Postmenopausal osteoporosis is very common in women. Currently, many kinds of new drugs are being developed for this disease. Postmenopausal osteoporosis is closely related to overactivity of osteoclasts in body. Shikonin is purple red naphthoquinone pigment extracted from lithospermum, which has anti-inflammation, antivirus, anticancer and other bioactivities. At the same time, it has been proved that shikonin can promote the proliferation and differentiation of osteoblasts, but its influence on osteoclasts and molecular mechanism are unknown. Our study showed that shikonin could inhibit the activity and formation of RANKL-mediated osteoclasts depending on dose without affecting the activity of bone marrow macrophages (BMM). In addition, we have also found that shikonin can inhibit the expression of specific marker gene of osteoclasts, including nuclear factor of activated T cells cytoplasmic 1 (NFATc1), cathepsin K (Ctsk), tartrate resistant acid phosphatase (TRAcP) and calcitonin receptor. Shikonin also could promote the proliferation of MC3T3-E1, increasing the expression of mRNA related to osteogenesis, like the expression of bone morphogenetic protein-2 (BMP-2), alkaline phosphatase (ALP), runt-related transcription factor 2 (Runx2) and osteocalcin (OCN). Luciferase reporter gene assay and Western blot analysis further indicated that shikonin could inhibit the activity of RANKL-induced NF-κB and NFAT receptors. Moreover, shikonin can also slow down bone loss of ovariectomized (OVX) mice by inhibiting the activity of osteoclasts. This work explains the molecular mechanism of shikonin in RANKL-mediated formation of osteoclasts, and reveals the potential of further developing shikonin into a new drug for prevention and treatment of postmenopausal osteoporosis.

绝经后骨质疏松症在女性中非常普遍。当前，针对该疾病的许多新药正在开发中。绝经后骨质疏松症与体内破骨细胞的过度活跃密切相关。紫草素是从紫草提取的紫红色萘醌颜料，具有抗炎，抗病毒，抗癌和其他生物活性。同时，已经证明了紫草素可以促进成骨细胞的增殖和分化，但是其对破骨细胞的影响和分子机制尚不清楚。我们的研究表明，紫草素可以根据剂量抑制RANKL介导的破骨细胞的活性和形成，而不会影响骨髓巨噬细胞（BMM）的活性。此外，我们还发现，紫草素可以抑制破骨细胞特异性标记基因的表达，包括激活的T细胞胞质1（NFATc1），组织蛋白酶K（Ctsk），酒石酸盐抗性酸性磷酸酶（TRAcP）和降钙素受体的核因子。紫草素还可以促进MC3T3-E1的增殖，增加与成骨相关的mRNA的表达，例如骨形态发生蛋白2（BMP-2），碱性磷酸酶（ALP），矮子相关转录因子2（Runx2）的表达。和骨钙素（OCN）。荧光素酶报告基因的测定和Western blot分析进一步表明，紫草素可以抑制RANKL诱导的NF-κB和NFAT受体的活性。此外，紫草素还可以通过抑制破骨细胞的活性来减缓卵巢切除（OVX）小鼠的骨质流失。这项工作解释了紫草素在RANKL介导的破骨细胞形成中的分子机制，