The bones of the human body support the structures of the body and provide protection for a person’s internal organs. Bone metabolic diseases are on the rise due to a significant increase in life expectancy over a short period of time. Therefore, we investigated the osteoblast differentiation promoting and osteoclastogenesis inhibitory activities of fermented Benincasa hispida cong. (HR1901-BS, HR1901-BSaf). We evaluated the alkaline phosphatase (ALP) activity of MC3T3-E1 mouse calvarial-derived osteoblasts. We also evaluated expression of ALP, osteocalcin (OCN), and runt-related transcription factor 2 (Runx2), which regulate osteoblast differentiation. To assess effects on osteoclast formation, tartrate-resistant acid phosphatase (TRAP) activity in RAW264.7 cells was analyzed. ALP activity increased by 121-136% and 140-156%, respectively in the presence of HR1901-BS and HR1901- BSaf. Expression of osteoblast differentiation factor also increased significantly. We also confirmed that HR1901-BS and HR1901-BSaf decreased TRAP activity in osteoclasts by 35-47% and 23-39%, respectively. Our results showed that fermented Benincasa hispida cong. (HR1901-BS, HR1901-BSaf) increase bone mineralization and osteoblast differentiation activity in MC3T3-E1 cells, and inhibit bone resorption activity in RAW264.7 cells. In conclusion, fermented Benincasa hispida cong. (HR1901-BS, HR1901-BSaf) can be used as an effective natural resource for preventing and treating bone-related diseases.

본 연구는 방사선 육종 차조기와 백출 복합물의 조골세포 분화 활성 및 파골세포 형성 억제를 조사하였다. 차조기와 백출 복합물은 MG-63 세포에서 ALP 활성 및 arlizarin red 염색을 확인하였고 조골세포 형성의 영향은 RAW 264.7 세포에서 TRAP 활성과 TRAP 염색을 진행하였다. 세포 독성 시험에서 차조기와 백출 복합물은 50 ㎍/㎖ 농도 이하에서 안전한 것으로 확인되었다. ALP 활성 및 골석회화 형성 능력은 대조군보다 활성이 낮았으나, 파골세포에서 TRAP 활성을 유의적으로 감소시켰으며, 효과적으로 TRAP(+) 다핵세포를 억제하였다. 따라서 차조기와 백출 복합물은 골 흡수 억제 활성을 향상시켜 뼈 관련 질환의 예방 및 치료에 효과적인 것으로 보여진다.

Dlx3 is a homeodomain protein and is known to playa role in development and differentiation of many tissues. Deletion of four base pairs in DLX3 (NT3198) is causally related to tricho-dento-osseous (TDO) syndrome (OMIM # 190320), a genetic disorder manifested by taurodontism, hair abnormalities, and increased bone density in the cranium. Although the observed defects of TDO syndrome involves bone, little is known about the role of Dlx3 in bone remodeling process. In this study, we examined the effect of wild type DLX3 (wtDlx3) expression on osteoclast differentiation and compared it with that of 4-BP DEL DLX3 (TDO mtDlx3). To examine whether Dlx3 is expressed during RANKL-induced osteoclast differentiation, RAW264.7 cells were cultured in the presence of receptor activator of nuclear factor-B ligand (RANKL). Dlx3 protein level increased slightly after RANKL treatment for 1 day and peaked when the fusion of prefusion osteoclasts actively progressed. When wtDlx3 and TDO mtDlx3 were overexpressed in RAW264.7 cells, they enhanced RANKL-induced osteoclastogenesis and the expression of osteoclast differentiation marker genes such as calcitonin receptor, vitronectin receptor and cathepsin K. Since osteoclast differentiation is critically regulated by the balance between RANKL and osteoprotegerin (OPG), we examined the effect of Dlx3 overexpression on expression of RANKL and OPG in C2C12 cells in the presence of bone morphogenetic protein 2. Overexpression of wtDlx3 enhanced RANKL mRNA expression while slightly suppressed OPG expression. However, TDO mtDlx3 did not exert significant effects. This result suggests that inability of TDO mtDlx3 to regulate expression of RANKL and OPG may contribute to increased bone density in TDO syndrome patients. Taken together, it is suggested that Dlx3 playa role as a positive regulator of osteoclast differentiation via up-regulation of osteoclast differentiation-associated genes in osteoclasts, as well as via increasing the ratio of RANKL to OPG in osteoblastic cells.

