氟砷联合染毒对成骨与破骨细胞共培养体系中TRAF-6/NF-κB1/NFATc1/TRAP mRNA表达的影响
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摘要
[目的]探讨氟、砷及氟砷联合染毒对小鼠颅顶前成骨细胞MC3T3-E1与小鼠单核巨噬细胞RAW 264.7共培养体系中肿瘤坏死因子相关受体因子6(TRAF-6)、核因子κB1(NF-κB1)、活化T细胞核因子1(NFATc1)及抗酒石酸酸性磷酸酶(TRAP)mRNA表达的影响。[方法]用成骨诱导剂诱导后的小鼠颅顶前成骨细胞MC3T3-E1与单核巨噬细胞RAW 264.7细胞建立共培养体系,培养7 d后,用TRAP染色鉴定破骨细胞。采用CCK-8法测定氟、砷不同浓度染毒对细胞增殖的影响,从而确定最终的实验染毒浓度。在共培养体系中,换用含不同浓度的氟化钠(0、0.1、0.4、1.6 mmol/L NaF,记为F0、F0.1、F0.4、F1.6)、亚砷酸钠(0、0.5、2.5、12.5μmol/L NaAsO_2,记为As0、As0.5、As2.5、As12.5)以及氟砷联合(上述剂量两两组合)的培养基分别培养24 h,实时荧光定量PCR法检测各染毒组TRAF-6、NF-κB1、NFATc1及TRAP mRNA的表达水平。[结果]成骨细胞与RAW 264.7细胞共培养7 d后,经TRAP染色可见细胞发生融合,体积变大,多核,说明RAW 264.7细胞已分化为多核的破骨细胞。氟单独染毒,与对照组比较(1.00±0.08、1.00±0.04、1.00±0.02、1.00±0.19),F1.6染毒组的TRAF-6、NF-κB1、NFATc1及TRAP mRNA表达水平均明显增加(1.74±0.16、2.04±0.09、1.33±0.06、3.24±0.29),差异均有统计学意义(P <0.05)。砷单独染毒,As12.5染毒组的TRAF-6和TRAP mRNA表达(1.24±0.06和1.26±0.08)高于对照组(1.00±0.08和1.00±0.19),而NF-κB1和NFATc1 mRNA表达(0.61±0.05和0.60±0.06)在As12.5组均低于对照组(1.00±0.04和1.00±0.02),差异均有统计学意义(P <0.05)。氟砷联合染毒,与F0.1组相比(0.80±0.02和0.59±0.03),NF-κB1和NFATc1的表达在F0.1As12.5组增加(1.11±0.02和1.07±0.05,P <0.05),但均低于氟、砷单独染毒之和;与F0.4比较(2.85±0.13,1.11±0.04,1.09±0.03),TRAF-6、NF-κB1和NFATc1表达在F0.4As12.5组降低(1.38±0.11,0.87±0.03和0.44±0.03,P <0.05);与F1.6比较(2.04±0.09和3.24±0.29),NF-κB1和TRAP mRNA表达在F1.6As12.5组均降低(1.24±0.07和2.09±0.22,P <0.05);与As0.5、As12.5组相比,TRAF-6、NF-κB1、NFATc1及TRAP mRNA在F1.6As0.5组和F1.6As12.5组表达均增加(P <0.05),但均低于氟、砷单独染毒之和。氟、砷对TRAF-6、NF-κB1、NFATc1及TRAP mRNA表达均有主效应作用(F=437.22、657.76、321.89及187.11;F=123.08、170.53、78.99及74.08,均P <0.05);氟砷联合染毒对TRAF-6、NF-κB1、NFATc1及TRAP mRNA表达存在交互作用(F=153.76、97.14、100.31、46.78,均P <0.05)。[结论]高氟可促进成骨细胞与破骨细胞共培养体系中TRAF-6、NF-κB1、NFATc1及TRAP mRNA的表达,从而达到增加破骨细胞分化成熟及骨吸收活性,氟、砷对共培养体系中各基因指标的影响均有主效应作用,氟砷联合染毒对共培养体系中相关基因表达的交互作用主要表现为砷对氟的拮抗作用。
[Objective] To investigate the effects of treatment protocols using fluoride, arsenic, and fluoridearsenic on the expressions of tumor necrosis factor-related receptor factor 6(TRAF-6), nuclear factor kappa B1(NF-κB1), nuclear factor of activated T cells cytoplasmic 1(NFATc1), and tartrate-resistant acid phosphatase(TRAP) mRNA in a co-culture system of mouse precranial osteoblasts MC3T3-E1 and mouse macrophages RAW 264.7. [Methods] After the induction of osteogenic inducer, mouse precranial osteoblasts MC3T3-E1 and monocytes Raw 264.7 were established as a co-culture system. After seven days of culture, osteoclasts were identified by TRAP staining. The effects of different concentrations of the drugs on cell proliferation were determined by CCK-8 method to determine the final experimental concentration. In co-culture system, the cells were cultured for 24 h with sodium fluoride(0, 0.1, 0.4, and 1.6 mmol/L NaF, designated as F0, F0.1, F0.4, and F1.6), sodium arsenite(0, 0.5, 2.5, and 12.5 μmol/L NaAsO_2, designated as As0, As0.5, As2.5, and As12.5), and combined fluoride-arsenite(combinations of 2 doses from previous 2 groups), respectively. The expressions of TRAF-6, NF-κB1, NFATc1, and TRAP mRNA in each group were detected by real-time fluorescence quantitative PCR.