骨保护素、核因子-κB受体活化因子配基在氟砷联合染毒大鼠骨骼毒性中的调控作用

Role of osteoprotegerin and receptor activator of nuclear factor-κB ligand in bone toxicity in rats co-exposed tofluoride and arsenite

目的 探讨骨保护素（OPG）与核因子-κB受体活化因子配基（RANKL）在氟砷联合染毒大鼠骨骼毒性中的调控作用。 方法 选取192只8周龄清洁级Wistar大鼠，体重为（200 ± 50）g，根据2 × 4析因实验设计按体重采用随机数字表法分成16组， 每组12只，雌雄各半。用氟化钠（NaF，空白对照0.0 mg／kg、低氟5.0 mg／kg、中氟10.0 mg／kg、高氟20.0 mg／kg）和亚砷酸 钠（NaAsO2，空白对照0.0 mg／kg、低砷2.5 mg／kg、中砷5.0 mg／kg、高砷10.0 mg／kg）灌胃染毒6个月。依据上述处理因素将 大鼠分为对照组（F0As0）、低氟组（F5.0As0）、中氟组（F10.0As0）、高氟组（F20.0As0）、低砷组（F0As2.5）、中砷组 （F0As5.0）、高砷组（F0As10.0）、低氟低砷组（F5.0As2.5）、低氟中砷组（F5.0As5.0）、低氟高砷组（F5.0As10.0）、中氟 低砷组（F10.0As2.5）、中氟中砷组（F10.0As5.0）、中氟高砷组（F10.0As10.0）、高氟低砷组（F20.0As2.5）、高氟中砷组 （F20.0As5.0）、高氟高砷组（F20.0As10.0）。采用酶联免疫吸附测定法（ELISA法）检测大鼠骨组织中OPG及RANKL蛋白表达水平 ，实时荧光定量PCR法检测OPG及RANKL mRNA表达水平。结果 与F0As0组［（2.678 ± 0.136）ng／mg、（29.658 ± 0.662）pg／ mg］比较，F5.0As0、F10.0As0、F20.0As0组骨组织中OPG［（2.857 ± 0.162）、（2.983 ± 0.272）、（3.117 ± 0.143）ng／ mg］、RANKL［（32.533 ± 0.999）、（32.698 ± 1.932）、（33.331 ± 1.140）pg／mg］蛋白表达水平随染氟剂量的升高均有 升高趋势；与F0As0组比较，F0As2.5、F0As5.0、F0As10.0组骨组织RANKL蛋白水平［（32.348 ± 2.838）、（31.589 ± 1.359） 、（28.843 ± 1.908）pg／mg］随染砷剂量的升高而先升高后下降（P均 〈 0.05）。与F0As0组（0.83 ± 0.19、0.92 ± 0.23） 比较，F5.0As0、F10.0As0、F20.0As0组骨组织OPG（1.14 ± 0.27、1.33 ± 0.39、1.69 ± 0.77）、RANKL mRNA水平（1.02 ± 0.21、1.17 ± 0.15、1.25 ± 0.31）随染氟剂量的升高均有升高趋势。氟对OPG、RANKL蛋白和mRNA的表达有主效应（F = 11.530 、21.765、6.320、3.543，P均 〈 0.05），氟砷联合对RANKL蛋白和mRNA、OPG蛋白表达存在交互拮抗作用（F = 9.496、2.217、 3.375，P均 〈 0.05）。结论 氟砷联合染毒对大鼠骨组织RANKL及OPG的影响中氟起主导作用，砷间接参与了氟致大鼠骨骼毒性作用 ，氟砷联合对OPG及RANKL表达的交互作用主要表现为拮抗作用。

Objective To analyze the role of osteoprotegerin （OPG） and receptor activator of nuclear factor-κB ligand （RANKL） in bone toxicity in rats co-exposed to fluoride and arsenite. Methods One hundred and ninety-two 8-week-old clean-grade Wistar rats weighing （200 ± 50） g were divided into 16 groups by weight using random number table method of 12 rats in each group by 2 × 4 factorial experimental design （half female and half male）, and treated with different doses of fluoride, arsenite and fluoride plus arsenite in deionized water （untreated control group containing 0.0 mg/kg fluoride and 0.0 mg/kg arsenite; low-, moderate-, and high-fluoride groups supplemented with 5.0, 10.0 and 20.0 mg/kg fluoride and 2.5, 5.0 and 10.0 mg/kg arsenite） for 6 months. Rats were divided into control （F0As0）, low fluorine （F5.0As0）, moderate fluoride （F10.0As0）, high fluoride （F20.0As0）, low arsenic （F0As2.5）, moderate arsenic （F0As5.0）, high arsenic （F0As10.0）, low fluorine and low arsenic（F5.0As2.5）, low fluorine and moderate arsenic （F5.0As5.0）, low fluorine and high arsenic （F5.0As10.0）, moderate fluorine and low arsenic （F10.0As2.5）, moderate fluorine and moderate arsenic （F10.0As5.0）, moderate fluorine and high arsenic （F10.0As10.0）, high fluorine and low arsenic （F20.0As2.5）, high fluorine and moderate arsenic （F20.0As5.0）, high fluorine and high arsenic （F20.0As10.0） groups. The protein expressions of OPG and RANKL in bone were measured via the enzyme-linked immunosorbent assay method. The mRNA expressions of OPG and RANKL were measured with quantitative real-time PCR. Results Compared with F0As0 [（2.678 ± 0.136） ng/mg, （29.658 ± 0.662） pg/mg], the protein expressions of OPG [（2.857 ± 0.162）, （2.983 ± 0.272）, （3.117 ± 0.143） ng/mg], and RANKL [（32.533 ± 0.999）, （32.698 ± 1.932）, （33.331 ± 1.140） pg/mg] in F5.0As0, F10.0As0, F20.0As0 were increased with increasing of fluoride doses; increased first and then decreased was observed in levels of RANKL protein [（32.348 ± 2.838）, （31.589 ± 1.359）, （28.843 ± 1.908） pg/mg] in F0As2.5, F0As5.0, F0As10.0 with increasing of arsenic doses （P 〈 0.05）. Compared with F0As0 （0.83 ± 0.19, 0.92 ± 0.23）, the mRNA expressions of OPG （1.14 ± 0.27, 1.33 ± 0.39, 1.69 ± 0.77） and RANKL （1.02 ± 0.21, 1.17 ± 0.15, 1.25 ± 0.31） in F5.0As0, F10.0As0, F20.0As0 were increased with increasing of fluoride dose. Fluoride had a significant effect on protein and mRNA expressions of OPG and RANKL （F = 11.530, 21.765, 6.320, 3.543, P 〈 0.05）. There was interaction between fluoride and arsenite on the expressions of RANKL protein and mRNA, OPG protein （F = 9.496, 2.217, 3.375, P 〈 0.05）. Conclusion When rat is co-exposed to fluorine and arsenic, fluorine plays a leading role in regulating RANKL and OPG, and arsenic is indirectly involved in the fluorine bone toxicity in rats, fluorine and arsenic has a antagonistic effect on OPG and RANKL expressions.