Shikimic Acid Inhibits Osteoclastogenesis in Vivo and in Vitro by Blocking RANK/TRAF6 Association and Suppressing NF-kappaB and MAPK Signaling Pathways.
Shikimic Acid Inhibits Osteoclastogenesis in Vivo and in Vitro by Blocking RANK/TRAF6 Association and Suppressing NF-kappaB and MAPK Signaling Pathways.
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DOI：
10.1159/000496039
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发表时间：
2018-01-01
期刊：
影响因子：
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通讯作者：
Su, Jiacan
中科院分区：
文献类型：
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作者：
Chen, Xiao;Li, Xiaoqun;Su, Jiacan
背景/目的：骨稳态与骨吸收破骨细胞和骨形成成骨细胞之间的平衡有关。由于骨生成减少或破骨细胞生成过多而导致的骨稳态失衡可导致诸如骨质疏松症、佩吉特病和类风湿性关节炎的病症。莽草酸是一种环己烷羧酸，据报道表现出药理学性质，包括抗炎和抗氧化活性。方法：首先，采用MTT法检测体外培养的成骨细胞的活性。抗酒石酸酸性磷酸酶（TRAP）和肌动蛋白环形成试验，以及免疫荧光染色，然后进行评估破骨细胞生成。通过蛋白质印迹法表征潜在的信号传导途径，并在过表达实验中进行验证。相关因素采用蛋白质印迹法、逆转录聚合酶链反应、电泳迁移率变动分析和免疫共沉淀法进行检测。结果：TRAP染色显示莽草酸对骨髓单核细胞和RAW264.7细胞的破骨细胞生成和陷窝吸收有明显抑制作用，肌动蛋白环形成实验显示莽草酸对破骨细胞的骨吸收功能有明显抑制作用。此外，莽草酸抑制核因子-κ B RANK/肿瘤坏死因子受体相关因子6（TRAF 6）的受体激活剂，抑制核因子-κ B和丝裂原活化蛋白激酶信号通路，并下调活化T细胞胞质1的核因子。破骨细胞生成生物标志物的表达，包括TRAF 6、降钙素受体、TRAP、组织蛋白酶K和基质金属蛋白酶-9，受到抑制。在体内，莽草酸也显着改善骨丢失，并防止破骨细胞在ovariectomized mice.CONCLUSION：莽草酸抑制破骨细胞生成和破骨细胞功能，通过阻断RANK配体诱导的募集TRAF 6，以及下游信号通路在体外。莽草酸还减少卵巢切除术诱导的破骨细胞生成和体内骨丢失。
BACKGROUND/AIMS: Bone homeostasis is associated with the balance between bone-resorbing osteoclasts and bone-forming osteoblasts. Unbalanced bone homeostasis as a result of reduced osteogenesis or excessive osteoclastogenesis can lead to disorders such as osteoporosis, Paget's disease, and rheumatoid arthritis. Shikimic acid is a cyclohexanecarboxylic acid, reported to exhibit pharmacological properties including anti-inflammatory and antioxidant activities. However, its effects on bone homeostasis remain unknown.METHODS: First, the in vitro MTT cell viability assay was performed. Tartrate-resistant acid phosphatase (TRAP) and actin ring formation assays, as well as immunofluorescence staining were then performed to evaluate osteoclastogenesis. Potential signaling pathways were characterized by western blotting and verified in overexpression experiments. Related factors were examined by western blotting, reverse transcription polymerase chain reaction, electrophoretic mobility shift assay, and co-immunoprecipitation. Ovariectomized mice were used for the in vivo study.RESULTS: TRAP staining showed that shikimic acid significantly inhibited osteoclastogenesis and pit resorption in bone marrow monocytes and RAW264.7 cells, and actin ring formation assays showed that shikimic acid suppressed the bone resorption function of osteoclasts. Furthermore, shikimic acid inhibited the receptor activator of nuclear factor-kappaB RANK/tumor necrosis factor receptor-associated factor 6 (TRAF6) association, suppressed nuclear factor-kappaB and mitogen-activated protein kinase signaling pathways, and downregulated nuclear factor of activated T-cell cytoplasmic 1. The expression of osteoclastogenesis biomarkers, including TRAF6, calcitonin receptor, TRAP, cathepsin K, and matrix metalloproteinase-9, was inhibited. In vivo, shikimic acid also significantly ameliorated bone loss and prevented osteoclastogenesis in ovariectomized mice.CONCLUSION: Shikimic acid inhibited osteoclastogenesis and osteoclast function by blocking RANK ligand-induced recruitment of TRAF6, as well as downstream signaling pathways in vitro. Shikimic acid also reduced ovariectomy-induced osteoclastogenesis and bone loss in vivo.