| ObjectiveDental fluorosis is a kind of enamel defect,which is caused by taking in overdosed fluoride during tooth development.The main reason of dental fluorosis is that the body takes in too much fluoride for a long time during the development of enamel.Although the etiology of dental fluorosis has been identified,the precise mechanism of dental fluorosis remains controversial.There are three direct effects on ameloblasts among these mechanisms.Firstly,for the proliferation of ameloblasts,micromolar concentration of fluoride promotes the proliferation,while millimolar concentration of fluroride inhibits their proliferation.Secondly,fluoride may regulate the differentiation of ameloblasts through the MAPK pathway.Thirdly,high fluoride promotes ameloblasts apoptosis through oxidative stress and endoplasmic reticulum stress.In the process of enamel matrix proteins formation,the addition of fluoride can also affect the formation of enamel indirectly.Furthermore,fluoride may change the structure and function of amelogenin,and which plays an important part in buffering the pH,thereby destroying the adjustment of the pH.However,it is still unclear how fluoride ions enter ameloblasts and cause a series of stress responses.The purpose of this research was to study the process of fluoride entering ameloblasts.The ameloblasts in the secretory stage and maturation stage were treated with high fluoride,and the changes of fluoride concentration in ameloblasts under high environmental fluoride were observed with a new type of fluoride fluorescence probe.At the same time,the expression of TGF-?1 and the chloride channel proteins ClC-5?ClC-7 and CFTR in the secretory stage and maturation stage ameloblasts under high environmental fluoride were detected by RT-qPCR and Western Blot.Then the possible mechanism of regulating the expression of chloride channel proteins ClC-5?ClC-7 and CFTR in the secretory stage and maturation stage ameloblasts was explored by examining the effects of exogenous TGF-?1 and its receptor inhibitors on the expression of chloride channel proteins ClC-5?ClC-7 and CFTR in ameloblasts under high environmental fluoride,which may provide a new idea for the prevention and treatment of dental fluorosis.Methods1 By using a new type of fluoride fluorescence probe,the fluorescence intensities of ameloblasts in the secretory stage and maturation stage were observed by laser scanning confocal microscope(LSCM)under high environmental fluoride.The expressions of TGF-?1 mRNA and protein in the secretory stage and maturation stage ameloblasts under high environmental fluoride were detected by RT-qPCR and Western Blot.2 The ameloblasts in the secretory stage and maturation stage were inoculated in culture dish,and the expressions of CIC-5?ClC-7 and CFTR mRNA and protein under high environmental fluoride were detected by RT-qPCR and Western Blot.3 The ameloblasts in the secretory stage and maturation stage were inoculated in culture dish,and were divided into groups according to the following conditions:the control group,the high fluoride group,the TGF-?1 group,the fluoride+TGF-?1 group,the TGF-?1+LY2157299 group.The expressions of ClC-5?ClC-7?CFTR mRNA and ClC-5?ClC-7?CFTR?p-Smad2 protein under different conditions were detected by RT-qPCR and Western Blot.Results1 The fluorescence intensities of the high fluoride group in the secretory stage and maturation stage ameloblasts were higher than those of the control group.TGF-?1 mRNA expression levels of the high fluoride group in the secretory stage and maturation stage ameloblasts were lower than those of the control group.Consistent with the RT-qPCR results,TGF-?1 protein expression levels of the high fluoride group in the secretory stage and maturation stage ameloblasts were lower than those of the control group.2 In the high fluoride group of the secretory stage ameloblasts,ClC-5 and ClC-7 mRNA expression levels were higher than those of the control group.Consistent with the RT-qPCR results,ClC-5 and ClC-7 protein expression levels in the high fluoride group of the secretory stage ameloblasts were higher than those of the control group.In the high fluoride group of the maturation stage ameloblasts,ClC-5?ClC-7 and CFTR mRNA expression levels were higher than those of the control group.Consistent with the RT-qPCR results,ClC-5?ClC-7 and CFTR protein expression levels in the high fluoride group were higher than those of the control group.3 In the high fluoride group of the secretory stage ameloblasts,ClC-5 and ClC-7 mRNA expression levels were higher than those of the control group.In the TGF-?1 group of the secretory stage ameloblasts,ClC-5 and ClC-7 mRNA expression levels were lower than those of the control group.In the fluoride+TGF-?1 group of the secretory stage ameloblasts,ClC-5 and ClC-7 mRNA expression levels were lower than those of the high fluoride group.In the TGF-?1+LY2157299 group of the secretory stage ameloblasts,ClC-5 and ClC-7 mRNA expression levels were higher than those of the TGF-?l group.Consistent with the RT-qPCR results,ClC-5 and ClC-7 protein expression levels of high fluoride group in the secretory stage ameloblasts were higher than those of the control group,p-Smad2 protein expression level was lower than that of the control group.In the TGF-?1 group of the secretory stage ameloblasts,ClC-5 and ClC-7 protein expression levels were lower than those of the control group,p-Smad2 protein expression level was higher than that of the control group.In the fluoride+TGF-?1 group of the secretory stage ameloblasts,ClC-5 and ClC-7 protein expression levels were lower than those of the high fluoride group,p-Smad2 protein expression level was higher than that of the high fluoride group.In the TGF-?1+LY2157299 group of the secretory stage ameloblasts,ClC-5 and ClC-7 protein expression levels were higher than those of the TGF-?1 group,p-Smad2 protein expression level was lower than that of the TGF-?1 group.In the high fluoride group of the maturation stage ameloblasts,ClC-5?ClC-7 and CFTR mRNA expression levels were higher than those of the control group.In the TGF-?1 group of the maturation stage ameloblasts,ClC-5?ClC-7 and CFTR mRNA expression levels were lower than those of the control group.In the fluoride+TGF-?1 group of the maturation stage ameloblasts,ClC-5?ClC-7 and CFTR mRNA expression levels were lower than those of the high fluoride group.In the TGF-?1+LY2157299 group of the maturation stage ameloblasts,ClC-5?ClC-7 and CFTR mRNA expression levels were higher than those of the TGF-?1 group.Consistent with the RT-qPCR results,ClC-5?ClC-7 and CFTR protein expression levels of high fluoride group in the maturation stage ameloblasts were higher than those of the control group,p-Smad2 protein expression level was lower than that of the control group.In the TGF-?1 group of the maturation stage ameloblasts,ClC-5?ClC-7 and CFTR protein expression levels were lower than those of the control group,p-Smad2 protein expression level was higher than that of the control group.In the fluoride+TGF-?1 group of the maturation stage ameloblasts,ClC-5?ClC-7 and CFTR protein expression levels were lower than those of the high fluoride group,p-Smad2 protein expression level was higher than that of the high fluoride group.In the TGF-?1+LY2157299 group of the maturation stage ameloblasts,ClC-5?ClC-7 and CFTR protein expression levels were higher than those of the TGF-?1 group,p-Smad2 protein expression level was lower than that of the TGF-?1 group.Conclusions1 The fluoride ions can accumulate in the secretory stage and maturation stage ameloblasts under high environmental fluoride.2 The expressions of TGF-?1 in the secretory stage and maturation stage ameloblasts can be inhibited under high environmental fluoride.3 The expressions of chloride channel proteins ClC-5,ClC-7 and CFTR in the secretory stage and maturation stage ameloblasts can be strengthened under high environmental fluoride.4 It is the first time to demonstrate that the expressions of chloride channel proteins ClC-5?ClC-7 and CFTR in the secretory and maturation stage ameloblasts can be regulated through TGF-?1 signaling pathway under high environmental fluoride. |
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