Screening Of Differentially Methylated Genes In Skeletal Fluorosis Of Rats With Different Types And Involvement Of Aberrant Methylation Of Cthrc1

Objective:Fluoride is a contaminant that is widely present in the environment.Fluorosis does extensive damage to the body,including bone phase and non-bone phase system damage.The etiology of skeletal fluorosis is clear,but the lesions are complex and diverse.At the same fluoride exposure,skeletal fluorosis can manifest as osteosclerotic,osteoporotic and osteomalacic phenotypes,and is influenced by nutritional factors.However,the existing hypotheses of the pathogenesis of skeletal fluorosis cannot explain well the different pathological manifestations of skeletal fluorosis and its logical relationship with nutritional factors.Recent studies have shown that DNA methylation is involved in the development and progression of skeletal fluorosis.In contrast,DNA methylation is dynamic throughout the life course and can be influenced by nutritional and environmental factors.We hypothesized that fluoride exposure under different nutritional states would lead to aberrant methylation of genes related to bone homeostasis,resulting in different skeletal fluorosis phenotypes.Therefore,in this study,we constructed rats with different types of skeletal fluorosis from which we screened differentially methylated genes and explored the role and mechanism of aberrant methylation of the Cthrc1 gene in the development of different types of skeletal fluorosis in rats.Our study deepens the understanding of the role of DNA methylation in regulating the formation of different types of skeletal fluorosis and provides new insights into the prevention and treatment strategies for patients with skeletal fluorosis.Methods:(1)Rat models with different types of skeletal fluorosis were constructed by intragastric poisoning of different nutritional diets combined with 20 mg/kg·bw Na F for 5 months.The dental fluorosis of rats was observed,the fluoride content in blood,urine and bone was detected by fluoride ion selective electrode method,and the serological nutritional indexes were detected by automatic biochemical analyzer.Serum bone metabolic markers were detected by ELISA,imaging changes of bone tissue were detected by Micro-CT,pathological changes were observed by H&E staining,and Western blotting was used to detect the expression of osteogenic and osteoclastic differentiation and functional proteins in bone tissue.After the rat models of different types of skeletal fluorosis were established successfully.Then m RNA sequencing was conducted on bone tissue and bone marrow,bioinformatics analysis was used to screen out significant differentially expressed genes closely related to bone development,and then targeted bisulfite sequencing was conducted on these genes to screen out key differentially methylated genes.The gene was verified in bone tissue by q RT-PCR and Western blotting.(2)After isolation of primary r BMSCs,surface markers were identified and osteogenic lipid differentiation was identified.then the methylation levels and expression levels of Cthrc1 in osteoblasts under different nutrient(normal nutrition α-MEM and customized lowcalcium and low-methionine α-MEM medium)fluoride exposures were identified by TBS,q RT-PCR and Western blotting.Afterwards,DNA pull-down combined with Western blotting was used to explore the enzymes causing aberrant methylation of Cthrc1 in osteoblasts under different nutritional fluorine exposure.To use targeted inhibitors of DNA methylase or demethylase to examine whether abnormal methylation of Cthrc1 alters its expression level and thereby participates in the differentiation and function of osteoblasts under different nutritional conditions of fluoride exposure.Lentivirus was used to construct r BMSCs with stable overexpression and knockdown of Cthrc1.CTHRC1 content in supernatant was detected by ELISA,ALP staining and activity determination,ARS staining and semi-quantitative.q RT-PCR and Western blotting were used to explore the effects of CTHRC1 exposure to different nutritional fluoride on the differentiation and function of osteoblasts.By using the agonists and inhibitors of Wnt3a/β-catenin signaling pathway,we investigate whether Wnt3a/β-catenin signaling pathway is involved in fluorosis of different phenotypes caused by abnormal methylation of Cthrc1.(3)After isolation of primary r BMMs,they were identified by surface markers and osteoclast differentiation.Then,the methylation and expression levels of Cthrc1 in osteoclasts exposed to different nutritional fluoride were identified by TBS,q RT-PCR,ELISA and Western blotting.r CTHRC1 was added to r BMMs during their differentiation into osteoblasts,and the effect of r CTHRC1 on osteoblast differentiation and function under different nutrient culture fluoride exposure was investigated by TRACP staining,and Western blotting.Transwell co-culture system was established,and r BMSCs with knockdown or overexpression of Cthrc1 were compared with normal r BMMs were cocultured in Transwell chambers,combined with ELISA to detect CTHRC1 content in supernatant,ALP staining and viability assay,ARS staining and semi-quantitative,q RTPCR and Western blotting to explore the effect of Cthrc1 on osteoblast differentiation and function under different nutrient fluorine exposure,combined with TRACP staining and Western blotting to investigate the effect of Cthrc1 on osteoblast differentiation and function under different nutrient culture fluoride exposure.Results:(1)There was no significant difference in re-exposure period among all groups.Dental fluorosis appeared in OS group and OP group from the second month of exposure,and after the end of exposure,severe dental fluorosis and fracture occurred,while no dental fluorosis appeared in NC group and MC group.The contents of blood fluoride,urine fluoride and bone fluoride in OS and OP groups were significantly higher than those in NC and MC groups;Compared with NC group,bone formation markers in OS