| Background:Chronic fluorosis is an endemic disease,the main cause of which is skeletal lesions due to excessive intake of fluorine.Up to now,the pathogenesis has been unclear.Skeletal changes of fluorosis,also known as skeletal fluorosis,include osteosclerosis,osteomalacia,osteoporosis,and periostealsoft tissuecalcification or ossification.Cbfa1 is an osteoblast-specific transcription factor and is essential for osteoblast differentiation and osteogenesis.In the past research,our group found that fluoride can significantly enhance the expression of Cbfa1 from mRNA to protein in FB,and it is confirmed that FB has osteogenesis under the action of fluoride.The study also found that the expression of other osteogenic related factors in the bone metabolism regulatory network in fluoride-follow fibroblasts was also significantly enhanced,suggesting that these osteogenic factors also play an important role in FB osteogenesis.In this study,the in vitro experimental method was used to further confirm the changes of various factors in FB under different conditions of fluorine exposure,to find out the relationship between various bone growth factors in fluorine-exposed FB,and to find out whether there are one or several important initiating factors that motivate or combine other factors to stimulate the osteogenesis process of FB,hoping to provide a scientific basis for the study of the pathogenesis of periosteal soft tissue ossification(calcification)in skeletal fluorosis.Methods:L929 cells were used as an object in this experiment.Fibroblasts were divided into two fluoride dosage groups(Group 1:the concentration of fluoride was designed as 0,0.0001,0.001,0.1,and 1 mg/LF~-;Group 2:the concentration of fluoride was designed as 0,2,5,10 mg/LF~-).CCK-8 method was used to test the cell viability;the early differentiation of the cells was observed by Alkaline phosphatase(ALP)stained with Gomori modified Calcium-Cobalt method;the Real-time PCR and Western-Blot were used to test the mRNA and protein of Cbfa1,BMP-2,OCN,TGF-?,IGF-1,FGF-2,PDGF-B,PTH,PTH-rp,CT and CaSR in fibroblasts exposed to different condition.Then it focused on correlation analysis of test results.Results:1.CCK-8 results showed that compared with the control group,2 mg/L F~-,5mg/L F~-could stimulate cell viability,while 10 mg/L F~-inhibited cell viability,and the cells proliferation activity decreased with the prolongation of fluoride exposure;2.Compared with the control group,the ALP activity of the 2 mg/L F~-group and the 5 mg/L F~-group was significantly enhanced,while the ALP activity of the 10mg/L F~-group was lower than that of the control group;3.Real-time PCR and Western Blot results were as follows:(1)the Cbfa1,BMP-2,and OCN mRNA and protein levels of fluoride-exposed fibroblasts were increased;(2)the TGF-?mRNA of fluoride-exposed fibroblasts showed no significant increase or even decreased significantly except 10 mg/L F~-group,but their protein expression levels were significantly up-regulated at each concentration of fluoride;(3)the expression of fibroblasts IGF-1 mRNA and protein were significantly up-regulated;(4)the expression of PDGF-B mRNA was significantly enhanced in the0.1 mg/L F~-group and 10 mg/L F~-group,and the expression of PDGF-B protein was significantly increased in the 1 mg/L F~-groupand 2 mg/L F~-group;(5)the expression of FGF-2 mRNA was all enhanced or significantly enhanced except for the 0.001mg/L F~-group;the expression of FGF-2 protein in the 2 mg/L F~-and 5 mg/L F~-groups was significantly enhanced;(6)the expression of PTH mRNA and protein were enhanced in most of the fluoride-exposed groups in this experiment;PTH-rp mRNA expression was significantly enhanced in the lower dosage groups,and significantly weakened in the higher dosage groups,while the expression of PTH-rp protein is opposite to that of mRNA;(7)the expression of CT and CaSR mRNA and protein is generally enhanced by fluoride;4.Correlation analysis results of various factors in fluoride-exposed fibroblasts(1)Cbfa1?BMP-2?OCN?TGF-?