氟化钠染毒致大鼠骨组织形态计量学及微小RNA-23a表达的变化
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摘要
[目的]探讨氟化钠染毒致大鼠骨组织形态计量学变化及对微小RNA(miR NA)-23a表达的影响。[方法 ]将96只SD大鼠按体重随机分为对照组(0 mg/L)、低氟组(50 mg/L)、中氟组(150 mg/L)、高氟组(250 mg/L)。采取饮水染毒方式,染氟至2、4、6个月时分别处死每组8只SD大鼠,收集大鼠左下肢及牙齿,采用灰化-氟离子电极法测定大鼠骨氟、牙氟含量;取大鼠股骨干骺端固定后脱钙,进行苏木精-伊红染色,光镜观察骨组织骨小梁、骺板、成骨细胞的形态学改变;取左前肢,剔除多余结缔组织,用液氮反复研磨成骨粉,使用RNAiso Plus提取骨粉总RNA,采用实时荧光定量PCR法测定miRNA-23a基因表达水平。[结果 ]除2个月时的低氟组外,不同时间各染毒组大鼠氟斑牙检出率均高于对照组(P <0.05)。随染毒时间的延长,各染氟组大鼠骨氟、牙氟含量呈上升的趋势。不同时期中氟组、高氟组大鼠骨小梁和各剂量组骺板面积均大于对照组(P <0.05),低氟组骨小梁面积均低于对照组(P <0.05)。随着染氟剂量的增加,骨小梁和骺板面积呈上升趋势;在2、4、6个月时各剂量组间差异有统计学意义(P <0.01)。各时期染氟大鼠成骨细胞数量均低于对照组(P <0.05),且随着剂量升高呈下降趋势。随着染毒时间的延长,各染氟组骨小梁、骺板面积以及成骨细胞数量均呈下降趋势。染氟2、4、6个月时,各染氟组大鼠miRNA-23a表达量均高于对照组(P <0.05);染氟6个月时,各染氟组大鼠miRNA-23a表达量均较2个月和4个月时升高,呈上升趋势。[结论 ]过量、长期的氟摄入会影响骨的形成和发育,推测miRNA-23a参与了氟中毒致大鼠体内骨转换异常的过程,提示miRNA-23a与氟致全身性危害具有潜在的关联性,但其参与机制尚未明确,有待进一步研究。
[Objective] To investigate the effects of different doses of sodium fluoride exposure on histomorphometric change of bone and miRNA-23 a expression in rats at different developmental stages.[Methods] Ninety-six SD rats were randomly divided into control(0 mg/L) and low(50 mg/L),moderate(150 mg/L), and high(250 mg/L) fluoride treated groups. The animals were executed after 2, 4, and 6 months of the treatment via drinking water to determine bone and dental fluoride levels in left lower limb and teeth by ashing-fluoride ion selective eletrode method. The rat femoral metaphysis samples were stained with hematoxylin-eosin(HE) after decalcification,and observed under optical microscope for evaluating morphological changes in trabeculae,epiphyseal plate, and osteoblasts. The expression levels of miRNA-23 a in total RNA extracted by RNAiso Plus from bone meal of left rat forelimb were detected by real-time fluorescence quantitative PCR. [Results] Except the low NaF group at 2 months, the dental fluorosis incidence rates of each NAF group at different time points were statistically higher than those of the control group(P <0.05). The bone and dental fluoride concentrations of each NaF group were increased over exposure time. At different time points, the trabecular area and epiphyseal area of both moderate and high NaF groups were statistically higher than those of the control group(P < 0.05), while the trabecular area of the low NaF group was statistically lower(P < 0.05). Both trabecular area and epiphyseal area showed rising trends with the increase of NaF dose, with statistical differences among the groups after 2, 4, and 6 months of treatment(P < 0.01). After the treatment for different time, the numbers of osteoblasts of each NaF group were statistically lower than that of the control group(P < 0.05), and showed a decreasing trend with the increase of NaF dose. The trabecular area, epiphyseal area, and number of osteoblasts all showed downward trends with prolonged time of the NaF treatment(P < 0.05). After the treatment for different time, the miRNA-23 a expression levels of each NaF group were statistically higher than that of the control group(P < 0.05); the expression level after 6 months was statistically higher than the levels after 2 and 4 months in each NaF group, showing an upward trend.[Conclusion] Excessive and long-term fluoride intake will affect bone formation and development, indicating that miRNA-23 a is involved in the abnormal bone transformation in rats caused by fluorosis, and that the systematic damage induced by fluoride exposure is potentially linked to miRNA-23 a, but the relevant mechanism is not yet clear and needs further study.
