Study On The Effect Of Betaine On Skeletal Fluorosis Based On DNA Methylation Of RANKL/OPG System

Pert 1.A case-control study on association of betaine and RANKL/OPG promoter DNA methylation with skeletal fluorosis Objective: Recent studies have demonstrated that DNA methylation plays a crucial role in the pathogenesis of fluorosis.Fluoride causes bone metabolic diseases by disrupting the expression of crucial gene Receptor activator of NF-k B-ligand(RANKL)and Osteoprotegerin(OPG)of bone remodel.However,the epigenetic mechanism of fluoride on RANKL/OPG expression is still unclear.The natural nutrient betaine has an important biological effect as an antioxidant and an efficient dietary methyl donor.A case-control study was conducted in this study to explore the relationship between betaine,RANKL/OPG promoter region DNA methylation and serum expression level and skeletal fluorosis risk.Methods: 218 skeletal fluorosis patients were collected from a coal-burning fluorosis area of Zhiin county Guizhou province,and 218 healthy participants who were 1:1 matched by gender and age(± 3 years)from the same district were recruited as control.The participants were 18-75-year-old natives of Zhijin county and those who have lived in there for at least 10 years.Fasting peripheral blood samples and first-spot morning urine were obtained from participants.A structure questionnaire was used to collect the socio-demographic factors,fluorosis-related lifestyle risk factors and history of diseases information though face-to-face interview.The 75-item food frequency questionnaire(FFQ)was used to investigate dietary consumption from all participants in the past one year.And then we conducted physical examinations on the participants and collected medical information.Urinary fluoride content was measured by the ion electrode method.The RANKL,OPG gene promoter DNA methylation levels were detected by Targeted Bisulfite Sequencing(TBS).Enzyme-linked immunosorbent assay(ELISA)was used to determine the serum expression level of RANKL,OPG.Results: 1.The association between dietary betaine and skeletal fluorosis risk.The urinary fluoride content was significantly higher in skeletal fluorosis patients as compared to controls(cases: median 1.46 mg/L,controls: median 1.18 mg/L,P<0.001),and the skeletal fluorosis patients had lower intake of betaine(cases:median 82.68 mg/day,controls: median 109.90 mg/day,P<0.001).After adjustment by basic socio-demographic,fluorosis related lifestyle risk factors,the ORs for skeletal fluorosis risk in the Q2-Q4,compared with the lowest quartile of uptakes,were 0.58(95% CI: 0.29-1.19),0.43(95% CI: 0.20-0.93)and 0.36(95% CI:0.13-0.96),(P-trend <0.05)for betaine.2.The relationship between RANKL,OPG gene promoter DNA methylation levels and serum expression levels and skeletal fluorosis.Significantly hypomethylation was observed in 1 CpG site of OPG promoter regions OPG(pos651)in skeletal fluorosis patients compared to controls(P<0.05).No significant changes in RANKL promoter CpG sites methylation were observed between skeletal fluorosis patients and healthy control participants.The serum RANKL,OPG level and RANKL/OPG ratio were significantly higher in skeletal fluorosis patients(median RANKL: 2728.64 pg/ml;OPG: 560.64 pg/ml;RANKL/OPG ratio: 4.30)compared to healthy control participants(median RANKL:1421.51 pg/ml;OPG: 332.66 pg/ml;RANKL/OPG ratio: 3.79)(P<0.05).The serum RANKL expression level and RANKL/OPG ratio were associated with lower skeletal fluorosis risk(RANKL: OR=1.04,95% CI: 1.01-1.08,P-trend<0.05;OPG:RANKL:OR=1.65,95% CI: 1.16-2.34,P-trend<0.01).No significant associations were observed between serum OPG level and skeletal fluorosis risk.Conclusion: 1.High dietary intake of betaine is associated with a lower risk of skeletal fluorosis;2.Fluoride exposure increase serum RANKL,OPG level and RANKL/OPG ratio in skeletal fluorosis patients.Fluorosis-induced hypomethylation of specific CpG site may plays a crucial role in the regulation of OPG expression in skeletal fluorosis patients.Party 2.Effect of betaine on RANKL/OPG promoter DNA methylation in fluoride-exposed rat and the antagonism of betaineObjective: A chronic fluorosis rat model and low,medium and high-dose betaine intervention groups were established to explore the mechanism of betaine’s effect on fluorosis based on RANKL/OPG gene DNA methylation in the promoter region.Methods: Sixty male SD rats were randomly divided into five experimental groups(n=12)by weights: control(drinking pure water),model(100 mg/L NaF in drinking water)and three betaine intervention groups(low dose group 100 mg/kg/d,medium dose group 200 mg/kg/d,high dose group 400 mg/kg/d).The betaine intervention groups were exposed to NaF as the model group and continues gavaged with betaine for 6 months.Betaine was orally administrated at the volume of 0.1 ml/20g/day to rats depend on their body mass.The ion-selective electrode method was used to detect the fluoride ion concentration in the rat urine,the TBS method was used to detect the RANKL/OPG gene promoter DNA methylation level in rat blood,serum concentrations of OPG were measured by ELISA,and the Western blot method was used to detect the protein expression of RANKL/OPG in the rat bone tissue.Results: The urinary fluoride concentration of rats in each exposure group was obviously higher than control group(P<0.001).The urinary fluoride content results showed no statistical difference between the model group and betaine intervention groups(P>0.05).The TBS showed that the methylation levels of 4 specific CpG sites pos1262(F=9.046,P=0.001),pos1289(F=19.766,P<0.001),pos1293(F=13.354,P<0.001),pos1295(F=5.375,P=0.007)in the OPG gene promoter region CpG island in blood of rats changed significantly among all the five experimental groups.As compared with the control group,the methylation of its 4 CpG sites(pos1262,pos1289,pos1293,and pos1295)in the model group all observed significant hypomethylation(P<0.05).While the betaine supplementation groups increase the methylation level of these CpG sites in a dose-dependent manner.In addition,the expression of OPG in blood of rats significantly increased in model group compared to the control group(F=7.168,P=0.001).A decrease in serum OPG expression was also observed with an increase of betaine supplementation.Western blot showed that chronic fluoride exposure caused the up-regulation of RANKL(F=17.019,P<0.001)and OPG(F=32.005,P<0.001)protein expression in rat bone tissues.The low,medium and high doses of betaine intervention corrected the abnormal expression of RANKL and OPG protein caused by fluoride exposure.Although chronic fluoride exposure significantly increased the expression of RANKL protein,no significant changes in the methylation level of the RANKL gene promoter region CpG island were observed.Conclusion: 1.Chronic fluoride exposure increased the expression of RANKL and OPG protein in rats bone tissue,caused hypomethylation in the specific CpG sites of the OPG gene promoter region CpG island and increased expression of OPG in the blood;2.Betaine supplementation reversed the hypomethylation of specific CpG sites in the blood OPG gene promoter region induced by fluoride exposure in a dose-dependent manner,and corrected abnormal expression of RANKL and OPG protein in the bone tissue and blood of fluorosis rats.