Glaucocalyxin A suppresses osteoclastogenesis induced by RANKL and osteoporosis induced by ovariectomy by inhibiting the NF-κB and Akt pathways.

Glaucocalyxin A (GLA), the most abundant active component of the aboveground sections of Rabdosia japonica (Burm. f.) Hara var. glaucocalyx (Maxim.) Hara, possesses various pharmacological activities, such as antioxidant, antithrombosis, anticoagulation, antibacterial, antitumor, anti-inflammatory activities. According to previous studies, inflammation is closely associated with osteoclast differentiation and activity. Although GLA has demonstrated effective anti-inflammatory properties, its effects on osteoclast differentiation remain unclear. To examine the possible inhibitory effects of GLA and its molecular mechanisms in osteogenesis induced by RANKL as well as ovariectomy (OVX)-induced osteoporosis (OP) in mice. Tartrate-resistant acid phosphatase (TRAP) staining, F-actin staining, and a bone resorption pit assay were applied for identifying the effects of GLA on the differentiation of osteoclasts and the function of bone resorption. The mRNA expression of the genes related to osteoclast differentiation was measured by quantitative PCR. Protein expression of nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1), c-fos and phosphorylation of inhibitor of nuclear factor kappa B (IκBα), protein kinase B (AKT), c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), and p38 in RANKL-induced osteoclasts was determined using western blotting. The effect of GLA on OP was studied using a mouse model of OVX. At nontoxic concentrations ≤0.5 μM in vitro, GLA suppressed the formation of osteoclasts induced by RANKL with the decreased number and area size of TRAP-positive multinuclear osteoclasts, and the resorption of bone function by reducing F-actin ring number and bone resorption pit areas. It also reduced the expression of the genes specific for osteoclasts, which included genes encoding NFATc1, cathepsin K, c-fos, TRAP, vacuolar-type ATPase d2, and dendritic cell-specific transmembrane protein. Moreover, GLA repressed NF-κB and Akt pathway activation induced by RANKL. Micro-CT analysis of femur samples indicated decreased bone loss and greater trabecular bone density after GLA treatment, which showed that GLA played a protective role by inhibiting bone loss in OVX-induced OP mice in vivo. Our study is the first to show that GLA has significant therapeutic potential in OP, which is the disease of osteoclast increase caused by estrogen deficiency.

Zhu M ，Shan J ，Xu H ，Xia G ，Xu Q ，Quan K ，Liu X ，Dai M ... -  《-》

Bergapten suppresses RANKL-induced osteoclastogenesis and ovariectomy-induced osteoporosis via suppression of NF-κB and JNK signaling pathways.

Bergapten (BP), derived from Cnidium monnieri (L.) Cusson, is an ingredient widely used in traditional Chinese medicine and has important biological and pharmacological activities. However, the effect of BP on ovariectomy-induced osteoporosis and the underlying mechanism are not entirely clear. In this study, we investigated the effects of BP on ovariectomy-induced osteoporosis and receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclastogenesis in vivo and in vitro, and explored the underlying mechanism. We found that BP treatment exerted beneficial effects on ovariectomy-induced osteoporosis in vivo. Further, BP attenuated osteoclastogenesis in bone marrow macrophages (BMMs) and RAW264.7 cells without any cytotoxicity. Additionally, BP specifically inhibited RANKL-induced NF-κB and JNK signaling,but did not suppress p38 and ERK. At the mRNA level, BP inhibits the OC-associated transcription factor NFATc1 and c-fos, thereby affecting the expression of OC differentiation-related genes. Moreover, BP disrupted the formation of F-actin rings, which are important for bone-resorbing activity, and impairs OC bone resorption. Therefore, BP may be a useful alternative therapy for post-menopausal osteoporosis.

Chen G ，Xu Q ，Dai M ，Liu X ... -  《-》

Vinpocetine inhibits RANKL-induced osteoclastogenesis and attenuates ovariectomy-induced bone loss.

Osteoporosis is a result of impaired bone formation and/or excessive bone resorption. Osteoclasts are the only cells in the body that have a bone resorption function. Inhibiting osteoclast activity and differentiation is a way to treat osteoporosis. The current pharmacological treatment for osteoporosis has many shortcomings, and more effective treatments are needed. Vinpocetine (Vinp), a derivative of the alkaloid vincamine, has been used to treat cerebrovascular disorders and cognitive impairment for a long time. Vinp inhibits mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB)-dependent inflammatory responses and oxidative damage in which osteoclasts are often involved. However, the effects of Vinp on the regulation of osteoclast activity remain unknown. In this study, we found that Vinp significantly inhibited receptor activator of NF-κB ligand (RANKL)-induced osteoclast and F-actin formation and decreased osteoclastic bone resorption in vitro. Vinp also suppressed the expression of osteoclast-specific genes, including NFATc1, c-Fos, tartrate-resistant acid phosphatase (TRAP), matrix metalloproteinase-9 (MMP-9), and cathepsin K (CTSK) at both the mRNA and protein levels. Vinp reduced activation of NF-κB, MAPK, and AKT signaling during osteoclastogenesis and prevented the production of reactive oxygen species with increased nuclear factor erythroid 2-related factor 2, heme oxygenase 1, and NAD(P)H:quinone acceptor oxidoreductase 1 expression. Animal experiments consistently demonstrated that Vinp treatment significantly attenuated ovariectomy-induced bone loss with a decrease in the osteoclast number and decreases in serum levels of RANKL, TRAP, interleukin-1β, and tumor necrosis factor-alpha, as well as increased serum levels of osteoprotegerin. Taken together, our findings reveal that Vinp may be a potential pharmacological choice for preventing and treating osteoporosis.

