Osteoporosis is a systemic skeletal disease characterized by reduced bone mineral density (BMD), which results in an increased risk of fracture. Melandrium firmum (Siebold & Zucc.) Rohrbach (MFR), 'Wangbulryuhaeng' in Korean, is the dried aerial portion of Melandrii Herba Rohrbach, which is a member of the Caryophyllaceae family and has been used to treat several gynecological conditions as a traditional medicine. However, to the best of our knowledge, the effect of MFR on osteoclast differentiation and osteoporosis has not been assessed. To evaluate the effects of MFR on osteoclast differentiation, tartrate‑resistant acid phosphatase staining, actin ring formation and bone resorption assays were used. Additionally, receptor activator of nuclear factor‑κB ligand‑induced expression of nuclear factor of activated T cell, cytoplasmic 1 (NFATc1) and c‑Fos were measured using western blotting and reverse transcription‑PCR. The expression levels of osteoclast‑related genes were also examined. To further investigate the anti‑osteoporotic effects of MFR in vivo, an ovariectomized (OVX) rat model of menopausal osteoporosis was established. Subsequently, the femoral head was scanned using micro‑computed tomography. The results revealed that MFR suppressed osteoclast differentiation, formation and function. Specifically, MFR reduced the expression levels of osteoclast‑related genes by downregulating transcription factors, such as NFATc1 and c‑Fos. Consistent with the in vitro results, administration of MFR water extract to OVX rats reduced BMD loss, and reduced the expression levels of NFATc1 and cathepsin K in the femoral head. In conclusion, MFR may contribute to alleviate osteoporosis‑like symptoms. These results suggested that MFR may exhibit potential for the prevention and treatment of postmenopausal osteoporosis.

Leonurus sibiricus L. (LS) is a medicinal plant used in East Asia, Europe and the USA. LS is primarily used in the treatment of gynecological diseases, and recent studies have demonstrated that it exerts anti‑inflammatory and antioxidant effects. To the best of our knowledge, the present study demonstrated for the first time that LS may promote osteoblast differentiation and suppress osteoclast differentiation in vitro, and that it inhibited lipopolysaccharide (LPS)‑induced bone loss in a mouse model. LS was observed to promote the osteoblast differentiation of MC3T3‑E1 cells and upregulate the expression of runt-related transcription factor 2 (RUNX2), a key gene involved in osteoblast differentiation. This resulted in the induction of the expression of various osteogenic genes, including alkaline phosphatase (ALP), osteonectin (OSN), osteopontin (OPN), type I collagen (COL1) and bone sialoprotein (BSP). LS was also observed to inhibit osteoclast differentiation and bone resorption. The expression levels of nuclear factor of activated T‑cells 1 (NFATc1) and c‑Fos were inhibited following LS treatment. NFATc1 and c‑Fos are key markers of osteoclast differentiation that inhibit receptor activator of nuclear factor‑κΒ ligand (RANKL)‑induced mitogen‑activated protein kinase (MAPKs) and nuclear factor (NF)‑κB. As a result, LS suppressed the expression of osteoclast‑associated genes, such as matrix metallopeptidase‑9 (MMP‑9), cathepsin K (Ctsk), tartrate‑resistant acid phosphatase (TRAP), osteoclast‑associated immunoglobulin‑like receptor (OSCAR), c‑src, c‑myc, osteoclast stimulatory transmembrane protein (OC‑STAMP) and ATPase H+ transporting V0 subunit d2 (ATP6v0d2). Consistent with the in vitro results, LS inhibited the reduction in bone mineral density and the bone volume/total volume ratio in a mouse model of LPS‑induced osteoporosis. These results suggest that LS may be a valuable agent for the treatment of osteoporosis and additional bone metabolic diseases.

