Rhinacanthin C Inhibits Osteoclast Differentiation and Bone Resorption: Roles of TRAF6/TAK1/MAPKs/NF-κB/NFATc1 Signaling

Rhinacanthin C is a naphthoquinone ester with anti-inflammatory activity, found in Rhinacanthus nasutus (L) Kurz (Acanthaceae). We found that rhinacanthin C inhibited osteoclast differentiation stimulated by the receptor activator of nuclear factor-κB ligand (RANKL) in mouse bone marrow macrophage cultures, although the precise molecular mechanisms underlying this phenomenon are unclear. In this study, we investigated the inhibitory mechanisms of rhinacanthin C in osteoclastogenesis. Rhinacanthin C suppressed RANKL-induced nuclear factor of activated T cells c1 (NFATc1) expression. Phosphorylation of ERK, JNK, and NF-κB, but not p38, was inhibited by rhinacanthin C, which also inhibited RANKL-stimulated TRAF6-TAK1 complex formation. Thus, the anti-osteoclastogenic effect of rhinacanthin C is mediated by a cascade of inhibition of RANKL-induced TRAF6-TAK1 association followed by activation of MAPKs/NF-κB; this leads to suppression of c-Fos and NFATc1, which regulate transcription of genes associated with osteoclast differentiation. In vivo, rhinacanthin C also reduced RANKL-induced osteoclast formation and bone resorption in mouse calvaria. Rhinacanthin C also suppressed LPS-stimulated osteoclastogenesis and bone resorption in vitro and in vivo. Rhinacanthin C may provide a novel therapy for abnormal bone lysis that occurs during inflammatory bone resorption.爵床素C（Rhinacanthin C）是一种具有抗炎活性的萘醌酯类化合物，存在于爵床（Rhinacanthus nasutus (L) Kurz，爵床科Acanthaceae）中。本研究团队在小鼠骨髓巨噬细胞培养实验中发现，爵床素C可抑制核因子κB受体活化因子配体（receptor activator of nuclear factor-κB ligand, RANKL）诱导的破骨细胞分化，但该现象的确切分子机制尚未阐明。本研究旨在探究爵床素C抑制破骨细胞生成的分子机制。实验结果显示，爵床素C可抑制RANKL诱导的活化T细胞核因子c1（nuclear factor of activated T cells c1, NFATc1）表达。爵床素C能够抑制ERK、JNK及NF-κB的磷酸化，但对p38的磷酸化无影响；同时还可阻断RANKL刺激下TRAF6-TAK1复合物的形成。综上，爵床素C的抗破骨细胞生成作用通过以下级联反应介导：先抑制RANKL诱导的TRAF6-TAK1结合，随后阻断丝裂原活化蛋白激酶（mitogen-activated protein kinases, MAPKs）/NF-κB信号通路的激活，最终抑制调控破骨细胞分化相关基因转录的c-Fos与NFATc1的表达。体内实验表明，爵床素C同样可减少小鼠颅盖骨中RANKL诱导的破骨细胞形成与骨吸收。此外，爵床素C在体外及体内均能抑制脂多糖（lipopolysaccharide, LPS）诱导的破骨细胞生成与骨吸收。爵床素C有望为炎症性骨吸收过程中出现的异常骨溶解提供全新的治疗策略。