破骨细胞分化增强参与慢性砷暴露导致的小鼠骨密度下降

Augmentation of osteoclast differentiation is involved in chronic arsenic exposure-induced decrease of bone mineral density

目的研究慢性饮水型砷暴露对小鼠长骨骨密度的影响及其相关机制。方法将5月龄雌性C57BL／6小鼠按体质量采用随机数字表法分为假手术组和摘除卵巢（去势手术）组，每组19只。每组分为3个亚组：①对照：饮用蒸馏水（rt=6）；②5mg／L砷处理：饮用蒸馏水中含三价无机砷（iAsⅢ）和五价无机砷（iAsⅢ）各2．5mg／L（n=7）；③20mg／L砷处理：饮用蒸馏水中含iAs“和iAsⅢ各10mg／L（n=6）。自由进食和饮水饲养3个月后，采用双能X射线检测小鼠股骨骨密度。体外实验处理条件：①0．00、0．25、0．50、0．75、1．00、1．50μmol／LiAsⅢ处理单核巨噬白血病（RAW264．7）细胞系或0．0、0．2、0．4、0．6、0．8、1．0μmol／LiAsⅢ处理原代培养小鼠骨髓造血干细胞6d，同时给予50μg／L核因子KB受体活化因子配体（RANKL）和30μg／L巨噬细胞集落刺激因子（M．CSF）进行诱导分化。②根据iAsⅢ处理RAW264．7细胞系诱导分化破骨细胞数量检测结果，选择0．6Ixmol／LiAsⅢ处理组，同时给予0（对照）、5、10mmol／L氧化抑制剂N-乙酰半胱氨酸（NAC）。采用抗酒石酸酸性磷酸酶（TRAP）染色观察iAsUI和NAC对破骨细胞分化的影响。结果①与假手术对照组[（84．44±4．40）mg／cm2]相比，假手术20mg／L砷处理组小鼠股骨骨密度[（80．04±4．06）mg／cm2]呈下降趋势，而去势手术对照组[（76．36±3．36）mg／cm2]明显下降（P〈0．05）；与去势手术对照组比较，去势手术5、20mg／L砷处理组未见明显变化[（77．74±4．91）、（75．56±3．71）mg／cm2,P均〉0．05]。②体外实验显示，在原代培养骨髓造血干细胞向破骨细胞诱导分化过程中，各iAsⅢ处理组破骨细胞数量比较，差异有统计学意义（F=1522，P〈0．05），0．50μmol／LiAsⅢ处理组破骨细胞数量达峰值；在RAW264．7细胞向破骨细胞诱导分化过程中，各iAsⅢ处理组破骨细胞数量比较，差异有统计学意义（F=1781，P〈0．05），0．6μmol／LiAsⅢ处理组破骨细胞数量达峰值；诱导分化低剂量iAsⅢ（0．6μmol／L）暴露RAW264．7细胞时，各NAC处理组破骨细胞数量比较，差异有统计学意义（F=1940，P〈0．05），且随NAC浓度升高，破骨细胞数量明显下降（109．33±3．06、56．00±2．65、22．67±0．58，P均〈0．05）。结论砷暴露对骨代谢的影响具有明显的卵巢功能依赖性，提示砷可能是通过干扰雌激素代谢或功能影响骨代谢。另外，砷暴露引发的氧化应激促进破骨细胞分化，且破骨细胞分化增强可能参与慢性砷暴露导致的骨密度下降，提示抗氧化剂的干预可能有效防治砷暴露引发的骨质疏松。

Objective To study the effects of chronic exposure to inorganic arsenic （iAs） in drinking water on bone mineral density （BMD） in mice and its underlying mechanisms. Methods Five-month-old female C57BL/6 mice were randomly divided into sham groups and ovarectomy （OVX） groups （n = 19 mice each group）, which were further randomly assigned into control group （distilled water） and iAs exposure groups [5 mg/L and 20 mg/L, inorganic arsenite （iAsⅢ）： inorganic arsenate （iAsv） = 1 ： 1]. Following 3 months of exposure to iAs, BMD of the mice were determined by the dual energy X-ray detector. RAW 264.7 cell line and bone marrow hematopoietic stem cells（BMHSC） primarily isolated from C57BL/6 mice were used to study the in vitro effects of iAs on osteoclast differentiation and underlying mechanisms. During differentiation induced by receptor activator of nuclear factor-K B ligand （RANKL, 50 μg/L） and macrophage colony-stimulating factor （M-CSF, 30 μg/L）, RAW 264.7 cell line were treated with 0.00, 0.25, 0.50, 0.75, 1.00, 1.50 μmol/L iAsⅢ, while BMHSC with 0.0, 0.2, 0.4, 0.6, 0.8, 1.0 μmol/L iAsⅢ for 6 days. Based on the effect of iAsnt on the differentiation of RAW cells, RAW 264.7 cell line were treated by 0.6 μmol/L iAsm combined with 0, 5, 10 mmol/L of N-acetyl-cysteine （NAC）. Tartrate resistant acid phosphatase （TRAP）-positive red-colored cells with 3 or more nuclei were considered mature osteoclast. Results The femoral BMD of the mice [（80.04 ± 4.06） mg/cm2] that had been exposed to 20 mg/L of iAs for 3 months was substantially decreased compared to that of sham control mice [（84.44 ± 4.40） mg/cm2]. As expected, the BMD of the OVX group [（76.36 ± 3.36） mg/cm2] was significant decreased compared to that of the sham control group （P 〈 0.05）. However, the BMD among the OVX groups showed no significant difference [5 mg/L： （77.74 ± 4.91） mg/cm2; 20 mg/L： （75.56 ± 3.71） mg/cm2, P 〉 0.05]. In vitro studies, the iAsm evidently affected the osteoclast differentiation in a concentration-dependent fashion. Low concentrations of iAs Ⅲ exposure significantly augmented osteoclast differentiation in the two cell models while high concentrations showed inhibitory effect. In RAW 264.7 cells, the number of osteoclasts in different groups was significantly different （F = 1 522, P 〈 0.05）, in the 0.50 μmo]/L iAsⅢ group the number of osteoclasts reached the peak. In the BMHSC, the number of osteoclasts in different groups was also significantly different （F = 1 781, P 〈 0.05）, in the 0.6 μmol/L iAsⅢ group the number of osteoclasts reached the peak. NAC pretreatment significantly abolished low-level iAsⅢ（0.6μmol/L）-induced augmentation of osteoclast differentiation in a concentration-dependent fashion （0 mmol/L： 109.33 ± 3.06; 5 mmol/L： 56.00 ± 2.65; 10 mmol/L： 22.67 ± 0.58, F = 1 940, P 〈 0.05）. Conclusions The inhibitory effect of iAs on bone metabolism is dependent on the availability of ovary function, suggesting that iAs may interfere with estrogen metabolism and/or function to disturb bone metabolism. Oxidative stress induced by iAs exposure stimulates osteoclast differentiation, and the increased osteoclast differentiation may be involved in the reduction of BMD caused by chronic iAs exposure. These preliminary findings suggest that antioxidant intervention may be an effective approach to prevent osteoporosis induced by chronic iAs exposure.