Dou C, Chen Y, Ding N, Li N, Jiang H, Zhao C, Kang F, Cao Z, Quan H, Luo F, Xu J, Dong S
Department of Biomedical Materials Science, School of Biomedical Engineering, Third Military Medical University, Gaotanyan Street No.30, Chongqing, 400038, China.
Department of Orthopedics, Southwest Hospital, Third Military Medical University, Chongqing, 400038, China.
Osteoporos Int. 2016 Jul;27(7):2335-2344. doi: 10.1007/s00198-016-3496-8. Epub 2016 Jan 25.
Xanthotoxin (XAT) is extracted from the seeds of Ammi majus. Here, we reported that XAT has an inhibitory effect on osteoclastogenesis in vitro through the suppression of both receptor activator of nuclear factor-κB ligand (RANKL)-induced ROS generation and Ca(2+) oscillations. In vivo studies showed that XAT treatment decreases the osteoclast number, prevents bone loss, and restores bone strength in ovariectomized mice.
Excessive osteoclast formation and the resultant increase in bone resorption activity are key pathogenic factors of osteoporosis. In the present study, we have investigated the effects of XAT, a natural furanocoumarin, on the RANKL-mediated osteoclastogenesis in vitro and on ovariectomy-mediated bone loss in vivo.
Cytotoxicity of XAT was evaluated using bone marrow macrophages (BMMs). Osteoclast differentiation, formation, and fusion were assessed using the tartrate-resistant acid phosphatase (TRAP) stain, the actin cytoskeleton and focal adhesion (FAK) stain, and the fusion assay, respectively. Osteoclastic bone resorption was evaluated using the pit formation assay. Reactive oxygen species (ROS) generation and removal were evaluated using dichlorodihydrofluorescein diacetate (DCFH-DA). Ca(2+) oscillations and their downstream signaling targets were then detected. The ovariectomized (OVX) mouse model was adopted for our in vivo studies.
In vitro assays revealed that XAT inhibited the differentiation, formation, fusion, and bone resorption activity of osteoclasts. The inhibitory effect of XAT on osteoclastogenesis was associated with decreased intracellular ROS generation. XAT treatment also suppressed RANKL-induced Ca(2+) oscillations and the activation of the resultant downstream calcium-CaMKK/PYK2 signaling. Through these two mechanisms, XAT downregulated the key osteoclastogenic factors nuclear factor of activated T cells c1 (NFATc1) and c-FOS. Our in vivo studies showed that XAT treatment decreases the osteoclast number, prevents bone loss, rescues bone microarchitecture, and restores bone strength in OVX mice.
Our findings indicate that XAT is protective against ovariectomy-mediated bone loss through the inhibition of RANKL-mediated osteoclastogenesis. Therefore, XAT may be considered to be a new therapeutic candidate for treating osteoporosis.
花椒毒素（XAT）从大阿米芹种子中提取。在此，我们报道XAT在体外通过抑制核因子κB受体活化因子配体（RANKL）诱导的活性氧（ROS）生成和Ca(2+)振荡对破骨细胞生成具有抑制作用。体内研究表明，XAT治疗可减少去卵巢小鼠的破骨细胞数量，预防骨质流失，并恢复骨强度。
破骨细胞过度形成以及由此导致的骨吸收活性增加是骨质疏松症的关键致病因素。在本研究中，我们研究了天然呋喃香豆素XAT对体外RANKL介导的破骨细胞生成以及体内去卵巢介导的骨质流失的影响。
使用骨髓巨噬细胞（BMMs）评估XAT的细胞毒性。分别使用抗酒石酸酸性磷酸酶（TRAP）染色、肌动蛋白细胞骨架和粘着斑激酶（FAK）染色以及融合试验评估破骨细胞分化、形成和融合。使用凹坑形成试验评估破骨细胞性骨吸收。使用二氯二氢荧光素二乙酸酯（DCFH-DA）评估活性氧（ROS）的生成和清除。然后检测Ca(2+)振荡及其下游信号靶点。采用去卵巢（OVX）小鼠模型进行体内研究。
体外试验表明，XAT抑制破骨细胞的分化、形成、融合和骨吸收活性。XAT对破骨细胞生成的抑制作用与细胞内ROS生成减少有关。XAT治疗还抑制RANKL诱导的Ca(2+)振荡以及由此产生的下游钙-CaMKK/PYK2信号的激活。通过这两种机制，XAT下调关键破骨细胞生成因子活化T细胞核因子c1（NFATc1）和c-FOS。我们的体内研究表明，XAT治疗可减少OVX小鼠的破骨细胞数量，预防骨质流失，挽救骨微结构，并恢复骨强度。
我们的研究结果表明，XAT通过抑制RANKL介导的破骨细胞生成对去卵巢介导的骨质流失具有保护作用。因此，XAT可被认为是治疗骨质疏松症的一种新的治疗候选药物。
