Bone homeostasis is an important physiological process that involved in the functional balance between bone depositing osteoblasts and bone resorption osteoclasts. Differentiation of these two types of cells are crucial to maintain the normal physiology of the bones [
1]. Osteoclasts are bone resorption multinucleated cells (MNCs), derived from hematopoietic stem cells of monocyte/macrophage lineage [
2]. The receptor activator of nuclear factor κ-B (NF-κB) ligand (RANKL) and the macrophage colony-stimulating factor (M-CSF) have been reported to be crucial for regulating osteoclast differentiation. RANKL is important for the differentiation and activation of osteoclasts, and M-CSF is responsible for survival and proliferation of osteoclasts precursors [
3,
4]. Binding of RANKL to RANK receptor could lead to the recruitment of tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6), and consequently activates not only NF-κB, but also mitogen-activated protein kinases (MAPKs) [
5‐
7]. This signaling cascade activates the nuclear factor of activated T cells c1 (NFATc1) and a member of activator protein 1 (AP-1) family c-Fos [
8,
9]. Subsequently, RANKL upregulates osteoclast-specific genes such as matrix metalloprotease-9 (MMP9) and Cathepsin K [
10]. These signal molecules ultimately lead to the survival, activation, and differentiation of actin rings and bone resorption by osteoclasts [
11]. Abnormal differentiation and dysfunction of osteoclasts, especially the excessive activity of osteoclasts, can lead to osteoporosis, osteoarthritis, and rheumatoid arthritis [
12].
In recent years, osteoporosis has been a serious public health problem. However, some clinically available therapies are effective but are limited due to their side effects [
13]. Natural products are studied as important sources of therapeutic drug molecules. We have previously reported that Ganghwaljetongyeum (GHJTY), a traditional decoction composed of 18 herbs, could be used for the treatment of fever, swelling, limitation of motion, joint pain, and inflammatory processes related to arthritis [
14,
15]. We selected five effective herbal constituents from GHJTY with greatest potential to enhance the efficacy and convenience of drug prescription through bioinformatics analysis and pharmacologic activity tests [
16]. The resulting concoction named as ChondroT, which comprised water extracts of
Ostericum koreanum (Maxim.) Kitag. (O),
Lonicera japonica Thunb. (L),
Angelica gigas Nakai (A),
Clematis manshurica Rupr. (C), and
Phellodendron amurense Rupr. (P) in a 6: 4: 4: 4: 3 ratio [
17]. ChondroT exhibited more significant chondroprotective effects and anti-inflammatory processes related to arthritis than GHJTY did [
17]. ChondroT also significantly demonstrated the efficacy of anti-osteoarthritis in a rat model of osteoarthritis induced by monosodium iodoacetate- or collagenase [
14,
18]. In addition, the efficacy and safety of ChondroT on knee-osteoarthritis were evaluated by randomized, double-blind, placebo-controlled, multicenter clinical trials [
19]. Recently, we demonstrated that ChondroT exhibited the anti-hyperuricemic effects by regulating xanthine oxidase activity and kidney mouse urate transporter 1 in a potassium oxonate-induced hyperuricemic mouse model [
20]. To further investigate the efficacy and mechanism of ChondroT as a therapeutic potential herbal medicine, we evaluated its function on bone disease in this study. Among its five constituent herbs,
Ostericum koreanum (Maxim.) Kitag.,
Angelica gigas Nakai and its major active decursin are found to possess anti-osteoclastogenic activity in bone marrow cells isolated from mice [
21‐
23]. Recently, the component phellodendrine from
Cortex Phellodendri Chinensis has been reported to have an obvious inhibitory effect on osteoclast differentiation and function [
24]. Therefore, the complex herbal medicine ChondroT has potential beneficial effects against osteoclastogenesis. The present study is aim to investigate anti-osteoclastogenic effects of ChondroT and its five constituent herbs in RANKL-activated primary precursor cells and the underlying signaling pathways involved.