Fluoride Inhibits The Proliferation And Differentiation Of C28/I2 Chondrocytes By Promoting The Nuclear Expression Of HDAC4 Downstream Of The Ihh/PTHrP Signaling Pathway

Objective:Fluoride exists widely in nature.Appropriate intake of fluoride can keep the hardness of teeth and bones and maintain the normal function of various systems.However,long-term excessive intake of fluoride can cause symptoms such as dental fluorosis and skeletal fluorosis,and cartilage tissue is one of the main pathological damage sites.It is known that growth plate chondrocytes undergo stages of proliferation,differentiation and hypertrophy during endochondral ossification,the cartilage is replaced by bone tissue,and the bone is finally elongated.Studies have shown that fluoride could impair the differentiation of chondrocytes and the normal mineralization of cartilage tissue,inhibit the process of endochondral ossification.PTHrP,Ihh,Mef2C,Runx2 and p38 all play a regulatory role in this process,but whether fluoride changes the expression of these molecules in chondrocytes needs to further study.Histone deacetylase 4(HDAC4),as a histone modification enzyme,is mainly expressed in chondrocytes.By transferring from the cytoplasm to the nucleus of cells,HDAC4 plays a role in deacetylation modification,regulates the functions of histones and non-histones,thereby regulating chondrocyte differentiation.Studies have shown that overexpression or deletion of HDAC4 could destroy the morphology and physiological function of cartilage tissue,but whether fluoride regulates cartilage formation through HDAC4 has not been reported.Therefore,in this study,C28/I2 chondrocytes were cultured with fluoride and detected the expression of chondrocyte proliferation and differentiation related molecules(Col2a1,Sox9 and Coll0a1),and Ihh/PTHrP signaling pathway related molecules(PTHrP,Ihh,Mef2C,Runx2 and p38).We also detected the expression and phosphorylation level of HDAC4 in chondrocytes.The purpose of this study was to explore the regulation of fluoride on the proliferation and differentiation of C28/I2 chondrocytes and its effect on Ihh/PTHrP signaling pathway including related molecules such as HDAC4,to further clarify the mechanism of fluoride-induced damage during cartilage development.Methods:In this study,C28/I2 chondrocytes were selected and treated with different concentrations of NaF for different times,and the optimal dose and time of fluoride treatment were selected by the CCK-8.Alcian blue staining was used to detect the expression of chondrocyte proteoglycan,and the expression of ACAN mRNA in chondrocytes was detected by qPCR.The qPCR and western blot were used to detect the mRNA and protein expression of chondrocyte proliferation related molecules including Col2al and Sox9 and differentiation related molecule Coll0a1.The mRNA and protein expression of Ihh/PTHrP signaling pathway related molecules including PTHrP,Ihh,Mef2C and Runx2 were detected by qPCR and western blot.The expression of HDAC4 in chondrocytes was detected by immunofluorescence,and the phosphorylation level of HDAC4 was detected by western blot.The level of p38 phosphorylation was detected by western blot.Results:1.The effect of fluoride on the viability of C28/I2 chondrocytes:Compared with the control group,the viability of C28/I2 chondrocytes which were treated with 10-6 mol/L,10-5 mol/L and 10-4 mol/L NaF for 72h did not change significantly,while the cell viability was significantly decreased after treating with 10-3 mol/L NaF for 72h(P<0.05).After treating the C28/I2 chondrocytes with 10-3 mol/L NaF for 24h,the cell viability did not change significantly compared with the control group,but the cell viability was significantly reduced after 48h and 72h treatment(P<0.05).In subsequent experiments,C28/I2 chondrocytes were treated with 10-3 mol/L NaF for 48h and 72h.2.The effect of fluoride on the expression of C28/I2 chondrocytes proteoglycan:Alcian blue staining in the NaF group was lighter than the control group.Compared with the control group,the expression of proteoglycan in C28/I2 chondrocytes after NaF treated was significantly reduced(P<0.05),and the expression of ACAN mRNA in chondrocytes was significantly decreased(P<0.05).3.The effect of fluoride on the expression of proliferation and differentiation related molecules in C28/I2 chondrocytes:The mRNA and protein expression of chondrocyte proliferation related molecule Col2a1 were both reduced in the NaF group compared with the control group(P<0.05),the expression of Sox9 mRNA had no significant changes after NaF treatment(P>0.05),but the Sox9 protein expression was significantly reduced in the NaF group(P<0.05).Compared with the control group,the mRNA expression of chondrocyte differentiation related molecule Col10a1 had no significant changes,but the protein expression was significantly decreased after treated with NaF(P<0.05).4.The effect of fluoride on Ihh/PTHrP negative feedback loop and downstream signal molecules in C28/I2 chondrocytes:Compared with the control group,the expression of PTHrP mRNA and protein in the NaF group were significantly increased(P<0.05).Both mRNA and protein expression of Ihh,Mef2C and Runx2 after NaF treatment were significantly reduced compared with control group(P<0.05).5.The effect of fluoride on the protein expression of HDAC4 in C28/I2 chondrocytes:Immunofluorescence results showed that HDAC4 was mainly expressed in the nucleus of C28/I2 chondrocytes,with a small amount of expression in the cytoplasm.The fluorescence intensity of HDAC4 in the nucleus of chondrocytes was increased after NaF treatment compared with the control group.The phosphorylation level of HDAC4 was decreased and p-HDAC4/HDAC4 was significantly decreased in the NaF group(P<0.05).6.The effect of fluoride on the protein expression of p38 in C28/I2 chondrocytes:In the NaF group,the phosphorylation level of p38 was declined and p-p38/p38 was significantly decreased compared with the control group(P<0.05).Conclusions:1.Fluoride inhibited the proliferation and differentiation of C28/I2 chondrocytes and decreased the expression of proteoglycan.2.Fluoride might promote the expression of PTHrP,inhibit the expression of Ihh,and inhibit the Ihh/PTHrP negative feedback loop,further reduce the phosphorylation level of HDAC4 and increase the nuclear expression of HDAC4,thereby declining the expression of Mef2C and Runx2,and inhibiting the differentiation of C28/I2 chondrocytes.3.Fluoride might increase the expression of PTHrP,inhibit the phosphorylation of p38,and further inhibit the expression of Mef2C.