[Objective] To investigate the effects of different doses of sodium fluoride exposure on histomorphometric change of bone and miRNA-23 a expression in rats at different developmental stages.[Methods] Ninety-six SD rats were randomly divided into control(0 mg/L) and low(50 mg/L),moderate(150 mg/L), and high(250 mg/L) fluoride treated groups. The animals were executed after 2, 4, and 6 months of the treatment via drinking water to determine bone and dental fluoride levels in left lower limb and teeth by ashing-fluoride ion selective eletrode method. The rat femoral metaphysis samples were stained with hematoxylin-eosin(HE) after decalcification,and observed under optical microscope for evaluating morphological changes in trabeculae,epiphyseal plate, and osteoblasts. The expression levels of miRNA-23 a in total RNA extracted by RNAiso Plus from bone meal of left rat forelimb were detected by real-time fluorescence quantitative PCR. [Results] Except the low NaF group at 2 months, the dental fluorosis incidence rates of each NAF group at different time points were statistically higher than those of the control group(P <0.05). The bone and dental fluoride concentrations of each NaF group were increased over exposure time. At different time points, the trabecular area and epiphyseal area of both moderate and high NaF groups were statistically higher than those of the control group(P < 0.05), while the trabecular area of the low NaF group was statistically lower(P < 0.05). Both trabecular area and epiphyseal area showed rising trends with the increase of NaF dose, with statistical differences among the groups after 2, 4, and 6 months of treatment(P < 0.01). After the treatment for different time, the numbers of osteoblasts of each NaF group were statistically lower than that of the control group(P < 0.05), and showed a decreasing trend with the increase of NaF dose. The trabecular area, epiphyseal area, and number of osteoblasts all showed downward trends with prolonged time of the NaF treatment(P < 0.05). After the treatment for different time, the miRNA-23 a expression levels of each NaF group were statistically higher than that of the control group(P < 0.05); the expression level after 6 months was statistically higher than the levels after 2 and 4 months in each NaF group, showing an upward trend.[Conclusion] Excessive and long-term fluoride intake will affect bone formation and development, indicating that miRNA-23 a is involved in the abnormal bone transformation in rats caused by fluorosis, and that the systematic damage induced by fluoride exposure is potentially linked to miRNA-23 a, but the relevant mechanism is not yet clear and needs further study.
引文
[1]OZSVATH D L.Fluoride and environmental health:a review[J].Rev Environ Sci Biotechnol,2009,8(1):59-79.
[2]李广生.进一步深入研究慢性氟中毒的发病机制[J].中华预防医学杂志,2003,37(4):225-226.
[3]秦治刚,杨小玉,刘乃杰.氟中毒大鼠股骨组织形态计量学指标测量[J].中国地方病防治杂志,2011,26(1):52-53.
[4]WANG C,LIAO H,CAO Z.Role of osterix and micrornas in bone formation and tooth development[J].Med Sci Monit,2016,22:2934-2942.
[5]Li T,Li H,Wang Y,et al.micro RNA-23a inhibits osteogenic differentiation of human bone marrow-derived mesenchymal stem cells by targeting LRP5[J].Int J Biochem Cell Biol,2016,72:55-62.
[6]ROUFAYEL R,KADRY S.Expression of miR-23a by apoptotic regulators in human cancer:a review[J].Cancer Biol Ther,2017,18(5):269-276.
[7]周贤锋,施静,骆乐.独立通气笼饲养大鼠的进食量及饮水量生理值测定[J].实用医院临床杂志,2015,12(5):72-74.
[8]王杰,陆丹,郑泽爱,等.氟化钠的毒性研究[J].卫生研究,1995,24(S1):39.
[9]李达圣,CUTRESS T W,李晓松,等.砷对大鼠氟牙症影响的实验研究[J].中华预防医学杂志,1996,30(6):354-356.
[10]季伯,刘建国,王凯,等.燃煤型氟斑牙动物模型复制的研究现状[J].口腔医学研究,2014,30(2):180-182,185.
[11]郭晓英,吴思思,何杨,等.亚慢性氟染毒对大鼠骨组织病理改变和β-连环蛋白表达的影响[J].卫生研究,2011,40(3):304-307.
[12]AUBIN J E,HEERSCHE J N.Osteoprogenitor cell differentiation to mature bone-forming osteoblasts[J].Drug Dev Res,2000,49(3):206-215.
[13]李广生,徐辉.当前氟中毒机制研究中的几个问题[J].中国地方病学杂志,2006,25(2):119-120.
[14]ZHONG D B,SUN L T,WU P F,et al.Effect of fluoride on proliferation,differentiation,and apoptosis of cultured caprine(goat)osteoblasts[J].Fluoride,2005,38(3):230-231.
[15]申筑,林少凯,喻茂娟,等.燃煤型氟中毒对大鼠骺板软骨组织形态学的影响[J].现代预防医学,2016,43(16):2917-2920,2925.
[16]DE CáSSIA ALVES NUNES R,CHIBA F Y,PEREIRA A G,et al.Effect of sodium fluoride on bone biomechanical and histomorphometric parameters and on insulin signaling and insulin sensitivity in ovariectomized rats[J].Biol Trace Elem Res,2016,173(1):144-153.
[17]NAIR M,BELAK Z R,OVSENEK N.Effects of fluoride on expression of bone-specific genes in developing Xenopus laevis larvae[J].Biochem Cell Biol,2011,89(4):377-386.