group and bone resorption markers in OP group were significantly increased.Micro-CT results showed osteosclerosis in OS group,osteoporosis in OP group,normal bone tissue in NC and MC groups,and osteosclerosis in OS group and osteoporosis in OP group.Western blotting results showed that the expression level of bone formation-related proteins was significantly higher in the OS The expression levels of bone formation-related proteins were significantly higher in the bone tissue of the OS group compared to the NC group,while the expression levels of bone resorption-related proteins were significantly higher in the OP group.the expression levels of bone formation-related proteins were significantly higher in the OS group compared to the NC group,while the expression levels of bone resorption-related proteins were significantly higher in the OP group.m RNA-Seq screened 60 significantly differentially expressed genes closely related to bone development from bone tissue,and TBS screened 8 significantly differentially methylated genes(Ostn,Siglec15,Cthrc1,Slc9b2,Mill1,Serpinh1,Dhh & Atp6v0a4)from these 60 genes.The downstream signalling pathway of Cthrc1 was screened for possible Wnt3a/β-catenin by raw signal analysis and verified by q RT-PCR and Western blotting,which showed that the trend of Wnt3a/β-catenin was consistent with that of Cthrc1.(2)The purity of r BMSCs was > 95%,ALP activity and mineralized nodules were significantly increased in r BMSCs after osteogenic differentiation compared to undifferentiated,and significant lipid droplets could be seen intracellularly after lipogenic differentiation.Cthrc1 methylation levels were significantly decreased and m RNA and protein expression levels were increased in NF group,while Cthrc1 methylation levels were significantly increased and m RNA and protein expression levels were decreased in LNF group.TET2 and DNMT1 were involved in the aberrant methylation of Cthrc1 gene in fluoride exposed osteoblasts under different culture conditions.The enhanced osteogenic differentiation induced by hypermethylation and hyperexpression of Cthrc1 was inhibited by TFMB-(S)-2-HG under normal nutritional conditions and fluoride exposure,while procainamide was able to salvage the decreased osteogenic differentiation caused by hypermethylation and hyperexpression of Cthrc1 under low nutritional conditions and fluoride exposure.After exposure to normal nutritional fluoride,the transcription and protein levels of Cthrc1 knockdown significantly decreased,and the osteogenic differentiation ability of r BMSCs was significantly weakened.In contrast,under low nutrient conditions and fluoride exposure,Cthrc1 overexpression expression could partially reverse the inhibited osteogenic differentiation.Under normal nutritional conditions and fluoride exposure,Cthrc1 promoted osteogenic differentiation by activating the Wnt3a/β-catenin signaling pathway.Under low nutrient conditions and fluoride exposure,Cthrc1 inhibited osteoblast differentiation and function but elevated the RANKL/OPG ratio secreted by osteoblasts by suppressing the Wnt3a/β-catenin signaling pathway.(3)The purity of r BMMs was > 85%.After osteoclastic differentiation of r BMMs,a large number of multinucleated cells were obviously visible under light microscope,and the number of TRACP-stained positive cells was > 90%.TBS results showed that the difference in methylation level of Cthrc1 gene between the fluoride-stained and control groups was not statistically significant.q RT-PCR results also showed that there was no significant difference between the groups.ELISA and Western blotting results were consistent with the q RT-PCR results,with no statistically significant differences between groups and significantly lower expression levels than in osteoblasts.Fluoride exposure increased TRACP activity in osteoblasts as well as the number of TRACP-positive cells under both normal and low nutrient conditions.In the indirect co-culture system,Cthrc1 knockdown inhibited the osteogenic differentiation ability of r BMSCs under normal nutrient fluoride exposure conditions,as evidenced by reduced alkaline phosphatase staining,reduced alkaline phosphatase activity,reduced calcium mineralization,downregulated expression of osteogenic markers(Runx2,Osterix,ALP,Col1α1,BGP)and reduced OPG/RANKL ratio.In contrast,under low nutrient and fluoride exposure conditions,overexpression of Cthrc1 partially reversed the inhibited osteogenic differentiation,as evidenced by enhanced alkaline phosphatase staining and activity,increased calcium mineralization,upregulated expression of osteogenic markers(Runx2,Osterix,ALP,Col1α1,RANKL,BGP)and increased OPG/RANKL ratio.Similarly,when CTHRC1 secretion was increased in the culture medium,osteoclast differentiation and function were significantly inhibited.Conversely,when CTHRC1 secretion was low in the culture medium,the expression levels of differentiation markers and functional markers of osteoclasts were increased.Conclusions:(1)Eight differential methylation genes(Ostn,Siglec15,Cthrc1,Slc9b2,Mill1,Serpinh1,Dhh& Atp6v0a4)were obtained from different types of skeletal fluorosis rats,which may be related to the occurrence and development of different types of skeletal fluorosis in rats.(2)The Cthrc1 gene showed hypomethylated and high expression in osteosclerotic skeletal fluorosis rats,and hypermethylated and low expression in osteoporotic/osteomalacic skeletal fluorosis rats.(3)Under normal nutritional conditions,Na F exposure induced hypomethylation and high expression of Cthrc1,inhibited osteoclast differentiation,and promoted osteoblast differentiation through activation of the Wnt3a/β-catenin signaling pathway,which was involved in osteosclerotic skeletal fluorosis.Under poor nutritional conditions,Na F exposure induced hypermethylation and low expression of Cthrc1 and promoted osteoclast differentiation through upregulation of the RANKL/OPG ratio,which was involved in the development of osteoporotic/osteomalacic skeletal fluorosis.