[Results] After co-culturing osteoblasts with RAW 264.7 cells for 7 days, the TRAP staining showed that the cells were fused, larger, and multinucleate, indicating that RAW 264.7 cells had differentiated into osteoclasts with multiple nuclei. As to only fluoride exposure, the expressions of TRAF-6, NF-κB1, NFATc1, and TRAP mRNA in the F1.6 group(1.74±0.16, 2.04±0.09, 1.33±0.06, and 3.24±0.29, respectively) were statistically higher than those in the control group(1.00±0.08, 1.00±0.04, 1.00±0.02, and 1.00±0.19, respectively)(P < 0.05). As to only arsenic exposure, the TRAF-6 and TRAP mRNA expressions in the As12.5 group(1.24±0.06 and 1.26±0.08) were higher than those in the control group(1.00±0.08 and 1.00±0.19), but the NF-κB1 and NFATc1 mRNA expressions in the As12.5 group(0.61±0.05 and 0.60±0.06) were significantly lower than those in the control group(1.00±0.04 and 1.00±0.02)(P < 0.05). As to combined exposure, compared with the F0.1 group(0.80±0.02 and 0.59±0.03), the expression of NF-κB1 and NFATc1 increased in the F0.1As12.5 group(1.11±0.02 and 1.07±0.05, P < 0.05), but they were lower than the sum of fluoride and arsenic exposure alone; compared with the F0.4 group(2.85±0.13, 1.11±0.04, and 1.09±0.03), the expressions of TRAF-6, NF-κB1 and NFATc1 decreased in the F0.4As12.5 group(1.38±0.11, 0.87±0.03, and 0.44±0.03, P < 0.05); compared with the F1.6 group(2.04±0.09 and 3.24±0.29), the NF-κB1 and TRAP mRNA expressions decreased in the F1.6As12.5 group(1.24±0.07 and 2.09±0.22, P < 0.05); compared with the As0.5 and As12.5 group, the TRAF-6, NF-κB1, NFATc1, and TRAP mRNA expressions increased in the F1.6As0.5 group and the F1.6As12.5 group(P < 0.05), but lower than the sum of fluoride and arsenic exposure alone. The main effects of fluoride and arsenic on the expressions of TRAF-6, NF-κB1, NFATc1, and TRAP mRNA were statistically significant(F=437.22, 657.76, 321.89, and 187.11; F=123.08, 170.53, 78.99, and 74.08, Ps < 0.05), while the combination of fluoride and arsenic also showed significant interactions on TRAF-6, NF-κB1, NFATc1, and TRAP mRNA expressions(F=153.76, 97.14, 100.31, and 46.78, Ps < 0.05).[Conclusion] In the co-culture system, high fluoride can up-regulate the expression levels of TRAF-6, NF-κB1, NFATc1, and TRAP mRNA, thereby enhance the differentiation, maturation, and bone resorption function of osteoclasts. Fluoride or arsenic alone have adverse effects on osteoclasts. The interaction of fluoride and arsenic with the expressions of osteoclast-related genes is mainly the antagonism of arsenic tofluoride.