We performed the present study to investigate whether Rehmannia glutinosa Libosch (RG) extracts (RGX) and Eleutherococcus senticosus Max (ES) extracts (ESX) play any roles in bone metabolism. We examined cellular activities of bone cells by measurement of osteoblastic cell viability, osteoprotegerin (OPG) secretion from osteoblasts, osteoclastogenesis, and osteoclastic activity. There is no cytotoxicity from osteoblasts after treatment with RGX and ESX. The secretion of OPG from the osteoblasts showed marked increases after treatment with RGX and ESX. In addition, RGX and ESX treatment decreased the number of tartrate-resistant acid phosphatase-positive multinucleated cells and the resorption areas. RGX and ESX, when mixed at optimal ratios, induced synergic effects, in vitro. OPB, which showed synergic effects, is the extract of natural ingredients RG and ES mixed at a raw material weight ratio of 4 : 1. It can be suspected that extracts of RG and ES mixtures contains active ingredients involved in bone tissue metabolism and may be effective in improving osteoporosis.

Although it has been known that TGF-β1 acts as a crucial cofactor in osteoclast differentiation, its mode of action is still unclear. In the present study, we studied the effect of TGF-β1 on the differentiation of osteoclast depending on the developmental stages. Murine bone marrow cells were induced to differentiate into mature osteoclasts in the presence of receptor activator of NF-xβ ligand (RANKL) and macrophage colony stimulating factor (M-CSF). In the early stage of the differentiation TRAP(-) mononuclear precursor cells were obtained from nonadherent M-CSF dependent bone marrow cells, which further differentiated into mature osteoclasts. TGF-β1 stimulated osteoclast differentiation, which was stronger when cells were stimulated by TGF-β1 in the early stage than the later differentiation. TGF-β1 increased the expression of RANK and synergistically stimulated RANKL-induced activation of NF-xβ MAP kinase in TRAP(-) mononuclear precursor cells. These results suggest that activation of osteoclast differentiation by TGF-β1 may be ascribed to the both increased expression and activation of RANK in the osteoclast differentiation, especially in the early stage of differentiation.

Background : Osteoclasts are differentiated from the monocytes/macrophages of hematopoietic cells, that excessive activities of bone-resorbing giant cells leads to pathological bone diseases such as osteoporosis (contained rheumatoid arthritis and autoimmune arthritis). Therefore, it is very important to suppress loss of bone mass by deactivation of osteoclast differentiation. In this context, we evaluated for the effects of black ginseng (BG) extract on TRAP activity, proliferation and differentiation in RANKL-induced osteoclastic RAW264.7 cells. Methods and Results : The aim of this study is to figure out the potential anti-osteoporosis effects and the underlying mechanism of BG extract in RANKL-induced osteoclastic RAW264.7 cells. The ginsenoside Rg3, Rg5, Rk1 and Rh4 content of BG was increased more than Red ginseng (RG). The extracts of BG markedly reduced the activity of tartrate-resistant acid phosphatase-positive (TRAP+) multinucleated cells from osteoclastic RAW264.7 cells, without cytotoxicity. BG clearly inhibited RANKL-induced osteoclast differentiation by decreased calcitonin and TRAP (p < 0.01). Furthermore, ginseonside Rg5 and Rk1 significantly inhibited TRAP activity in formation of osteoclastic differentiation (p < 0.01). It is also found that Ginseonside Rg5 and Rk1 mixture more inhibits osteoclast differentiation activity. Conclusion : Our results suggest that Black ginseng extract has an anti-osteoporosis effects in bone disease when administered as a food supplement and has potential as a therapeutic agent for osteoporosis.

This study investigated the effects of pressurized steam-treated Corni Frutus (PSC) extract on osteoblast differentiation and osteoclast formation. The osteoblast differentiation effect of the extract was evaluated by measuring cellular alkaline phosphatase (ALP) activity, cell matrix ALP staining, alizarin Red S staining and von Kossa staining on proliferating MC3T3-E1 osteoblast cells. The results confirmed that ALP activity, cell matrix ALP staining, alizarin Red S staining and von Kossa staining were all increased as proliferation increased from 1 to 14 days, without cytotoxicity. The osteoclast formation effect of the PSC extract was evaluated by measuring the cellular tartrate-resistant acid phosphatase (TRAP) activity and cell matrix TRAP staining on receptor activator of nuclear factor kappa-B ligand (RANKL)-induced RAW264.7 osteoclast cells. Treating RAW264.7 cells with RANKL for 7 days increased matrix staining for TRAP and cellular TRAP activity. The PSC extract decreased these changes in a concentration-dependent manner. Therefore, PSC is expected to be a natural source for developing health functional foods and medicinal agents to prevent bone-related diseases, such as osteoporosis, by increasing osteoblast differentiation and reducing osteoclast activity.

