| Objective:Fluorine is one of the important chemical elements in the human body and is widely distributed in nature.However,long-term excessive intake of fluoride can cause chronic systemic diseases,mainly fluorosis and dental fluorosis,namely fluorosis.Patients with dental fluorosis may show different degrees of enamel development defects,such as spots,plaques and accompanied by different degrees of enamel formation disorders and other symptoms,but the specific mechanism has not been fully elucidated.Several studies have shown that fluoride can affect enamel mineralization by inhibiting the expression of specific genes in the maturation phase of ameloblasts.It has been shown that activation ofβ-catenin in ameloblasts causes mineralization abnormalities and ultimately leads to mineralized incisal enamel hypomineralization.However,whether fluoride affects ameloblasts differentiation via PPARγupstream of Wnt and the circadian rhythm has not been reported.Therefore,we selected mouse LS8 cells for in vitro culture and established an in vivo fluorosis model in SD rats to observe whether fluoride inhibits ameloblasts differentiation and to investigate the role of Wnt/β-catenin signaling pathway and its upstream PPARγand circadian rhythm in this process.This study provides a new direction to elucidate the study of defective enamel formation caused by fluoride and provides new ideas for the prevention and treatment of dental fluorosis.Methods:In vivo experiments:6-week-old Sprague-Dawley(SD)male rats were purchased from the Experimental Animal Center of China Medical University under license No.CMU2022028.30 rats were randomly divided into 3 groups according to their body weight,i.e.,control,50 ppm F~-and 100 ppm F~-groups,with 10 rats in each group.After 3 days of acclimatization feeding,the control group was given distilled water and the poisoned group was given distilled water containing 50 ppm F~-and 100ppm F~-,respectively,and each group was given a free diet and water for 6 weeks of the poisoning cycle.Urine was collected and the urine fluoride content was measured.After anesthesia with an intraperitoneal injection of duration(20%,0.75 ml/100g),and the serum was collected.The blood fluoride content was measured by the fluoride ion selective electrode method.After stripping the mandible,the length was measured with vernier calipers and recorded,and the stripped mandible was fixed in 4%paraformaldehyde fixative for 2 d.The mandible was decalcified with 15%EDTA decalcification solution for 3-4 months until the end point of decalcification(the mandible was soft without obvious resistance to needle pricking),dehydrated in gradient alcohol(70%ethanol,80%ethanol,90%ethanol and 95%ethanol for 12 h each,100%ethanol I and II for 1.5 h each),and dehydrated in xylene(70%ethanol,80%ethanol,90%ethanol and 95%ethanol for 1.5 h each).After 1.5 h of gradient alcohol dehydration(70%ethanol,80%ethanol,90%ethanol and 95%ethanol for 12h,100%ethanol I and II for 1.5 h each),xylene I and II were transparent for about 30min each(to the extent that the tissue was transparent to the naked eye).Immunohistochemistry was performed to detect the expression of proliferation and differentiation-related genes,stromal degradation-related genes,Wnt signaling pathway-related genes,PPARγand clock genes.In vivo experiments:mouse ameloblasts were selected and cultured in high sugar culture medium with 10%fetal bovine serum,100 U/ml penicillin,and 100 g/ml streptomycin,and the medium was changed every two days.CCK8 was used to detect cell survival and determine the staining experiment and dose;immunofluorescence was used to detectβ-catenin entry into the nucleus;Western Blot and PCR were used to detect proliferation and differentiation-related the m RNA and protein expression of genes related to proliferation and differentiation,matrix degradation-related genes,Wnt signaling pathway-related genes,PPARγand clock genes were detected by Western Blot and PCR;after adding DKK1,an inhibitor of Wnt signaling pathway,the m RNA and protein expression were detected by Western Blot to detect the protein expression of proliferation and differentiation-related genes and Wnt signaling pathway-related genes;Western Blot to detect proliferation and differentiation-related genes after