Zhu M ，Liu H ，Sun K ，Liu J ，Mou Y ，Qi D ，Zhou C ，Abudunaibi M ，Tasiken B ，Li J ，Cheng H ，Huang H ... -  《-》

Caffeic acid 3,4-dihydroxy-phenethyl ester suppresses receptor activator of NF-κB ligand–induced osteoclastogenesis and prevents ovariectomy-induced bone loss through inhibition of mitogen-activated protein kinase/activator protein 1 and Ca2+–nuclear fact

Receptor activator of NF-κB ligand (RANKL) stimulation leads to the activation of mitogen-activated protein kinase (MAPK)/AP-1 and Ca2+–nuclear factor of activated T-cells cytoplasmic 1 (NFATc1) signaling pathways in osteoclastogenesis. Targeting these pathways has been an encouraging strategy for bone-related diseases, such as postmenopausal osteoporosis. In this study, we examined the effects of caffeic acid 3,4-dihydroxy-phenethyl ester (CADPE) on osteoclastogenesis. In mouse bone marrow monocytes (BMMs) and RAW264.7 cells, CADPE suppressed RANKL-induced osteoclast differentiation and actin-ring formation in a dose-dependent manner within non–growth inhibitory concentrations at the early stage, while CADPE had no effect on macrophage colony-stimulating factor (M-CSF)-induced proliferation and differentiation. At the molecular level, CADPE inhibited RANKL-induced phosphorylation of MAPKs, including extracellular signal-regulated kinases 1/2 (ERK1/2), p38, and c-Jun N-terminal kinase (JNK), without significantly affecting the NF-κB signaling pathway. CADPE abrogated RANKL-induced activator protein 1 (AP-1)/FBJ murine osteosarcoma viral oncogene homolog (c-Fos) nuclear translocation and activation. Overexpression of c-Fos prevented the inhibition by CADPE of osteoclast differentiation. Furthermore, CADPE suppressed RANKL-induced the tumor necrosis factor receptor associated factor 6 (TRAF6) interaction with c-src tyrosine kinase (c-Src), blocked RANKL-induced the phosphorylation of protein kinase B (AKT), and inhibited RANKL-induced Ca2+ oscillation. As a result, CADPE decreased osteoclastogenesis-related marker gene expression, including NFATc1, TRAP, cathepsin K, and c-Src. To test the effects of CADPE on osteoclast activity in vivo, we showed that CADPE prevented ovariectomy-induced bone loss by inhibiting osteoclast activity. Together, our data demonstrate that CADPE suppresses osteoclastogenesis and bone loss through inhibiting RANKL-induced MAPKs and Ca2+-NFATc1 signaling pathways. CADPE is a novel agent in the treatment of osteoclast-related diseases, such as osteoporosis.

Wu X ，Li Z ，Yang Z ，Zheng C ，Jing J ，Chen Y ，Ye X ，Lian X ，Qiu W ，Yang F ，Tang J ，Xiao J ，Liu M ，Luo J ... -  《-》

Ethanol extract of Cyathulae Radix inhibits osteoclast differentiation and bone loss.

Cyathulae Radix, a traditional Chinese medicine and a common vegetable, boasts a history spanning millennia. It enhances bone density, boosts metabolism, and effectively alleviates osteoporosis-induced pain. Despite its historical use, the molecular mechanisms behind Cyathulae Radix's impact on osteoporosis remain unexplored. In this study, we investigated the effects and mechanisms of Cyathulae Radix ethanol extract (CEE) in inhibiting osteoporosis and osteoclastogenesis. Eight-week-old female mice underwent ovariectomy and were treated with CEE for eight weeks. Micro-computed tomography (micro-CT) assessed histomorphometric parameters, bone tissue staining observed distal femur histomorphology, and three-point bending tests evaluated tibia mechanical properties. Enzyme-linked immunosorbent assay (ELISA) measured serum estradiol (E2), receptor activator for nuclear factor B ligand (RANKL), and osteoprotegerin (OPG) levels. Osteoclastogenesis-related markers were analyzed via Western blotting (WB) and quantitative real-time polymerase chain reaction (qRT-PCR). Additionally, CEE effects on RANKL-induced osteoclast formation and bone resorption were investigated in vitro using tartrate-resistant acid phosphatase (TRAP) staining, qRT-PCR, and WB assay. Compared with the ovariectomy (OVX) group, CEE treatment enhanced trabecular bone density, maximal load-bearing capacity, and various histomorphometric parameters. Serum E2 and OPG levels significantly increased, while Receptor activator of nuclear factor-κB (RANK) decreased in the CEE group. CEE downregulated matrix metallopeptidase 9 (MMP-9), Cathepsin K (CTSK), and TRAP gene and protein expression. In bone marrow macrophages (BMMs), CEE reduced mature osteoclasts, bone resorption pit areas, and MMP-9, CTSK, and TRAP expression during osteoclast differentiation. Compared with DMSO treatment, CEE markedly inhibited RANK, TNF receptor associated factor 6 (TRAF6), Proto-oncogene c-Fos (c-Fos), Nuclear factor of activated T-cells cytoplasmic 1 (NFATc1) expressions, and Extracellular regulated protein kinases (ERK), c-Jun N-terminal kinase (JNK), NF-kappa B-p65 (p65) phosphorylation in osteoclasts. In conclusion, CEE significantly inhibits OVX-induced osteoporosis and RANKL-induced osteoclastogenesis, potentially through modulating the Estrogen Receptor (ER)/RANK/NFATc1 signaling pathway.

Shi L ，Ren L ，Li J ，Liu X ，Lu J ，Jia L ，Xie B ，Tang S ，Liu W ，Zhang J ... -  《-》