[18]GUO Q,CHEN Y,GUO L,et al.miR-23a/b regulates the balance between osteoblast and adipocyte differentiation in bone marrow mesenchymal stem cells[J].Bone Res,2016,4:16022.
[19]MAO J,LV Z,ZHUANG Y.MicroRNA-23a is involved in tumor necrosis factor-αinduced apoptosis in mesenchymal stem cells and myocardial infarction[J].Exp Mol Pathol,2014,97(1):23-30.
[20]MA S,LIU M,XU Z,et al.A double feedback loop mediated by microRNA-23a/27a/24-2 regulates M1 versus M2 macrophage polarization and thus regulates cancer progression[J].Oncotarget,2016,7(12):13502-13519.
[21]HASSAN M Q,GORDON J A,BELOTI M M,et al.A network connecting Runx2,SATB2,and the miR-23a~27a~24-2cluster regulates the osteoblast differentiation program[J].Proc Natl Acad Sci USA,2010,107(46):19879-19884.
[2]李广生.进一步深入研究慢性氟中毒的发病机制[J].中华预防医学杂志,2003,37(4):225-226.
[3]秦治刚,杨小玉,刘乃杰.氟中毒大鼠股骨组织形态计量学指标测量[J].中国地方病防治杂志,2011,26(1):52-53.
[4]WANG C,LIAO H,CAO Z.Role of osterix and micrornas in bone formation and tooth development[J].Med Sci Monit,2016,22:2934-2942.
[5]Li T,Li H,Wang Y,et al.micro RNA-23a inhibits osteogenic differentiation of human bone marrow-derived mesenchymal stem cells by targeting LRP5[J].Int J Biochem Cell Biol,2016,72:55-62.
[6]ROUFAYEL R,KADRY S.Expression of miR-23a by apoptotic regulators in human cancer:a review[J].Cancer Biol Ther,2017,18(5):269-276.
[7]周贤锋,施静,骆乐.独立通气笼饲养大鼠的进食量及饮水量生理值测定[J].实用医院临床杂志,2015,12(5):72-74.
[8]王杰,陆丹,郑泽爱,等.氟化钠的毒性研究[J].卫生研究,1995,24(S1):39.
[9]李达圣,CUTRESS T W,李晓松,等.砷对大鼠氟牙症影响的实验研究[J].中华预防医学杂志,1996,30(6):354-356.
[10]季伯,刘建国,王凯,等.燃煤型氟斑牙动物模型复制的研究现状[J].口腔医学研究,2014,30(2):180-182,185.
[11]郭晓英,吴思思,何杨,等.亚慢性氟染毒对大鼠骨组织病理改变和β-连环蛋白表达的影响[J].卫生研究,2011,40(3):304-307.
[12]AUBIN J E,HEERSCHE J N.Osteoprogenitor cell differentiation to mature bone-forming osteoblasts[J].Drug Dev Res,2000,49(3):206-215.
[13]李广生,徐辉.当前氟中毒机制研究中的几个问题[J].中国地方病学杂志,2006,25(2):119-120.
[14]ZHONG D B,SUN L T,WU P F,et al.Effect of fluoride on proliferation,differentiation,and apoptosis of cultured caprine(goat)osteoblasts[J].Fluoride,2005,38(3):230-231.
[15]申筑,林少凯,喻茂娟,等.燃煤型氟中毒对大鼠骺板软骨组织形态学的影响[J].现代预防医学,2016,43(16):2917-2920,2925.
[16]DE CáSSIA ALVES NUNES R,CHIBA F Y,PEREIRA A G,et al.Effect of sodium fluoride on bone biomechanical and histomorphometric parameters and on insulin signaling and insulin sensitivity in ovariectomized rats[J].Biol Trace Elem Res,2016,173(1):144-153.
[17]NAIR M,BELAK Z R,OVSENEK N.Effects of fluoride on expression of bone-specific genes in developing Xenopus laevis larvae[J].Biochem Cell Biol,2011,89(4):377-386.
[18]GUO Q,CHEN Y,GUO L,et al.miR-23a/b regulates the balance between osteoblast and adipocyte differentiation in bone marrow mesenchymal stem cells[J].Bone Res,2016,4:16022.
[19]MAO J,LV Z,ZHUANG Y.MicroRNA-23a is involved in tumor necrosis factor-αinduced apoptosis in mesenchymal stem cells and myocardial infarction[J].Exp Mol Pathol,2014,97(1):23-30.
[20]MA S,LIU M,XU Z,et al.A double feedback loop mediated by microRNA-23a/27a/24-2 regulates M1 versus M2 macrophage polarization and thus regulates cancer progression[J].Oncotarget,2016,7(12):13502-13519.
[21]HASSAN M Q,GORDON J A,BELOTI M M,et al.A network connecting Runx2,SATB2,and the miR-23a~27a~24-2cluster regulates the osteoblast differentiation program[J].Proc Natl Acad Sci USA,2010,107(46):19879-19884.