| Objective:Fluorine is the essential trace element for the humans.But excessive ingestion of fluoride can cause endemic fluorosis.Epidemiology survey indicated that the bone growth and the bone age of children and adolescents in high-fluorine areas was delayed;The results of animal and organ culture studies showed that fluorine can inhibit the longitudinal growth of bone by inhibiting the process of endochondral bone formation.Animal and organ culture studies showed that fluorine can inhibit the longitudinal growth of bone by inhibiting the process of endochondral bone formation.Endochondral ossification is one of the main ways of bone formation.The process of endochondral bone formation occurs at the primary and secondary ossification centers separated by the growth plate.And this process is the main force to driving the longitudinal growth of the bone.Endochondral ossification is tightly controlled by circulating systemic hormones and locally produced signaling factors,such as growth hormone(GH)and insulin-like growth factor(IGF-I),Indian hedgehog(Ihh)/parathyroid hormone–related protein(PTHrP),fibroblast growth factors(FGFs)signaling pathway.In addition the transcription factors Sox9 and Runx2 play major roles in the process of endochondral bone formation.Epidermal growth factor receptor(EGFR)is also an important regulator of cartilage metabolism,and it regulates the proliferation and differentiation of chondrocytes and the process of osteoclastogenesis.However,it has not been reported whether fluoride can inhibit the process of endochondral osteogenesis by affecting EGFR signaling pathway.Therefore we established the fluorosis rat models to observe the effects of different concentrations of fluorine on the chondrocyte in the growth plate cartilage,include chondrocyte proliferation,differentiation,hypertrophy,matrix mineralization,invasion of blood vessels,osteoclastogenesis and other processes of endochondral ossification.At the same time,explore whether fluorine regulates endochondral ossification through affecting EGFR signaling pathways.The ultimate goal is to provide clues to elucidate the mechanism of fluoride inhibition of long bone growth and provide basis for the prevention and treatment of skeletal fluorosis.Methods:In this study,21 days-old male Sprague-Dawley rats(60±10g)were randomly divided into four groups,exposed to 0(control),50,100,150 ppm fluoride(from NaF)in drinking water for 12 weeks.At the end of the exposure period,the body length,femur length,and tibia length were measured.The left tibia bone were fixed and embedded to prepare paraffin sections.Taken the right tibia bone growth plate cartilage to observe the effect of fluoride on ultrastructure of chondrocytes under the electron microscopy.The IGF-I contents in serum were assayed by ELISA method;Paraffin sections of the tibia were stained by hematoxylin-eosin(HE)and Alcian blue/Van Gesion double staining to observe the histomorphology of the growth plate cartilage;BrdU staining was used to detect the proliferation of chondrocytes,and TRAP staining was used to detect osteoclastogenesis;Besides,paraffin sections were stained by immunohistochemistry staining to detect the expression of related proteins during the process of endochondral ossification in the growth plate cartilage(COL II,COL X,Sox9,Runx2,Mig6,p-EGFR,MMP-13,VEGF,RANKL,OPG).Results:1.Effects of fluoride on body weight,body length and food utilization in Rats:Compared with the control group,the body weight in each fluorine group were lower than the control group;From the 6th week,the body weight of 150 ppm F~-group rats was significantly lower than control,the difference was statistically significant(P<0.05);After the end of the fluorine exposure period,the body length of150 ppm F~-group rats was significantly lower than the control,the difference was statistically significant(P<0.05);The length of tibia in 150 ppm F~-group rats was significantly lower than that in control group,50 ppm F~-group and 100 ppm F~-group,and the difference was statistically significant(P<0.05);The length of femur in 100ppm F-group was significantly lower than control,and the difference was statistically significant.(P<0.05);Compared with the control group and 50 ppm F~-group,the femur length in 150 ppm F~-group rats was significantly decreased,and the difference was statistically significant(P<0.05);There was no significant difference in food utilization rate among rats in each group.2.Effects of fluoride on IGF-I levels in rats serum:The levels of IGF-I in each group were lower than control,and decreased with the increase of the fluoride concentration,the 150 ppm F~-group had a significant decrease,the difference was statistically significant(P<0.01).3.Effects of fluoride on morphology of rat growth plate cartilage:The growth plate boundary of the control group is clear,and the chondrocytes of the growth plate are arranged in columns,The trabecular bones are arranged in parallel on the chondrocyte column.In the fluorine exposure groups,the boundary of the growth plate at the epiphyseal was disordered,and the trabecular bone became shorter,thinner,sparsely arranged,and the trabecular bone fracture occurred.The height of the growth plate and the height of the proliferation zone in the fluorine groups were significantly higher than those in the control group(P<0.05);The height of growth plate hypertrophy zone was significantly increased in 150 ppm F~-group rats,and the difference was statistically significant(P<0.05);There was no significant difference in the ratio of height between proliferation zone and hypertrophic zone of growth plate in rats of each group;The number of hypertrophic chondrocytes in growth plate was slightly increased in fluorine group;The longitudinal diameter of hypertrophic chondrocytes in the growth plate of rats in the fluorine groups increased with the increase of fluoride concentration,and the 150 ppm F~-group had the most significant,the difference was statistically significant(P<0.05).4.Effects of fluoride on the ultrastructure of chondrocytes:The ultrastructure of chondrocytes in the control group showed regular nuclear morphology,uniform chromatin distribution,and clear and neat endoplasmic reticulum;In the fluorine-treated group,the nuclear morphology of chondrocytes showed irregular changes,chromatin accumulated around the nucleus,endoplasmic reticulum dilatation,mitochondrial swelling,and lysosomes increased;In the 150ppm F~-group,there was a pre-apoptotic phenomenon,endoplasmic reticulum expanded into pools,and lysosomes increased in chondrocytes.5.Effects of fluoride on the proliferation of rat growth chondrocyte cells:Compared with the control group,the proliferation of growth plate chondrocytes decreased in the fluorine group,and the BrdU positive cell rate decreased with the increase of the fluoride concentration,the 150 ppm F~-group was significantly reduced(P<0.05).6.Effects of fluoride on rat chondrocyte-associated proteins in growth plates:Compared with the control group,the expression levels of COLII,p-EGFR,MMP-13,Runx2,VEGF protein were decreased in the fluorine group,and the expression of COX,Sox9,Mig-6,OPG protein were increased.7.Effects of fluoride on osteoclast formation in rat growth plates:Compared with the control group,the number of osteoclasts in the fluoroine groups gradually decreased with the increase of the fluoride concentration,the 150 ppm F~-group was significantly reduced(P<0.05).Conclusion:1.Fluoride may inhibit bone growth by reducing systemic hormone IGF-I expression.2.Fluoride can inhibit the proliferation and cause chondrocyte differentiation disorders,and inhibit osteoclastogenesis through the inhibition of EGFR signaling pathway,thereby affecting the endochondral osteogenesis process.3.Fluoride may upregulate Sox9 and downregulate transcription factors Runx2 to inhibit cartilage matrix degradation,mineralization,and invasion of blood vessels by thereby inhibiting endochondral ossification. |
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