雌激素缺乏环境下铁蓄积对骨吸收及破骨细胞分化的影响

Effect of iron accumulation on bone resorption and osteoclast differentiation in the absence of estrogen

目的在雌激素缺乏状态下,探讨转录因子κB (nuclear factor-kappa B,NF-κB)信号通路在高铁环境影响骨吸收及破骨细胞分化过程中的作用。方法将小鼠随机分为假手术组(SHAM组)、去势组(OVX组)、高铁去势组(F+OVX组)。酶联免疫吸附法(enzyme linked immunosorbent assay,ELISA)检测血清Ⅰ型胶原C端肽(C-terminal peptide of type I collagen,CTX),股骨硬组织切片行普鲁士蓝铁染色,逆转录聚合酶链反应(reverse transcription-polymerase chain reaction,RT-PCR)检测胫骨基因酸性磷酸酶(tartrate-resistant acid phosphatase 5,Acp5)、组蛋白酶K (cathepsin K,Ctsk)表达差异。培养小鼠单核细胞株RAW264. 7向破骨细胞分化,模拟动物实验分为雌激素正常组(E2+组)、雌激素缺乏组(E2-组)、高铁雌激素缺乏组(F+E2-组),抗酒石酸酸性磷酸酶(tartrate-resistant acid phosphatase,TRAP)染色及噬骨馅窝实验观察破骨细胞分化情况,提取胞浆、胞核蛋白,Western-Blot检测各组胞浆磷酸化抑制因子(p-inhibitor of NF-κB,p-IκBα)、亚基p65蛋白水平及胞核蛋白亚基p65的差异。结果动物实验中,F+OVX组股骨普鲁士蓝染色铁沉积较SHAM组及OVX组更明显。SHAM组、OVX组、F+OVX组血清CTX水平分别为(2 588±661. 9)、(9 624±1 140)、(14 506±914. 9) pmol/L,Acp5表达水平分别为(9. 7±2. 7)%、(36. 1±4. 5)%、(55. 4±4. 4)%,Ctsk表达水平分别为(11. 1±3. 3)%、(53. 0±4. 7)%、(66. 1±4. 2)%;与SHAM组比较,OVX组血清CTX、基因Acp5、Ctsk表达水平升高(P分别为0. 000、0. 001、0. 000),F+OVX组较OVX组进一步升高(P分别为0. 000、0. 006、0. 023)。细胞实验中,E2-组TRAP阳性细胞及噬骨馅窝数分别为(142. 329±12. 699)和(31. 667±4. 320),较E2+组的(45. 875±7. 972)和(9. 500±2. 074)增加,F+E2-组分别为(468. 782±49. 015)和(84. 167±8. 010),进一步增加(均P=0. 000)。Western-Blot结果显示,E2-组胞核蛋白p65、胞浆蛋白p-IκBα分别为(50. 1±6. 0)%和(2. 0±0. 0)%,均较E2+组的(2. 2±1. 1)%和(0. 8±0. 0)%升高(P分别为0. 000和0. 042),F+E2-组分别为(69. 3±7. 1)%和(22. 2±2. 1)%,均较E2-组进一步升高(P分别为0. 023和0. 000)。结论雌激素缺乏合并铁蓄积可促进破骨活性,加重骨量丢失,该影响可能与NF-κB信号通路有关。

Objective To explore the role of nuclear factor-kappa B (NF-kappa B) signaling pathway in the process of bone resorption and osteoclast differentiation under the condition of estrogen deficiency in high iron environment. Methods Mice were randomly divided into sham operation group (SHAM group), ovariectomy group (OVX group) and iron + ovariectomy group (F+OVX group). Serum C-terminal peptide of type Ⅰ collagen (CTX) was detected by ELISA. Femoral tissue sections were stained with Prussian blue iron. Tibial tartrate-resistant acid phosphatase 5 (Acp5) and cathepsin K (Ctsk) were detected by reverse transcription-polymerase chain reaction (RT-PCR). Mouse monocyte RAW264.7 was cultured and differentiated into osteoclasts. Animal experiments were simulated and divided into normal estrogen group (E 2 + group), estrogen deficiency group (E 2 - group) and ferrugin deficiency group (F+ E 2 - group). Tartrate-resistant acid phosphatase (TRAP) staining and bone-piercing experiment were used to observe the differentiation of osteoclasts, extract cytoplasmic and nuclear proteins. Western-Blot was used to detect the levels of p-inhibitor of NF-κB (p-IκBα) and subunit p65 in each group.Results In vitro experiment, iron deposition was more obvious in F+OVX group than that in SHAM group and OVX group.In SHAM group, OVX group, and F+OVX group, the level of serum CTX was (2 588±661.9),(9 624±1 140), and (14 506±914.9)pmol/L, respectively;the level of Acp5 was (9.7±2.7)%,(36.1±4.5)%, and (55.4±4.4)%, respectively;the level of Ctsk was (11.1±3.3)%,(53.0±4.7)%, and (66.1±4.2)%, respectively. Compared with SHAM group, the levels of serum CTX, gene Acp5 and Ctsk in OVX group increased (P was 0.000, 0.001 and 0.000, respectively), and those in F+OVX group increased further (P was 0.000, 0.006 and 0.023, respectively). In vivo experiment, the number of TRAP positive cells and bone-phage fillings in E 2 - group[(142.329±12.699) and (31.667±4.320), respectively] was higher than that in E 2 +group [(45.875±7.972) and (9.500±2.074), respectively], and further increased in F+E 2 - group [(468.782±49.015) and (84.167±8.010), all P=0.000]. Western-Blot results showed that the levels of nucleoprotein p65 and cytoplasmic protein p-IκBα in E 2 - group [(50.1±6.0)% and (2.0±0.0)%] were higher than those in E 2 + group [(2.2±1.1)% and (0.8±0.0)%, P was 0.000 and 0.042, respectively], and those in F+E 2 - group [(69.3±7.1)% and (22.2±2.1)%] were higher than those in E 2 - group (P was 0.0023 and 0.000, respectively). ConclusionEstrogen deficiency combined with iron accumulation can promote osteoclasts activity and aggravate bone loss, which may be related to NF-kappa B signaling pathway.