| Objective: Fluoride is an essential trace element for the humans.An appropriate amount of fluorine can maintain normal physiological functions of the bodies.Excessive fluoride can cause fluorosis.Epidemiological investigation has showed the rate of bone-age of children in high-fluoride areas were delayted and their height,weight and bust were lower than those of normal children of the same age.Previous animal study and organ culture study of our research group have found that fluorine inhibited the growth of long bone by inhibiting endochondral ossification.Endochondral ossification is the main way of bone formation.This process is affected by a series of hormone,cytokine and signal factors.Mammalian target of rapamycin(m TOR),a serin/threonine kinase,is the key regulator of cell growth,proliferation,differentiation,survival and autophagy.However,The underlying mechanisms that fluorine inhibited the chondrogenesis remain unclear.In the study,we used an organ culture system and the mouse ATDC5 cell lines to evaluate the effects of fluoride on the proliferation and differentiation of chondrocytes and the role of PI3K/AKT/m TOR signaling pathway in fluoride-induce bone impairment.Methods:In this study,tibias were isolated from E20 fetal rats and cultured inα-MEM supplemented with 0.2% BSA,100U/ml penicillin,100ug/ml streptomycin,0.05mg/ml vitamin C and 1m M sodium glycerophosphate.The left and right tibias were treated separately with or without 10-4M Na F.After 7 days treatment,the tibias were fixed in 4% paraformaldehyde,embedded in paraffin and cut in 5-μm longitudinal sections for further studies.The expressions of PCNA and p-S6 were detected by immunohistochemical analysis.The ATDC5 cells were cultured in DMEM supplemented with 5% FBS,100U/ml penicillin,100ug/ml streptomycin,ITS and treated with or without 10-3M Na F for 3,5,7,14,21 days.Cell viability was detected by CCK8 assay and the morphological changes of ATDC5 cells were observed by phase-contrast micrographs.The proteoglycan synthesis and the formation of cartilage nodules were examined by alcian blue staining.The expression of chondrocyte differentiation-related genes(Aggrecan,Sox9,Col II,Ihh, PTHr P and Col X)and m TOR signaling pathway-related genes(PI3K,AKT,m TOR,S6K1,4EBP1,LC3 II / I,Beclin1,P62)were detected by western blot and real-time PCR.MHY1485(m TOR activator)and Rapamycin(m TOR inhibitor)were used to explore the role of PI3K/AKT/m TOR signaling in fluoride-induced cartilage damage.Results:1.Effects of Na F on chondrocyte proliferation and m TOR activity in tibia :the percentage of PCNA-positive cells and p S6-positive cells was significantly decreased in Na F-treated group compared with control group(P?<?0.01).2.Effects of Na F on ATDC5 cells viability and cell morphology: the viability of cells was dramatically decreased at the dose of 10-3 M Na F added(P?<?0.01).The cells were grown-well and structure of cells was clearly in control group.In contrast,the growth of cells was inhibited,the shapes of cells was changed and the number of viable cells were decreased in Na F-treated group.3.Effects of Na F on the differentiation of ATDC5 cells: ATDC5 cells were cultured in DMEM containing ITS for 5,7,14 and 21 days.ATDC5 cells proliferated and formed a confluent monolayer from 0 to 5 days,the formation of cartilage nodules gradually increased in a time-dependent manner from 14 to 21 days.The alcian blue staining intensity and number of nodules were increased with time in control cells,but they were reduced in Na F-treated cells compared with control cells.The m RNA expression of Aggrecan,Sox9,Col II,Ihh,PTHr P and Col X in Na F-tread cells were significantly reduced compared with the control cells(P?<?0.05).The protein expression of Sox9 and Col II were significantly decreased in Na F-tread group compared with the control group(P?<?0.05).4.Effects of Na F on PI3K/AKT/m TOR signaling pathway in ATDC5 cells: the m RNA expressions of m TOR,S6k1 and 4EBP1 were significantly decresed and the realitve protein expression of p-PI3K/t-PI3 K,p-AKT/t-AKT,p-m TOR/t-m TOR,p-S6K1/t-S6K1 and p-4EBP1/t-4EBP1 was significantly reduced after treatment with Na F compared with control group(P<0.05).5.Effects of Na F on autophagy-related genes expression in ATDC5 cells: Na F was markedly promoted the protein expression of LC3II/I and Beclin1 but depressed P62(P<0.05).6.MHY1485 upregulates the Na F inhibition of the m TOR singnaling pathway in ATDC5 cells: Na F treatment decreased significantly p-m TOR,p-S6K1 and p-4EBP1 expression in ATDC5 cells(P<0.05).Moreover,after m TOR signaling activited by MHY1485,the p-m TOR,p-S6K1 and p-4EBP1 expressions were significantly increased(P<0.05).The p-m TOR,p-S6K1 and p-4EBP1 expressions have no significant difference between MHY1485 and Na F co-treatment groups and the controls.The p-m TOR,p-S6K1 and p-4EBP1 expressions in MHY1485 and Na F co-treatment groups were dramatically increased compared with the Na F exposure groups(P<0.05).7.Na F-induced autophagy via the down-regulation of phosphorylation of m TOR in ATDC5 cells: the P62 expression was significantly decreased in Na F-treated cells.Moreover,MHY1485 increased significantly the P62 expressions.After m TOR signaling inhibited by rapamycin,the P62 expressions were significantly decreased(P<0.05).The P62 expressions in MHY1485 and Na F co-treatment groups were significantly decreased compared with the Na F exposure groups(P<0.05).However,the result of LC3II/LC3 I and Beclin-1 was opposed with that of P62.The P62,LC3II/LC3 I and Beclin1 expressions have no significant difference between MHY1485 and Na F co-treatment groups and the controls.8.Na F inhibited differentiation via the down-regulation of phosphorylation of m TOR in ATDC5 cells: the m RNA expressions of Aggrecan,Sox9,Col II,Ihh,PTHr P and Col X were significantly decreased in Na F-treated cells compared with control cells(P<0.05).MHY1485 treatment increased markedly Aggrecan,Sox9,Col II,Ihh,PTHr P and Col X m RNA expression(P<0.05).The Sox9,Col II,Ihh,PTHr P and Col X m RNA expressions have no significant difference between MHY1485 and Na F co-treatment groups and the controls.The Sox9,Col II,Ihh,PTHr P and Col X m RNA expressions in MHY1485 and Na F co-treatment groups were dramatically increased compared with the Na F exposure groups(P<0.05).The result of Sox9,Col II,Ihh,PTHr P and Col X m RNA expression was agreed with that of Sox9 and Col II protein expression.Conclusion:1.Fluorine inhibits the proliferation and differentiation and induced aotophagy of chondrocytes by inhibiting the PI3 K / AKT / m TOR signaling pathway. |
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