[Objective] To investigate the effects of treatment protocols using fluoride, arsenic, and fluoridearsenic on the expressions of tumor necrosis factor-related receptor factor 6(TRAF-6), nuclear factor kappa B1(NF-κB1), nuclear factor of activated T cells cytoplasmic 1(NFATc1), and tartrate-resistant acid phosphatase(TRAP) mRNA in a co-culture system of mouse precranial osteoblasts MC3T3-E1 and mouse macrophages RAW 264.7. [Methods] After the induction of osteogenic inducer, mouse precranial osteoblasts MC3T3-E1 and monocytes Raw 264.7 were established as a co-culture system. After seven days of culture, osteoclasts were identified by TRAP staining. The effects of different concentrations of the drugs on cell proliferation were determined by CCK-8 method to determine the final experimental concentration. In co-culture system, the cells were cultured for 24 h with sodium fluoride(0, 0.1, 0.4, and 1.6 mmol/L NaF, designated as F0, F0.1, F0.4, and F1.6), sodium arsenite(0, 0.5, 2.5, and 12.5 μmol/L NaAsO_2, designated as As0, As0.5, As2.5, and As12.5), and combined fluoride-arsenite(combinations of 2 doses from previous 2 groups), respectively. The expressions of TRAF-6, NF-κB1, NFATc1, and TRAP mRNA in each group were detected by real-time fluorescence quantitative PCR.[Results] After co-culturing osteoblasts with RAW 264.7 cells for 7 days, the TRAP staining showed that the cells were fused, larger, and multinucleate, indicating that RAW 264.7 cells had differentiated into osteoclasts with multiple nuclei. As to only fluoride exposure, the expressions of TRAF-6, NF-κB1, NFATc1, and TRAP mRNA in the F1.6 group(1.74±0.16, 2.04±0.09, 1.33±0.06, and 3.24±0.29, respectively) were statistically higher than those in the control group(1.00±0.08, 1.00±0.04, 1.00±0.02, and 1.00±0.19, respectively)(P < 0.05). As to only arsenic exposure, the TRAF-6 and TRAP mRNA expressions in the As12.5 group(1.24±0.06 and 1.26±0.08) were higher than those in the control group(1.00±0.08 and 1.00±0.19), but the NF-κB1 and NFATc1 mRNA expressions in the As12.5 group(0.61±0.05 and 0.60±0.06) were significantly lower than those in the control group(1.00±0.04 and 1.00±0.02)(P < 0.05). As to combined exposure, compared with the F0.1 group(0.80±0.02 and 0.59±0.03), the expression of NF-κB1 and NFATc1 increased in the F0.1As12.5 group(1.11±0.02 and 1.07±0.05, P < 0.05), but they were lower than the sum of fluoride and arsenic exposure alone; compared with the F0.4 group(2.85±0.13, 1.11±0.04, and 1.09±0.03), the expressions of TRAF-6, NF-κB1 and NFATc1 decreased in the F0.4As12.5 group(1.38±0.11, 0.87±0.03, and 0.44±0.03, P < 0.05); compared with the F1.6 group(2.04±0.09 and 3.24±0.29), the NF-κB1 and TRAP mRNA expressions decreased in the F1.6As12.5 group(1.24±0.07 and 2.09±0.22, P < 0.05); compared with the As0.5 and As12.5 group, the TRAF-6, NF-κB1, NFATc1, and TRAP mRNA expressions increased in the F1.6As0.5 group and the F1.6As12.5 group(P < 0.05), but lower than the sum of fluoride and arsenic exposure alone. The main effects of fluoride and arsenic on the expressions of TRAF-6, NF-κB1, NFATc1, and TRAP mRNA were statistically significant(F=437.22, 657.76, 321.89, and 187.11; F=123.08, 170.53, 78.99, and 74.08, Ps < 0.05), while the combination of fluoride and arsenic also showed significant interactions on TRAF-6, NF-κB1, NFATc1, and TRAP mRNA expressions(F=153.76, 97.14, 100.31, and 46.78, Ps < 0.05).[Conclusion] In the co-culture system, high fluoride can up-regulate the expression levels of TRAF-6, NF-κB1, NFATc1, and TRAP mRNA, thereby enhance the differentiation, maturation, and bone resorption function of osteoclasts. Fluoride or arsenic alone have adverse effects on osteoclasts. The interaction of fluoride and arsenic with the expressions of osteoclast-related genes is mainly the antagonism of arsenic tofluoride.
引文
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[3]AKBAL A,YILMAZ H,TUTKUN E.Arsenic exposure associated with decreased bone mineralization in male[J].Aging Male,2014,17(4):256-258.
[4]HU Y C,CHENG H L,HSIEH B S.Arsenic trioxide affects bone remodeling by effects on osteoblast differentiation and function[J].Bone,2012,50(6):1406-1415.
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[8]张禄锴,马剑雄,赵杰,等.唑来膦酸抑制RAW264.7细胞系分化的最佳浓度体外实验[J].中华老年骨科与康复电子杂志,2018,4(1):4-8.
[9]LI J,WAN Z,LIU H,et al.Osteoblasts subjected to mechanical strain inhibit osteoclastic differentiation and bone resorption in a co-culture system[J].Ann Biomed Eng,2013,41(10):2056-2066.
