The methylation of the RANKL/OPG system is involved in fluorosis, and the effect of betaine supplementation

Abstract This study aims to explore the role of DNA methylation in the promoter region of the RANKL/OPG on fluorosis and the effect of betaine supplementation. A case-control study of 82 paired cases and controls was conducted among residents in coal-burning fluorosis areas in Guizhou Province, China. RANKL and OPG levels in serum of particpants were measured using ELISA. In addition, 60 SD rats were randomly divided into five groups ( n = 12): control group, model group, low-dose betaine group, medium-dose betaine group, and high-dose betaine group. 20 pairs case-control were randomly selected from 82 matched pairs to assess gene-specific DNA methylation. The methylation levels of RANKL/OPG in the human and rat whole blood were determined using targeted bisulfite sequencing (TBS). The expression of RANKL and OPG proteins in the femur tissues of rats was detected by Western blotting. The serum RANKL and OPG levels, and their ratios were elevated in the fluorosis patients compared with healthy individuals. A CpG site (pos651) in the OPG promoter exhibited hypomethylation in fluorosis patients, but this did not retain statistical significance after FDR correction( q > 0.05). Furthermore, betaine supplementation produced a dose-dependent reduction in dental fluorosis. Notably, dental fluorosis was present in 100% of the fluoride-exposed model group. Moreover, DNA methylation of CpG sites at pos1262, pos1289, pos1293, and pos1295 in the OPG gene of rat was reduced in the model group compared with the control group, and the methylation level was significantly restored in the betaine intervention group ( P < 0.05). Additionally, long-term fluoride exposure upregulated the expression of RANKL and OPG proteins in bone tissues of the rats however, whereas betaine supplementation could reverse these changes dose-dependently ( P < 0.05). Fluorosis may be associated with aberrant methylation of CpG sites in the OPG promoter. And betaine may potentially alleviate fluoride-induced bone changes, potentially through the regulation of the OPG/RANKL axis via epigenetic mechanisms.