去铁胺对去势伴铁蓄积小鼠骨量的作用及机制

The effect and relative mechanism of deferoxamine on bone mass in ovariectomized mice companied with iron accumulation

目的探讨铁螯合剂甲磺酸去铁胺（DFO）对去势后枸橼酸铁铵干预的雌性小鼠骨量的影响及其作用机制。方法选取2月龄C57BL／6雌性小鼠24只，随机分为去势（OVX）组、去势＋铁剂干预组（OVX＋Fe组）、去势＋铁剂＋去铁胺组（OVX＋Fe＋DFO组）。3组均行双侧卵巢切除术，后两组在去势后1周采用每周0．12g／kg的枸橼酸铁铵干预6周，干预方法为腹腔注射，每周干预3次，OVX组采用同样方式和频次的生理盐水干预。随后OVX＋Fe＋DFO组采用每周4000mg／kgDFO干预2周，干预方法为腹腔注射，一日两次，其他两组给予同样方式和频次的生理盐水干预。干预结束后处死小鼠，微计算机断层扫描技术（Micro—CT）观察股骨远端骨微结构，普鲁士蓝染色观察股骨局部铁蓄积，实时荧光定量聚合酶链反应（Real-timePCR）检测骨组织骨保护素（OPG）、核因子-κB受体活化因子配体（RANKL）的表达，酶联免疫吸附试验法（ELISA）检测小鼠血清铁蛋白、OPG和RANKL水平。结果血清铁蛋白水平方面，OVX＋Fe组[（408．90±17．20）μg/ml]明显高于OVX组[（87．53±3．73）μg/ml]和OVX＋Fe＋DFO组[（162．30±10．58）μg／ml，P=0．000]。普鲁士蓝染色结果显示股骨铁蓄积OVX组〈OVX＋Fe＋DFO组〈OVX＋Fe组。OVX＋Fe组[（0．09±0．02）mg／mm^3]比OVX组[（0．13±0．01）mg／mm^3]的骨密度低（P=0．000），OVX＋Fe＋DFO组[（0．12±0．01）mg／mm^3]比OVX＋Fe组[（0．09±0．02）mg／mm^3]的骨密度高（P=0．000）。OVX＋Fe组（0．75±0．15）骨组织OPG基因表达水平低于OVX组（1．00±0．20）和OVX＋Fe＋DFO组（0．89±0．15，P=0．019、0．012），OVX＋Fe组（2．20±0．12）骨组织RANKL基因表达水平高于OVX组（1．00±0．13）和OVX＋Fe＋DFO组（1．30±0．20，P=0．000），OVX＋Fe组（2．8±0．4）骨组织RANKL／OPG的表达比值高于OVX组（1．0±0．5）和OVX＋Fe＋DFO组（1．4±0．3，P=0．000）。OVX＋Fe组[（1．22±0．18）μg/L]血清OPG含量低于OVX组[（2．33±0．33）μg/L]和OVX＋Fe＋DFO组[（2．18±0．25）μg/L，P=0．000、0．015]，OVX＋Fe组[（484．30±22．87）ng／L]血清RANKL含量高于OVX组[（216．40±21．18）ng／L]和OVX＋Fe＋DFO组[（289．70±20．76）ng／L，P=0．000、0．017]。结论铁蓄积可能通过影响RANKL／核因子-κB受体活化因子（RANK）／OPG轴促进去势小鼠骨量丢失，DFO可以减少小鼠全身及骨组织局部铁蓄积从而恢复骨量。

Objective To observe the effect of iron chelator deferoxamine mesylate （DFO） on ovariectomized mice plus iron intervention, and to explore the relative mechanism. Methods 8 - weeks old C57BL/6J female mice were underwent oophorectomy and randomly divided into ovariectomized （OVX） group, ovariectomized ＋ iron intervention group （ OVX ＋ Fe group） and ovariectomized ＋ iron ＋ deferoxam- ine group （ OVX ＋ Fe ＋ DFO group）. After two weeks, the OVX ＋ Fe group mice and the OVX ＋ Fe ＋ DFO group mice were treated with 0. 12 g/kg per week ferric ammonium citrate intervention four weeks via intra- peritoneal injection three times a week, while the OVX group mice were treated with saline via the same methods and frequency. Afterwards, the OVX ＋ Fe ＋ DFO group mice were administrated with 4 000 mg/kg per week of DFO for two weeks via intraperitoneal injection twice a day, while mice in the other two groups administrated with saline in the same manner and frequency. Mice were sacrificed at the end of the administration. The serum ferritin, osteoprotegerin （OPG） or receptor ativator for nuclear factor- κB ligand （RANKL） level were detected by enzyme - linked immuno sorbent assay. Distal femur three - dimen- sional analysis was detected by micro computed tomography （ Micro - CT）. The prussian blue staining was conducted to observe bone iron deposition in femur. Real -time quantitative polymerase chain reaction （Real- time PCR） was conducted to analyze the expression of OPG and RANKL in bone tissue. Results The serum ferritin in OVX ＋ Fe group [ （408.90 ± 17.20） μg/ml] was significantly higher than the other groups [ OVX group （87. 53 ± 3.73 ） μg/ml and OVX ＋ Fe ＋ DFO group （ 162. 30 ± 10. 58） μg/ml, respectively] （P = 0. 000）. Besides, prussian blue staining showed that the iron deposition in bone of OVX ＋ Fe group mice was the most, and in bone of OVX group mice was the least. These results suggested that iron accumulation could increase systemic and local bone tissue iron accumulation, while DFO could reduce the iron accumulation no matter in serum and in local bone tissue. The bone mass in the OVX ＋ Fe group [ （0.09 ± 0. 02） mg/mm3 ] was significantly lower than the OVX groups [ （0. 13 ± 0. 01 ） mg/mm3, P = 0. 000]. However, after administrated with DFO, the bone mass in the OVX ＋ Fe ＋ DFO group [ （0. 12 ± 0. 01 ） mg/mm3 ] was significantly increased compared with OVX ＋ Fe group [ （0.09 ± 0. 02） mg/mm3, P =0. 000]. The bone tissue OPG expression in OVX ＋ Fe group （0. 75 ±0. 15） was down regulated when compared with the OVX group （1.00 ± 0. 20, P = 0. 019, O. 012）. But after administrated with DFO （0. 89 ± 0. 15 ） , the bone tissue OPG expression was increased significantly compared with the OVX ＋ Fe group （0.75 0.15, P =0. 000）. The bone tissue RANKL expression and RANKL/OPG ratio in OVX ＋ Fe group （2. 20 ± 0. 12 and 2. 80 ＋ 0. 40, respectively） was up regulated when compared with the OVX group （ 1.00±0. 13 and 1.00 ± 0. 50, respectively, P = 0. 000）. But after administrated with DFO, the bone tissue RANKL expression （ 1.3 ± 0. 2） and RANKL/OPG ratio （ 1.4 ± 0. 3 ） was decreased signifi- cantly compared with the OVX ＋ Fe group （P =0. 000, 0. 015）. The levels of OPG and RANKL in serum were as the same as the results of real - time PCR （P = 0. 000, 0. 017）. Conclusion Iron accumulation decreased bone mass in OVX mice through affecting the RANKL/receptor ativator for nuclear factor - κB （RANK）/OPG axis, while DFO might reduce iron accumulation and partially restored this effect.