The Effects Of Fluoride On Nrf2Signal Pathway In Mouse Ameloblasts

Background and ObjectiveFluorosis is a chronic disease, caused by chronic ingestion of high concentrationof fluoride through the drinking water, food and air intake. Fluoride has an importanteffect on human’s health. Appropriate fluoride is beneficial to accelerate bone-growthas well as to prevent caries. However, chronic excessive intake of fluoride may causeskeletal fluorosis, dental fluorosis so far as to the pathological changes in multi-organmetabolic disorders including the central nervous system, liver, kidney and endocrinesystem. Dental fluorosis not only cause serious damage to teeth but also affected theappearance. The continuous intake of excessive fluoride in the mineralization stage ofthe teeth growth is the underlying etiology of dental fluorosis. However, pathogenesisof dental fluorosis has not been fully elucidated.Ameloblasts play the key role in thetooth formation process. The Ameloblasts are the most sensitive cells to fluorosis. Thefollowing dental fluorosis mechanisms have been proposed: Fluoride increasesapoptosis on ameloblasts; Fluoride interferes with the synthesis and secretion ofenamel matrix; Fluoride inhibits ameloblasts proliferation; Therefore, ameloblasts isan important object in the study for pathogenesis of dental fluorosis. And our researchhave carried out the studies of fluoride on injury mechanism in osteoblast.In recent years, experts had been studying the relationship between fluorine andapoptosis at the molecular level. Apoptosis plays an important role in toothdevelopment. Oxidative stress is a central link in apoptosis. As a nuclear transcription factor NF-E2-related factor2and the cytoplasmic adapter protein Kelch-like ofECH-associated protein1(Keap1) is a central regulator of cell oxidative stress.Transcription factor Nrf2binds to the antioxidant response element (ARE), codingantioxidant enzymes and phase II expression of drug metabolizing enzymes incytoprotection and antioxidant defense, increasing the body resistance andmaintaining the body to survive.In summary, this study used NaF as exposure factor to treat normal mouseameloblasts cultured in vitro, investigated the effects of fluoride on oxidative stress,DNA damage, cell proliferation and apoptosis as well as the role of the Nrf2signalingpathway on the basis of the oxidative damage caused by fluoride in mouseameloblasts. A new experimental evidence for the mechanism of the dental fluorosiswould be provided by exploring the protective effects on the Nrf2signaling pathwayof the damage caused by fluoride in ameloblasts. Oxidative stress is generallyconsidered to be an important mechanism of fluorosis. The study also investigated theeffects of Lycium barbarum polysaccharide(LBP), Oligomeric Proantho Cyanidins(OPC), selenium and zinc on the DNA damage caused by fluoride in mouseameloblasts, to provide the scientific significance for the prevention of fluorosis.Part I Effects of fluoride on oxidative stress, cell proliferation andapoptosis in mouse ameloblastsObjective To determine the fluoride-exposed doses and explore the effects of fluorideat different concentrations on oxidative stress, cell proliferation, DNA damage andapoptosis of mouse ameloblasts.Methods After established and improved the method of mouse ameloblasts culture invitro, cell proliferation activity was measured by MTT assay after the ameloblastswere incubated with sodium fluoride in vitro. The contents of malonyl-dialdehyde(MDA) and activities of superoxide dismutase(SOD), glutathione peroxid-ase(GSH-Px) were measured. DNA damage in ameloblast was determined by singlecell gel (SCG) electrophoresis or comet assay, and Elisa was employed to measure the level of8-OHdG. Apoptosis and cell cycle in ameloblasts were detected by flowcytometry.Results The improved cell culture method had good repetition characterisrtic. Usedameloblasts to establish the cell model in vitro for fluorosis had feasibility. Theexperiment demonstrated that fluoride influenced the proliferation of ameloblasts.Fluoride could increase the content of MDA and reduce the level of SOD and GSH.DNA damage results showed that: Compared with the control group, tail rate, taillength, olive tail moment, tail moment, DNA content in tail were increased in theconcentration of0.25-4.00mmol/L