(1)There was a significant positive correlation between the expression of Cbfa1protein and BMP-2 protein in the 1 mg/L F~-group.(2)There was a significant positive correlation between the protein expression levels of Cbfa1 and OCN in FB in the 5 mg/L F~-group.(3)There was a positive correlation between Cbfa1 and OCN protein expression in the 10 mg/L F~-group.(4)There was a negative correlation between BMP-2 and OCN protein expressionin only 1 mg/L F~-group.(5)The relationship between TGF-?and CBfa1,BMP-2,OCN was not obvious(2)IGF-1?FGF-2 and PDGF-B(1)There was a positive correlation between IGF-1 and PDGF-B protein expressionin the 5 mg/L and 10 mg/L F~-groups.(2)The expression of IGF-1 and FGF-2 mRNA was positively correlated in the 2mg/L F-group,but negatively correlated in the 0.001 mg/L F-group and 10 mg/L F~-group.(3)FGF-2 and PDGF-B mRNA were positively correlated in the 0.0001 mg/L,0.1mg/L and 5 mg/L groups,and their protein expression was positively correlated in the2 mg/L group,but negatively correlated in the 0.1 mg/L group.(4)The expression of TGF-?and PDGF-B mRNA was positively correlated in the2 mg/L and 5 mg/L groups,and their protein expression was positively correlated in the 0.001 mg/L group,but negatively correlated in the 1 mg/L group.(3)PTH?PTH-rp?CT and CasR(1)PTH and CaSR mRNA expression were positively correlated in the 0.1 mg/L and 1 mg/L groups,and negatively correlated in the 0.0001 mg/L and 0.001 mg/L groups.(2)PTH-rp and CaSR mRNA were positively correlated in the 0.001 mg/L and10 mg/L F-groups,and negatively correlated in the 0.0001 mg/L and 0.1 mg/L groups,and their protein expressionwas positively correlated in the 0.0001 mg/L,0.001 mg/L,and 2 mg/L F~-groups,and negatively correlated in the 5 mg/L F~-group.(3)The PTH-rp and CT mRNA expressionwas negatively correlated in the 0.001mg/L F~-group,and their protein expressionwas positively correlated in the 10 mg/L F~-group,but negatively correlated in the 0.001 mg/L and 0.1 mg/L F~-groups.(4)PTH and PTH-rp protein expression were negatively correlated in the 10mg/L F~-group,and there mRNA expression was positively correlated in the 0.0001mg/L F~-group,and negatively correlated in the 0.001 mg/L and 0.1 mg/L F~-groups.Conclusion:1.Fluoride significantly stimulates fibroblast proliferation and osteogenic phenotype expression.(1)Fluoride significantly stimulates the proliferation and differentiation of fibroblasts;(2)The expression of ALP in the fluoride-exposed fibroblasts was significantly enhanced;(3)Fluoride stimulated the expression from mRNA to proteinof FB Cbfa1,BMP-2 and OCN.2.Expression of IGF-1,FGF-2 and PDGF-B in fluoride-exposed fibroblast(1)Fluoride concentration affects the expression of IGF-1 mRNA and protein in fibroblasts,most of which were significant up-regulated;(2)Fluoride significantly stimulated the expression of FGF-2 and PDGF-B in fibroblasts.3.PTH,PTH-rp,CT and CaSR expression in fluoride-exposed fibroblasts(1)After exposed in fluoride for 48 h,PTH mRNA and protein expression in fibroblasts were enhanced in most of the fluoride-exposed groups,and PTH-rp mRNA was significantly enhanced in the lower dosage groups,and significantly weakened in the higher dosage groups;PTH-rp protein was just the opposite;(2)Fluoride mainly enhanced CT in fibroblasts;(3)Fluoride mainly enhanced the FB CaSR mRNA and protein expression.4.Correlation analysis between Cbfa1 and other related factors(1)Cbfa1 has intricate interrelationships with osteogenic proteins,bone growth factors,and many factors associated with osteogenesis.This correlation is affected by the concentration of fluorine,and their response is different or even opposite in terms of mRNA and protein expression;(2)There are complex and unclear interrelationships between osteogenesis-related factors. |
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