treating cells with the antioxidant resveratrol,the expression of the genes related to proliferation and differentiation,matrix degradation-related genes,Wnt signaling pathway-related genes,PPARγand clock genes were detected by Western Blot after treatment with the antioxidant resveratrol.Results:In vivo experiments:1.Effect of fluorine on blood and urine fluoride in rats:compared with the control group,fluorine significantly inhibited the concentration of fluoride ions in the blood and urine of rats(P<0.05).2.Effect of fluorine on proliferation and differentiation of rat ameloblasts:compared with the control group,fluorine significantly inhibited the expression of proliferation marker and differentiation marker proteins in rat mandibular ameloblasts(P<0.05).3.The effect of fluorine on matrix degradation:compared with the control group,fluorine significantly inhibited the expression of matrix lectin marker(MMP9)protein in rat mandibular ameloblasts(P<0.05).4.The effect of fluorine on Wnt/β-catenin signaling pathway in ameloblasts:compared with the control group,fluorine activated the expression of Wnt3a andβ-catenin protein and inhibited the expression of CXXC5protein(P<0.05).5.Effect of fluorine on PPARγand clock gene-related molecules in ameloblasts:fluorine inhibited the expression of PPARγ,Clock,Bmal1 and Per2proteins compared with the control group.In vitro assays:1.Effect of fluorine on LS8cell proliferation and differentiation and matrix degradation:compared with the control group,fluorine significantly inhibited the expression of LS8 proliferation marker and differentiation marker and matrix degradation marker protein and m RNA expression(P<0.05).2.Effect of fluorine on Wnt/β-catenin signaling pathway in ameloblasts:compared with control group the effect of fluorine on Wnt3a,β-catenin protein and m RNA expression,and the inhibition of CXXC5 protein expression(P<0.05).3.Effect of fluorine on PPARγand clock gene-related molecules in ameloblasts:compared with the control group,fluorine inhibited the expression of PPARγ,Clock,Bmal1 and Per2 proteins and m RNA(P<0.05).4.Immunofluorescence detection ofβ-catenin entry:fluorine significantly increased the entry ofβ-catenin compared with the control(P<0.05).5.After adding DKK1,an inhibitor of Wnt signaling pathway,DKK1 pretreatment improved the fluorine-induced increase in Wnt3a andβ-catenin protein expression,as well as the fluoride-induced decrease in CXXC5,RUNX2 and MMP9 expression(P<0.05).6.Flow cytometry assay of reactive oxygen content in LS8 cells:fluorine increased the reactive oxygen content in LS8 cells compared with the control group(P<0.05).7.Treatment with the antioxidant resveratrol upregulated fluorine-inhibited proliferation differentiation and matrix degradation in LS8 cells:after the exogenous addition of resveratrol,the expression of PCNA,RUNX2 and MMP9 proteins were significantly higher in the Na F+RES(Resveratrol)group cells compared with the fluoride-stained group(P<0.05).8.Antioxidant resveratrol treatment inhibited Wnt/β-catenin signaling pathway-related molecules in fluorine-activated LS8 cells:after exogenous addition of resveratrol,Wnt3a andβ-catenin protein expression were significantly lower in the Na F+RES group compared with the fluorine-stained group(P<0.05).9.Antioxidant resveratrol treatment up-regulated PPARγand clock gene-related molecules in fluorine-suppressed LS8 cells related to:after exogenous addition of resveratrol,PPARγ,Clock,Bmal1 and Per2 protein expressions were significantly higher in Na F+RES group cells compared with the fluoride-stained group(P<0.05).Conclusion:Fluoride inhibits the expression of PPARγand clock genes through activation of the Wnt/β-catenin signaling pathway,which in turn downregulates the expression of molecules related to proliferation differentiation and matrix degradation in rat ameloblasts and LS8 cells.Pretreatment of LS8 cells with or without Na F with the antioxidant resveratrol partially rescued the circadian rhythm disturbance induced by oxidative stress,inhibited the activation of the Wnt signaling pathway,partially restored the effects on ameloblasts differentiation and matrix degradation,and protected the cells from fluoride oxidative damage. |
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