[10]宋晓甜,洪峰.氟砷联合暴露对大鼠成骨细胞增殖分化及β-连环蛋白表达的影响[J].中华地方病学杂志,2016,35(5):344-349.
[11]ZHAO Q,WANGX,LIUY,etal.NFATc1:functions in osteoclasts[J].Int J Biochem Cell Biol,2010,42(5):576-579.
[12]WONG B R,BESSER D,KIM N,et al.TRANCE,a TNF family member,activates Akt/PKB through a signaling complex involving TRAF6 and c-Src[J].Mol Cell,1999,4(6):1041-1049.
[13]TAKAYANAGI H.Induction and activation of the transcription factor NFATc1(NFAT2)integrate RANKL signaling in terminal differentiation of osteoclasts[J].Dev Cell,2002,3:889-901.
[14]YAMAGUCHI N,KUKITA T,LI Y J,et al.Adiponectin inhibits induction of TNF-alpha/RANKL-stimulated NFATc1 via the AMPK signaling[J].FEBS Lett,2008,582(3):451-456.
[15]王会.CaMKⅡγ信号干扰对破骨细胞分化及相关基因NFATc1、TRAP、c-Src表达的影响[D].广州:华南理工大学,2017.
[16]华坤,卜丽莎,李广生.氟化物对体外培养的大鼠破骨细胞活性的影响[J].中华预防医学杂志,2003,37(4):256-258.
[17]曾奇兵,喻仙,杨鋆,等.氟砷污染对暴露人群骨代谢交互作用[J].中国公共卫生,2012,28(11):1480-1482.
[2]毕华银.氟骨症基础毒理学研究进展[J].地方病通报,1992(3):126-130.
[3]AKBAL A,YILMAZ H,TUTKUN E.Arsenic exposure associated with decreased bone mineralization in male[J].Aging Male,2014,17(4):256-258.
[4]HU Y C,CHENG H L,HSIEH B S.Arsenic trioxide affects bone remodeling by effects on osteoblast differentiation and function[J].Bone,2012,50(6):1406-1415.
[5]PEI J,LI B,GAO Y,et al.Relationship between fluoride exposure and osteoclast markers during RANKL-induced osteoclast differentiation[J].Environ Toxicol Pharmacol,2016,46:241-245.
[6]MATSUO K,IRIE N.Osteoclast-osteoblastcommunication[J].Arch Biochem Biophys,2008,473(2):201-209.
[7]杨德鸿,金大地,陈建庭,等.共育体系中成骨细胞和破骨细胞生物学特性观察[J].中华骨科杂志,2001,21(11):676-680.
[8]张禄锴,马剑雄,赵杰,等.唑来膦酸抑制RAW264.7细胞系分化的最佳浓度体外实验[J].中华老年骨科与康复电子杂志,2018,4(1):4-8.
[9]LI J,WAN Z,LIU H,et al.Osteoblasts subjected to mechanical strain inhibit osteoclastic differentiation and bone resorption in a co-culture system[J].Ann Biomed Eng,2013,41(10):2056-2066.
[10]宋晓甜,洪峰.氟砷联合暴露对大鼠成骨细胞增殖分化及β-连环蛋白表达的影响[J].中华地方病学杂志,2016,35(5):344-349.
[11]ZHAO Q,WANGX,LIUY,etal.NFATc1:functions in osteoclasts[J].Int J Biochem Cell Biol,2010,42(5):576-579.
[12]WONG B R,BESSER D,KIM N,et al.TRANCE,a TNF family member,activates Akt/PKB through a signaling complex involving TRAF6 and c-Src[J].Mol Cell,1999,4(6):1041-1049.
[13]TAKAYANAGI H.Induction and activation of the transcription factor NFATc1(NFAT2)integrate RANKL signaling in terminal differentiation of osteoclasts[J].Dev Cell,2002,3:889-901.
[14]YAMAGUCHI N,KUKITA T,LI Y J,et al.Adiponectin inhibits induction of TNF-alpha/RANKL-stimulated NFATc1 via the AMPK signaling[J].FEBS Lett,2008,582(3):451-456.
[15]王会.CaMKⅡγ信号干扰对破骨细胞分化及相关基因NFATc1、TRAP、c-Src表达的影响[D].广州:华南理工大学,2017.
[16]华坤,卜丽莎,李广生.氟化物对体外培养的大鼠破骨细胞活性的影响[J].中华预防医学杂志,2003,37(4):256-258.
[17]曾奇兵,喻仙,杨鋆,等.氟砷污染对暴露人群骨代谢交互作用[J].中国公共卫生,2012,28(11):1480-1482.