Zhang H, Yang F, Wang A F, Jia P, Wang X, Yuan Y, Zhang P, Xu Y J
Department of Orthopedics, the Second Affiliated Hospital of Soochow University, Osteoporosis Institute of Soochow University, Suzhou 215004, China.
Zhonghua Yi Xue Za Zhi. 2018 Apr 17;98(15):1183-1188. doi: 10.3760/cma.j.issn.0376-2491.2018.15.012.
To investigate the effects of hepcidin on iron, osteoclasts and bone mass in iron accumulation mice. Eight-week old C57/BL6 male mice were divided into control group (CTR group), high iron group (Fe group) and iron reduction group (Hepc group). CTR group and Fe group were wild-type mice, Hepc group were hepcidin gene overexpression mice.Fe group and Hepc group were injected intraperitoneally with iron, and the mice in the CTR group were injected intraperitoneally with the same amount of saline.Three groups of mice were injected with tamoxifen to induce hepcidin overexpression.Eight weeks later, specimens of three groups of mice were collected and the serum levels of hepcidin, ferritin and carboxy-terminal telopeptides (CTX) were measured by enzyme linked immunosorbent assay (ELISA). Frozen section and Prussian blue stain were carried out to detect bone iron.Reverse transcriptase polymerase chain reaction (RT-PCR) was used to detect osteoclast related gene expression[cytokinin (CTK), matrix metalloprotein (MMP9), protein tyrosine kinase 2β(PTK2β), cathepsin K (CTSK)]. Tartrate resistant acid phosphatase (TRAP) staining was used to observe the proliferation and differentiation of osteoclasts.The osteoclast activity was examined by lacunar resorption pits.The microstructure of the femur was measured by micro-computed tomography (Micro-CT). Variance analysis was applied to compare the differences among groups. The levels of serum hepcidin in CTR, Fe and Hepc group were (508±42), (728±82) and (1 423±85) ng/L, respectively; serum ferritin were (355±26), (1 270±35) and (801±23) μg/L, respectively; CTX were (4.4±0.5), (13.9±1.4) and (8.5±0.6) mg/L, respectively; there were significant differences in the up-mentioned indexes among the three groups (=129.6, 781.7, 77.3, all <0.05). The genes expression level of CTK, MMP9, PTK2β, TRAP, CTSK in the Hepc and CTR group were all significantly lower than those in Fe group (=39.6, 8.0, 5.4, 19.5, 8.8, all <0.05). The results of TRAP staining and pits formation also showed that osteoclast proliferation and activity in group Hepc reduced significantly when compared with those in Fe group (=4.295, 7.557, both <0.05). The overexpression of hepcidin can down-regulate the content of ferritin in iron-accumulating mice and inhibit the proliferation, differentiation and activity of osteoclasts, and improve the bone mass.
为研究铁调素对铁蓄积小鼠铁、破骨细胞及骨量的影响。将8周龄C57/BL6雄性小鼠分为对照组（CTR组）、高铁组（Fe组）和铁还原组（Hepc组）。CTR组和Fe组为野生型小鼠，Hepc组为铁调素基因过表达小鼠。Fe组和Hepc组腹腔注射铁，CTR组小鼠腹腔注射等量生理盐水。三组小鼠均注射他莫昔芬以诱导铁调素过表达。8周后，收集三组小鼠标本，采用酶联免疫吸附测定（ELISA）法检测血清铁调素、铁蛋白和羧基末端肽（CTX）水平。进行冰冻切片和普鲁士蓝染色检测骨铁。采用逆转录聚合酶链反应（RT-PCR）检测破骨细胞相关基因表达[细胞因子（CTK）、基质金属蛋白酶（MMP9）、蛋白酪氨酸激酶2β（PTK2β）、组织蛋白酶K（CTSK）]。采用抗酒石酸酸性磷酸酶（TRAP）染色观察破骨细胞的增殖和分化。通过骨吸收陷窝检测破骨细胞活性。采用显微计算机断层扫描（Micro-CT）测量股骨微观结构。采用方差分析比较各组间差异。CTR组、Fe组和Hepc组血清铁调素水平分别为（508±42）、（728±82）和（1423±85）ng/L；血清铁蛋白分别为（355±26）、（1270±35）和（801±23）μg/L；CTX分别为（4.4±0.5）、（13.9±1.4）和（8.5±0.6）mg/L；三组上述指标差异均有统计学意义（=129.6、781.7、77.3，均<0.05）。Hepc组和CTR组CTK、MMP9、PTK2β、TRAP、CTSK基因表达水平均显著低于Fe组（=39.6、8.了、5.4、19.5、8.8，均<0.05）。TRAP染色和陷窝形成结果也显示，与Fe组相比，Hepc组破骨细胞增殖和活性显著降低（=4.295、7.557，均<0.05）。铁调素过表达可下调铁蓄积小鼠铁蛋白含量，抑制破骨细胞增殖、分化和活性，改善骨量。
