the expressions of four miRNA were up-regulated. Target gene analysis of the 10 selected mouse osteoblastic apoptosis-related miRNA reveals their involvement of the functions of inhibiting or promoting apoptosis, which has certain theoretical significance for early identification of skeletal fluorosis. The involved signaling pathways include the Wnt signaling pathway, ubiquitin-regulated proteolysis, Toll signaling pathway, TNF signaling pathway, pluripotent stem cell signaling pathway, MAPK signaling pathway, phosphatidylinositide metabolism, FoxO signaling pathway, ErbB signaling pathway, autophagy, and so forth.