Effect Of Fluoride On Proliferation And Differentiation In ATDC5 Cell And The Regulatory Role Of Circadian Clock Signal Pathway

Objective:Fluoride is an essential trace element for human growth and development.Proper amount of fluoride can enhance the anti-caries ability of teeth,but long-term intake of excessive fluoride can cause endemic fluorosis.Because bone tissue is the main target organ of fluoride,skeletal fluorosis caused by excessive fluoride is the most representative,and the damage to cartilage is one of its basic manifestations.Chondrogenesis is regulated by hormones,cytokines and local signaling pathways.Circadian clock refers to the daily 24-hour rhythm of physiology and behavior,which is composed of positive and negative regulatory feedback loops generated by external time cues.The positive feedback loop is composed of transcription factors Clock and Bmal1 and the negative feedback loop is composed of transcription factors Per and Cry.However,the effects of fluoride on the proliferation and differentiation of chondrogenesis and whether fluoride regulates chondrogenesis through circadian clock signaling pathway have not been reported.Therefore,we preliminarily explored the effects of fluoride on the differentiation process of ATDC5 cells in vitro and the role of circadian clock signaling pathway by neonatal rats infected during pregnancy and feeding and mouse ATDC5 cells.This study is intended to provide clues to elucidate the mechanism of fluoride inhibiting long bone growth and theoretical basis for the prevention and treatment of skeletal fluorosis.Methods:In this study,the protein expression of Bmal1 in rat growth plate cartilage was detected by immunohistochemical staining on fluorosis Wistar primary weaning rats during pregnancy and lactation.We determined the dose at which fluoride inhibited the proliferation of ATDC5 cells by CCK8.We established an in vitro differentiation model for fluorosis under the action of insulin-transferrin-sodium selenite inducer.Proteoglycan synthesis was detected by Acian blue staining,and mRNA and protein expressions of chondrogenic differentiation related markers?Sox9,Col2a1,Aggrecan,Ihh,Col10a1?and circadian clock signaling pathway related factors?Bmal1,Clock,Per1,Cry1?were detected by real-time quantitative PCR and western blot.ATDC5stably transfected cell lines were established in the presence of Bmal1 overexpressed lentivirus,and mRNA and protein expressions of chondrogenic differentiation markers?Sox9,Col2a1,Aggrecan,Col10a1?were detected by real-time quantitative PCR and western blot.Results:1.Effect of fluoride on Bmal1 protein expression in growth plate chondrocytes of rats:Compared with the control group,the expression level of Bmal1 protein in growth plate of rats in NaF-treated group was significantly decreased?P<0.05?.2.Effects of fluoride on ATDC5 cell proliferation and proteoglycan:Compared with the control group,low dose of fluoride promoted cell proliferation,while high dose of 10^-3M NaF inhibited cell proliferation?P<0.05?,and 10^-3M NaF inhibited proteoglycan synthesis in chondrocytes?P<0.05?.3.The influence of fluoride on the differentiation marker factors of ATDC5 cells:During the proliferation and pre-hypertrophy stage of ATDC5 cells,compared with the control group,fluoride inhibited the mRNA and protein expression of the transcription factor Sox9?P<0.05?,and inhibited the mRNA and protein expression of the proliferation marker Col2a1?P<0.05?.In the hypertrophy stage of ATDC5 cells,compared with the control group,fluoride promoted the expression of mRNA and protein of the transcription factor Sox9?P<0.05?to prevent further cell differentiation and inhibit the expression of mRNA and protein of differentiation markers Ihh and Col10a1?P<0.05?.Consistent with Alcian blue staining,fluoride inhibited Aggrecan mRNA expression?P<0.05?.4.Influence of fluoride on Bmal1 and circadian clock signaling pathways in ATDC5 cells:Compared with the control group,fluoride inhibited mRNA and protein expression of core clock factor Bmal?P<0.05?,inhibited mRNA and protein expression of Clock?P<0.05?,and promoted mRNA and protein expression of downstream Per1 and Cry1?P<0.05?.5.Effects of fluoride on ATDC5 cells of circadian rhythm:Compared with control group,in NaF-treated group cell differentiation factor?Col2a1,Ihh,Col10a1?and circadian clock signaling pathways related factor?Bmal1,Clock,Per1,Cry1?protein rhythmicity were suppressed,and fluoride inhibited the protein expression of Col2a1,Ihh,Col10a1,Bmal1 and Clock,promoted Per1 and Cry1 protein expression.6.Effects of lentivirus overexpression of Bmal1 on the differentiation of ATDC5 cells after Na F-treatment:After overexpression of Bmal1,the inhibitory effect of fluoride on mRNA and protein levels of Sox9,Aggrecan,Col2a1 and Col10a1 in ATDC5 cells were completely reversed,and Bmal1 significantly promoted the differentiation of chondrocytes compared with the control group?P<0.05?.Conclusion:1.Fluoride inhibited the proliferation of ATDC5 cells and delayed the differentiation of ATDC5 cells,thus hindered the chondrogenesis process.2.Fluoride inhibited the expression of positive feedback loop factor of circadian clock and promoted the expression of negative feedback loop factor,thus disrupted the circadian rhythm of ATDC5 cells.3.Fluoride inhibited chondrogenesis and differentiation of ATDC5 cells by down-regulating transcription factor Bmal1 and interfering with circadian clock signaling pathway.