Cilostazol Attenuates Ovariectomy-Induced Bone Loss by Inhibiting Osteoclastogenesis

BackgroundCilostazol has been reported to alleviate the metabolic syndrome induced by increased intracellular adenosine 3’,5’-cyclic monophosphate (cAMP) levels, which is also associated with osteoclast (OC) differentiation. We hypothesized that bone loss might be attenuated via an action on OC by cilostazol.Methodology and Principal FindingsTo test this idea, we investigated the effect of cilostazol on ovariectomy (OVX)-induced bone loss in mice and on OC differentiation in vitro, using μCT and tartrate-resistant acid phosphatase staining, respectively. Cilostazol prevented from OVX-induced bone loss and decreased oxidative stress in vivo. It also decreased the number and activity of OC in vitro. The effect of cilostazol on reactive oxygen species (ROS) occurred via protein kinase A (PKA) and cAMP-regulated guanine nucleotide exchange factor 1, two major effectors of cAMP. Knockdown of NADPH oxidase using siRNA of p47phox attenuated the inhibitory effect of cilostazol on OC formation, suggesting that decreased OC formation by cilostazol was partly due to impaired ROS generation. Cilostazol enhanced phosphorylation of nuclear factor of activated T cells, cytoplasmic 1 (NFAT2) at PKA phosphorylation sites, preventing its nuclear translocation to result in reduced receptor activator of nuclear factor-κB ligand-induced NFAT2 expression and decreased binding of nuclear factor-κB-DNA, finally leading to reduced levels of two transcription factors required for OC differentiation.Conclusions/SignificanceOur data highlight the therapeutic potential of cilostazol for attenuating bone loss and oxidative stress caused by loss of ovarian function.背景：已有研究表明，西洛他唑（cilostazol）可缓解因细胞内3',5'-环腺苷单磷酸（cAMP）水平升高诱发的代谢综合征，而该通路亦与破骨细胞（OC）分化密切相关。本研究推测，西洛他唑可通过作用于破骨细胞，进而减轻骨丢失。 方法与主要结果：为验证该假说，本研究分别采用显微CT（μCT）与抗酒石酸酸性磷酸酶染色法，探讨了西洛他唑对小鼠卵巢切除术（OVX）诱导骨丢失的体内作用，以及对体外破骨细胞分化的影响。实验结果显示，西洛他唑可阻断卵巢切除术诱发的骨丢失，并降低体内氧化应激水平；同时亦可减少体外培养中破骨细胞的数量与活性。西洛他唑对活性氧（ROS）的调控作用，经由cAMP的两大主要效应分子——蛋白激酶A（PKA）与cAMP调节型鸟苷酸交换因子1介导实现。使用靶向p47phox的小干扰RNA（siRNA）敲低NADPH氧化酶后，西洛他唑对破骨细胞生成的抑制作用被削弱，提示西洛他唑减少破骨细胞生成的部分机制，与活性氧生成受损相关。西洛他唑可促进活化T细胞核因子胞质1（NFAT2）在PKA磷酸化位点的磷酸化，阻碍其核易位，进而降低核因子κB受体激活剂配体诱导的NFAT2表达，同时减弱核因子κB与DNA的结合能力，最终使破骨细胞分化所需的两种转录因子水平下调。 结论与意义：本研究结果证实，西洛他唑可用于缓解卵巢功能丧失诱发的骨丢失与氧化应激，凸显了其临床治疗潜力。