| Objectives:1.A population-based study was conducted to evaluate the dietary riboflavin intake in the Chinese adult population,to investigate the differences in bone health status among people with different dietary riboflavin intake levels,and then to analyze the association between riboflavin dietary intake and bone health.2.To analyze the effects of riboflavin nutritional status on bone mineral density and bone microstructure by constructing animal models with different riboflavin nutritional status.Compare the differences of bone metabolic indexes and related gene and protein expression levels under different riboflavin nutritional status to explore the mechanism of riboflavin nutritional status affecting bone health.Methods:1.The association between dietary riboflavin intake and bone health in the Chinese adult population.Participants were recruited for questionnaires,physical examinations and laboratory biochemical examinations.Food frequency questionnaire combined with Chinese food composition database was used to calculate dietary riboflavin intake.Heel bone mineral density was measured by ultrasound absorptiometry.Multiple logistic regression model was used to analyze the association between riboflavin intake and bone health after adjusting for confounding factors.2.The Effects of riboflavin nutritional status on bone mineral density in rats.Rat models with different riboflavin nutritional status were established by diet intervention with different riboflavin content（<1mg/kg、3mg/kg、6mg/kg）.The contents of riboflavin and its derivatives in serum and liver were measured by high performance liquid chromatography,and the nutritional level of riboflavin was evaluated in combination with glutathione reductase activity.Femoral sections were stained to observe the changes of bone microstructure and collagen cross-linking.Bone mineral density was measured by dual-energy X-ray.3.Effect of riboflavin nutritional status on bone metabolism in rats.The levels of IL-1,IL-6 and TNF-αwere detected by ELISA to analyze the inflammation of rats.SOD and MDA were measured to evaluate the oxidative stress response.Determination of estrogen levels by ELISA.Serum calcium and phosphorus were measured,and25-（OH）2-D3,PTH,and CT were measured by ELISA to evaluate calcium and phosphorus metabolism.BALP,BGP,TRACP-5b,CTX-Ⅰand NTX were measured by ELISA to evaluate bone formation and resorption.The levels of T3,T4,TSH and Vitamin K were measured by ELISA to evaluate the influence of other factors.4.Effect of riboflavin nutritional status on the expression of related genes and proteins.q PCR was used to detect the m RNA expression levels of RUNX2,OPG,RANK,RANKL,WNT,β-Catenin,C-JUN and C-FOS.Western blot was performed to detect the protein expression of RUNX2,OPG,RANK,RANKL,WNT,β-Catenin,C-JUN,and C-FOS.The differences between different groups were compared.Results:1.The results of a cross-sectional study on the association between riboflavin intake and the prevalence of osteoporosis showed that dietary riboflavin intake was inversely associated with the prevalence of osteoporosis,and the OR value of the highest quartile of riboflavin intake was significantly lower than that of the lowest quartile of riboflavin intake.However,the association between riboflavin intake and osteoporosis prevalence showed different results in men and women.No significant results were found in men.Riboflavin intake was negatively correlated with the prevalence of osteoporosis in women,and the OR values of the highest and the third quartile of riboflavin intake were significantly lower than those of the lowest quartile of riboflavin intake.In addition,the results of the two sensitivity analyses were consistent with those of the previous analyses.2.The results of a population-based cross-sectional study on the association between riboflavin intake and the prevalence of low bone mineral density showed that the OR of low bone mineral density in the highest quartile group was significantly lower than that in the lowest quartile group after multivariate adjustment.The results of gender stratification analysis showed that there was no significant difference in men,but in women,the OR value of the prevalence of low bone mineral density gradually decreased with the increase of riboflavin intake,and the OR value of the highest quartile group was significantly lower than that of the lowest quartile group.Two sensitivity analyses also supported the primary results.3.The lack of riboflavin dietary intake in Wistar rats can lead to decreased appetite,decreased food intake,slow weight gain,decreased serum glutathione reductase activity,decreased riboflavin and FMN content in serum and liver,decreased bone mineral density of total body and femur,changed bone histomorphology,and reduced bone collagen content.In addition,insufficient dietary intake of riboflavin can lead to a decrease in the content of FMN in serum and liver,a significant decrease in total bone mineral density.4.Riboflavin deficiency resulted in increased serum and bone IL-1 levels,increased serum TNF-αlevels,increased serum and bone peroxidation product MDA levels,increased blood calcium levels,decreased blood phosphorus levels,decreased25-（OH）2-D3 levels,increased PTH levels,decreased CT levels,and decreased BALP levels in rats.In addition,insufficient riboflavin intake can lead to increased serum IL-1and MDA levels,increased blood calcium levels,decreased blood phosphorus levels,decreased 25-（OH）2-D3 levels,and decreased BALP levels.5.Riboflavin deficiency decreased the m RNA expression levels of RUNX2,OPG,C-JUN and C-FOS,and increased the expression levels of RANK,RANKL andβ-Catenin.Comparative analysis of related protein expression levels revealed that riboflavin deficiency decreased the expression levels of RUNX2,OPG,WNT,C-JUN and C-FOS,and increased the expression levels of RANK and RANKL;while insufficient riboflavin intake decreased the expression levels of OPG and increased the expression levels of RANK and RANKL.Conclusion:1.Dietary riboflavin intake was negatively associated with the prevalence of osteoporosis and lower mineral density in Chinese adult population.In addition,the above associations were significant only in women but not in men.The results of our population-based study suggest that women are more sensitive to riboflavin intake in maintaining normal BMD.2.Riboflavin intake deficiency can lead to decreased appetite,slow growth,decreased riboflavin nutritional level in the body,decreased bone mineral density and altered bone histomorphology in rats.3.Riboflavin intake deficiency resulted in increased inflammation levels and oxidative stress damage in rats.Insufficient intake of riboflavin resulted in elevated blood calcium,decreased blood phosphorus,and significant changes in indicators regulating calcium and phosphorus homeostasis in rats,and imbalance in calcium and phosphorus homeostasis.Insufficient dietary riboflavin intake also caused a significant decrease in BALP,a marker of bone formation.These results suggest that insufficient dietary intake of riboflavin may lead to inflammation and oxidative stress,imbalance of calcium and phosphorus homeostasis,inhibition of bone formation,and consequently decrease of bone mineral density.4.Riboflavin deficiency decreased the expression levels of RUNX2,OPG,C-JUN and C-FOS,and increased the expression levels of RANK and RANKL.This suggests that riboflavin deficiency downregulates the expression of RUNX2,OPG,C-JUN and C-FOS and upregulates RANK and RANKL,leading to an increase in the binding ratio of RANK and RANKL,which promotes osteoclast differentiation and delays osteoclast apoptosis,directly or indirectly affecting the proliferation and differentiation of osteoblasts,inhibiting bone formation and increasing bone resorption,leading to a Bone formation is inhibited and bone resorption is increased,leading to a decrease in bone mineral density. 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