Lycopus lucidus (LL) is a perennial herb that is traditionally used in Asia to treat edema, wound healing, and gynecological diseases such as irregular menstruation and menstrual pain. We hypothesized that LL would decrease the risk of developing osteoporosis, which is a condition related to gynecological diseases. In this study, we aimed to investigate the effect of a water extract of LL on osteoclastogenesis in vitro and osteoporosis in vivo. In vitro study, we used RAW 264.7 cells as osteoclast precursor cell. Osteoclast differentiation was induced by receptor activator nuclear factor-kappa B ligand (RANKL). We investigated the effect of LL on RANKL-induced osteoclastogenesis, tartrate-resistant acid phosphatase (TRAP) activity, and osteoclast-related genes. In vivo study, we used ovariectomized- (OVX-) induced osteoporosis rat model. OVX-induced Sprague-Dawley rats were randomly separated into sham, OVX, 17β-estradiol (100 μg/kg), wLL-L (15.2 mg/kg), and wLL-H (152 mg/kg) groups. Drugs were administered orally once daily for 9 weeks. wLL inhibited the formation of TRAP-positive osteoclasts; TRAP activity; pit formation; transcription factors (the nuclear factor of activated T-cell cytoplasmic 1 and c-fos); and osteoclast-related genes such as TRAP, carbonic anhydrase II, cathepsin K, osteoclast-associated receptor, and the d2 isoform of the vacuolar ATPase Vo domain. Also, wLL prevented loss of the trabecular area in the OVX femur without change of estrogen level. These results indicate that wLL is able to inhibit osteoclastogenesis and protect bone loss in the OVX-induced osteoporosis model without the influence of hormones like estrogen.

Bone homeostasis is maintained by osteoclasts that absorb bone and osteoblasts that form bone tissue. Menopausal osteoporosis is a disease associated with aging and hormonal changes due to menopause causing abnormal activation of osteoclasts, resulting in a decrease in bone density. Existing treatments for osteoporosis have been reported to have serious side effects, such as jawbone necrosis and breast and uterine cancer; therefore, their use by patients is decreasing, whilst studies focusing on alternative treatments are increasingly popular. Solanum nigrum Line (SL) has been used as a medicinal plant that possesses several pharmacological effects, such as anti‑inflammatory and hepatotoxic protective effects. To the best of our knowledge, however, its effects on osteoporosis and osteoclasts have not been demonstrated previously. In the present study, the anti‑osteoporotic effect of SL was investigated using a postmenopausal model of osteoporosis in which Sprague‑Dawley rat ovaries were extracted. In addition, the inhibitory effects on osteoclast differentiation and function of SL was confirmed using an osteoclast model treated with receptor activator of NF‑κB ligand (RANKL) on murine RAW 264.7 macrophages. In vivo experiments showed that SL reduced the decrease in bone mineral density and improved changes in the morphological index of bone microstructure, such as trabecular number and separation. In addition, the number of tartrate resistant acid phosphatase‑positive cells in the femur and the expression levels of nuclear factor of activated T‑cells cytoplasmic 1 (NFATc1) and cathepsin K protein were inhibited. In vitro, SL suppressed RANKL‑induced osteoclast differentiation and bone resorption ability; this was mediated by NFATc1/c‑Fos, a key transcription factor involved in osteoclast differentiation, ultimately inhibiting expression of various osteoclast‑associated genes. These experimental results show that SL may be an alternative treatment for osteoporosis caused by abnormal activation of osteoclasts in the future.

Postmenopausal osteoporosis is caused by an imbalance between osteoclasts and osteoblasts and causes severe bone loss. Osteoporotic medicines are classified into bone resorption inhibitors and bone formation promoters according to the mechanism of action. Long-term use of bisphosphonate and selective estrogen receptor modulators (SERMs) can cause severe side effects in postmenopausal osteoporosis patients. Therefore, it is important to find alternative natural products that reduce osteoclast activity and increase osteoblast formation. Sparganii Rhizoma (SR) is the dried tuberous rhizome of Sparganium stoloniferum Buchanan-Hamilton and is called "samreung" in Korea. However, to date, the effect of SR on osteoclast differentiation and the ovariectomized (OVX)-induced bone loss model has not been reported. In vitro, tartrate-resistant acid phosphatase (TRAP) staining, western blots, RT-PCR and other methods were used to examine the effect of SR on osteoclast differentiation and osteoblasts. In vivo, we confirmed the effect of SR in a model of OVX-induced postmenopausal osteoporosis. SR inhibited osteoclast differentiation and decreased the expression of TNF receptor-associated factor 6 (TRAF6), nuclear factor of activated T cells 1 (NFATc1) and c-Fos pathway. In addition, SR stimulates osteoblast differentiation and increased protein expression of the bone morphogenetic protein 2 (BMP-2)/SMAD signaling pathway. Moreover, SR protected against bone loss in OVX-induced rats. Our results appear to advance our knowledge of SR and successfully demonstrate its potential role as a osteoclastogenesis-inhibiting and osteogenesis-promoting herbal medicine for the treatment of postmenopausal osteoporosis.