NaF, difference were statistically significant. The8-OHdG levels in ameloblasts was significantly increased at0.50,1.00,4.00mmol/l(P<0.05). The percentage of G2/M and G0/G1stage cells increasedsignificantly in the2.00mmol/L NaF group(P<0.05). Fluoride at4.00mmol/Lincreased the cell number of S phase and G2/M phase. A significant increase inapoptosis rate of ameloblasts was observed after exposure to4.00mmol/LNaF(P<0.05).Conclusion After the cells were treated for24hours, fluoride inhibited theproliferation of ameloblasts significantly. Fluoride could induce oxidative stress andcause DNA damage. A certain dose of fluoride could arrest the cells in S phase,decrease cell viability, change cell cycle and trigger apoptosis.Part II The effects of fluoride on Nrf2signal pathway in mouseameloblastsObjective To investigate the protective effect of Nrf2signaling pathway andtert-butylhydroquinone on cytotoxicity induced by fluoride in ameloblasts in vitro, theinfluences of Nrf2proteins and expression of HO-1, NQO1, Nrf2mRNA in culturedameloblasts in vitro were explored at the different doses of fluoride.Methods RT-PCR was applied to measure the quantity of HO-1, NQO1, Nrf2mRNAin ameloblasts which were exposed to different does of fluoride. Western-blotting wasapplied to measure the content of intracellular Nrf2protein. Results Fluoride could continuous induce Nrf2mRNA and Nrf2protein expression.The mRNA expression level of Nrf2gene increased significantly in the1.00,4.00mmol/L NaF group(P<0.05). The mRNA expression level of HO-1wassignificant increased in0.50,1.00,2.00and4.00mmol/L NaF, and the mRNAexpression level of NQO-1was significant increased in1.00mmol/L NaF. NaF canincrease Nrf2protein expression in cytoplasm. Nrf2protein expression in nucleus wasincreased than the control group. tBHQ pretreatment and then exposure to fluoride,the level of Nrf2, HO-1, NQO1mRNA and Nrf2protein expression was significantlyhigher than a separate fluoride group. Nrf2protein expression in cytoplasm wasincreased than in nucleus. The mRNA expression level of Nrf2was significantincreased in (0.00,0.25,0.50,2.00,4.00mmol/L) NaF than that of control group(P<0.05). The mRNA expression level of HO-1was significant increased in (0.00,0.50,2.00,4.00mmol/L) NaF than that of control group (P<0.05). The mRNAexpression level of NQO1was significant increased in (0.25,4.00mmol/L) NaF thanthat of control group (P<0.05). The nuclear/cytoplasm ratio was significant increasedin (0.25,1.00,2.00,4.00mmol/L) NaF than that of control group (P<0.05).Conclusion Fluoride under certain dosage could induce Nrf2gene and proteinexpression, to increase the mRNA expression level of HO-1and NQO1. tBHQpretreatment could increase the mRNA expression level of Nrf2, HO-1and NQO1,also increase the Nrf2protein in cytoplasm.Part Ⅲ Studies on the effects of antioxidant agents on preventionand treatment of fluorosisObjective To investigate the effects of LBP, OPC, selenium and zinc on the DNAdamage caused by fluoride in mouse ameloblasts.Methods Ameloblasts adherent cultured24h with different concentrations of antioxi-dants:100mg/L,200mg/L,400mg/L of LBP;10mg/L,25mg/L,50mg/L of OPC;2.5μmol/L,5μmol/L,10μmol/L of Na2SeO3;5μmol/L,10μmol/L,20μmol/L of ZnSO4.The medium was changed by adding different doses of NaF. Single cell gelelectrophoresis assay was used to test the DNA damage rate in mouse ameloblasts.Results LBP, OPC, selenium and zinc treatment group in olive tail moment wassignificantly lower than that of control group (P<0.05).400mg/L LBP treatmentgroup in olive tail moment was significant decrease in0.25,0.50,2.00,4.00mmol/LNaF than100mg/L,200mg/L LBP treatment group(P<0.05);10mg/L OPC treatmentgroup in olive tail moment was significant decrease in0.00,0.25,0.50,1.00mmol/LNaF than25mg/L,50mg/L OPC treatment group(P<0.05);2.5μmol/L Se treatmentgroup in olive tail moment was significant decrease in0.00,0.25,0.50,1.00mmol/LNaF than5μmol/L,10μmol/L Se treatment group(P<0.05);10mg/L Zn treatmentgroup in olive tail moment was significant decrease in0.00,0.25,0.50,1.00,2.00,4.00mmol/L NaF than5μmol/L,20μmol/L Zn treatment group(P<0.05).Conclusion The certain dosage LBP, OPC, selenium and zinc could antagonize DNAdamage caused by fluoride. It was suggested that anti-oxidation have a significantprotective effect on fluorosis in mouse ameloblasts.