Background : Capsaicin is an active component of chili peppers, which are plants belonging to the genus Capsicum. Research reported capsaicin has antioxidant and anti-inflamatory activity and osteoclast lineages are very susceptible to oxidative stress, as osteoclasts are produced by increased-generation of intracellular ROS and osteoclasts are activated by ROS. Therefore, our study was evaluated the influence of intracellular oxidative stress such as increased ROS level on RANKL-mediated osteoclast differenciation. Methods and Results : Capsaicin showed a good free radical scavenging activity at in-vitro antioxidant activity. The inhibitory effect of osteoclast differentiation on capsaicin was confirmed. Osteoclast differentiation from murine macrophage RAW 264.7 cells was induced by RANKL. The effect of capsaicin on receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL)-induced osteoclast differentiation was demonstrated using a tartrate-resistant acid phosphatase (TRAP) assay and TRAP staining. Capsaicin showed an inhibitory effect on TRAP activity. The TRAP staining showed that the number of TRAP positive osteoclasts was reduced in capsaicin-treated cells. Conclusion : Capsaicin revealed an inhibitory effect in osteoclast differentiation induced by RANKL. These results suggest that capsaicin may have a beneficial effect for the prevention or treatment of osteoclast caused bone diseases such as osteoporosis.

Background : Osteoclasts as multinucleated cells originate from hematopoietic monocyte/ macrophage precursor cell, shows the bone absorption through the commitment, differentiation, fusion, and bone resorption stages by regulation of M-CSF and RANKL. It has been reported a significant negative correlation between the increase of oxidative stress and the bone density, and when RANKL reaction to the osteoclasts precursor cells is mainly generated ROS is due to increased activity of NADPH oxidase1 (NOX1), and these ROS act as a factor which promotes osteoclasts differentiation. Thus, RANKL signaling process is important that excessive osteoclast formation and differentiation inhibited through the regulation of each step. Methods and Results : F3570 ethanol extract showed relatively high activity at in-vitro antioxidant activity. F3570 water extract inhibited ROS generation in RAW 264.7 cells stimulated with H2O2 and RANKL, even at low concentrations. The inhibitory effect of osteoclast differentiation on F3570 water extract was confirmed that shown through NF-κB pathway, MAPK pathway including ERK and JNK. F3570 ethanol extract is considered to be regulated by the p38 MAPK and the other signaling pathway. Also, F3570 both water and ethanol extract were significantly reduced gene expression such as TRAP, calcitonin receptors and integrin β3 of RANKL-induced mature osteoclast in the bone resorption stage. Conclusion : Through this study, F3570 extract revealed an outstanding inhibitory effect and signaling mechanisms in osteoclast differentiation induced by RANKL. These results suggest that F3570 is bone diseases associated with aging or osteoporosis caused by menopause in an aging society is expected to be a superior candidate for the treatment or the prevention

Osteoporosis induces a bone mineral density loss due to imbalance of bone homeostasis that is achieved by osteoclasts (which are involved in bone resorption) and osteoblasts (which are involved in bone formation). Thus, this study was performed to evaluate the effects of hot water extract of the Achyranthes bidentata Blume (ABB) and Panax ginseng (Gin) on osteoclast and osteoblast differentiation. In this study, there was no cytotoxicity by ABB, 50 and 100 μg/ml of Gin significantly decreased cell viability of RANKL-induced osteoclast in RAW264.7 cell (p < 0.01). But, it was 50 μg/ml of ABB and Gin mixtures increased due to protective action of ABB. Furthermore, Gin contained groups (Gin, ABB and Gin mixtures) were inhibitory effects on osteoclast differentiation and bone resorption, and increased in osteoblast differentiation activity. Gin clearly inhibited RANKL-induced osteoclast differentiation by decreased calcitonin and TRAP (p < 0.01). Also, these extracts significantly increased calcium accumulation formation of osteoblastic differentiation reagents-induced osteoblast in MC3T3-E1 cell (p < 0.05). These results suggest that ABB and Gin mixtures may be a potential as drug for the treatment of osteoporosis.