Osteoporosis is characterized by a decrease in bone microarchitecture with an increased risk of fracture. Long-term use of primary treatments, such as bisphosphonates and selective estrogen receptor modulators, results in various side effects. Therefore, it is necessary to develop alternative therapeutics derived from natural products. Crataegus pinnatifida Bunge (CPB) is a dried fruit used to treat diet-induced indigestion, loss of appetite, and diarrhea. However, research into the effects of CPB on osteoclast differentiation and osteoporosis is still limited. In vitro experiments were conducted to examine the effects of CPB on RANKL-induced osteoclast differentiation in RAW 264.7 cells. Moreover, we investigated the effects of CPB on bone loss in the femoral head in an ovariectomized rat model using microcomputed tomography. In vitro, tartrate-resistant acid phosphatase (TRAP) staining results showed the number of TRAP-positive cells, and TRAP activity significantly decreased following CPB treatment. CPB also significantly decreased pit formation. Furthermore, CPB inhibited osteoclast differentiation by suppressing NFATc1, and c-Fos expression. Moreover, CPB treatment inhibited osteoclast-related genes, such as Nfatc1, Ca2, Acp5, mmp9, CtsK, Oscar, and Atp6v0d2. In vivo, bone mineral density and structure model index were improved by administration of CPB. In conclusion, CPB prevented osteoclast differentiation in vitro and prevented bone loss in vivo. Therefore, CPB could be a potential alternative medicine for bone diseases, such as osteoporosis.

Background and Objective: Bupleurum falcatum (BF) extract, a natural product with anti-inflammatory properties, has been traditionally used to treat menopausal symptoms, but its role in osteoporosis, another serious health concern of menopausal women, remains unknown. Here we investigated whether and how BF prevents estrogen deficiency-induced bone loss using both in vitro and in vivo models. Methods: Female Sprague-Dawley rats were ovariectomized (OVX) and subjected to oral BF treatment daily for 8 weeks. Additionally, pre-osteoclastic RAW 264.7 cells were employed to evaluate the effects of BF and its underlying mechanism on receptor activator of nuclear factor kappa-B ligand (RANKL)-induced osteoclast formation in vitro. Results: A high dose of BF partially prevented ovariectomy (OVX)-induced bone loss and reduced the levels of tartrate-resistant acid phosphatase (TRAP) in serum and osteoclast numbers in femurs of OVX rats. Furthermore, BF clearly inhibited RANKL-induced osteoclast differentiation and bone resorption activity in RAW 264.7 cells. BF also inhibited the osteoclastogenic transcription factors c-Fos and nuclear factor of activated T cells c1 (NFATc1) and, consequently, downregulated the expression of osteoclast marker genes. Moreover, BF upregulated interferon-β (IFN-β)/inducible nitric oxide synthase (iNOS)/nitric oxide (NO) signaling, even though it had no impact on mitogen-activated protein kinases (MAPK) or NF-κB. The inhibition of osteoclast formation by BF was abrogated by iNOS-specific inhibitors. Consistent with cellular studies, BF upregulated iNOS protein expression in femurs from OVX rats. Conclusion: Taken together, our results indicate that BF partially prevented estrogen deficiency-induced bone loss with anti-osteoclastogenic activity potentially due to enhanced iNOS/NO signaling.