• Journal of Acupuncture Research
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• 제34권2호
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• pp.1-18
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• 2017

Objectives : The purpose of this study was to evaluate the effects of water extracts of Eucommiae cortex (EC), Psoraleae semen (PS), and their combination on receptor activator of nuclear factor-kappa-B ligand (RANKL)-induced osteoclast differentiation. Methods : We assayed the protein expression levels of nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1), c-Fos, mitogen-activated protein kinases (MAPKs), and ${\beta}-actin$ in cell lysates using western blotting. Similarly, mRNA expression levels of NFATc1, c-Fos, tartrateresistant acid phosphate (TRAP), and glyceraldehyde-3-phosphate dehydrogenase, spermatogeni (GAPDHS) from bone marrow macrophages (BMMs) were analyzed using reverse transcription-polymerase chain reaction (RT-PCR). Furthermore, we determined the anti-osteoporotic effects of the water extracts of EC, PS, and their combination in a lipopolysaccharide (LPS)-induced bone-loss mouse model. Results : The in vitro data revealed showed that the combination of EC and PS extract showed a more remarkable inhibition of osteoclast differentiation than each herb did alone. The combination downregulated the induction of c-Fos, NFATc1, and TRAP by suppressing the phosphorylation of p38 and c-Jun N-terminal kinases (JNKs) and inhibiting nuclear factor kappa-light-chain-enhancer of activated B cells ($NF-{\kappa}B$). Lastly, the in vivo data showed that PS reduced the LPS-induced bone erosion. Conclusion : The result of this study suggests that EC and PS could be potential therapeutic agents for bone loss diseases such as osteoporosis.

• 한국식품영양학회지
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• 제29권2호
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• pp.178-185
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• 2016

This study was designed to develop and to qualify a coffee alternative beverage using a mixture of coffee beans and roasted black beans (Rhynchosia nulubilis). Therefore, the total isoflavone content (TIC), total phenol content (TPC), antioxidant activity, anti-inflammatory activity, NFATc1 (Nuclear factor of activated T-cells c1) expression in RANKL (receptor activator of nuclear factor kappa-B ligand)-stimulated RAW264.7 cells and sensory evaluation were measured for 5 different Cb (coffee bean)-RoS (roasted seomoktae) mixture extracts (Cb100RoS0, Cb75RoS25, Cb50RoS50, Cb25RoS75, and Cb0RoS100). Cb0RoS100 had the highest TIC ($516.83{\pm}36.61mg/100g$) and TPC ($18.11{\pm}1.77mg$ TAE/100 g) along with the highest antioxidant activity as measured by DPPH radical scavenging activity ($73.55{\pm}8.11%$) and ABTS radical scavenging activity ($63.27{\pm}7.27%$). Also, Cb0RoS100 showed the highest anti-inflammatory activity as measured by NO production ($13.57{\pm}2.21{\mu}M$) and PGE2 production ($3.25{\pm}0.21ng/mL$). The more the RoS ratio was increased in the mixtures of Cb-RoS, the more the NFATc1 protein expression was decreased in RANKL-stimulated RAW264.7 cells. In case of sensory evaluation, Cb50RoS50 had the highest scores for flavor, delicate flavor and overall quality, which were similar to those in Cb alone (Cb100RoS0). We suggest that the use of RoS replacement instead of Cb in/as a coffee alternative beverage may help to reduce the risk of caffeine-related bone loss and/or bone disease by effectively blocking NFATc1 expression in RANKL-stimulated RAW264.7 cells compared with Cb alone.

• 생약학회지
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• 제44권3호
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• pp.257-262
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• 2013

Osteoporosis is a disease of bones that leads to an increased risk of fracture. In osteoporosis, the bone mineral density is reduced, bone microarchitecture deteriorates, and the amount and variety of proteins in bone are altered. $It^{\circ}{\emptyset}s$ caused by the imbalance between born resorption and born formation. Recently natural products from plants have been extensively studied as therapeutic drugs to treat and prevent various diseases. Wheat bran is the hard outer layers of wheat grain and produced as a by-product of milling in the production of refined grains. In oriental medicines, Bu So Maek (Tritici Immaturi Semen) with wheat bran has been used as bronchitis, sedatives and anti-sweating effects. However effects of wheat bran butanol fraction (WBB, 50 ${\mu}g/ml$) in osteoclast differentiation remains unknown yet. Thus we investigated the effects of WBB on RANKL induced osteoclast differentiation. WBB inhibited osteoclast differentiation by downregulating the RANKL-induced activations of MAP kinases. Moreover mRNA expression of osteoclast-mediating molecules such as c-Fos, NFATc1 and DC-STAMP were attenuated by WBB during osteoclast differentiation. The finding of this study show that WBB and its components might prevent osteoclast-related bone loss.

• 생명과학회지
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• 제29권10호
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• pp.1104-1110
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• 2019

최근 노령인구의 증가로 고령화 사회로 접어들면서 골다공증과 같은 골 대사 질환이 사회적 문제로 대두되고 있다. 파골세포는 골 흡수 역할을 하는 세포이며 골 흡수가 강하게 일어나는 경우 골다공증이 유발된다. 그러나 현재 사용 중인 골 흡수 억제제는 장기간 사용 시 부작용이 발생할 수 있어, 파골세포 분화 억제에 효과가 있는 새로운 소재 개발이 필요한 실정이다. 따라서 본 연구에서는 풀무치 에탄올 추출물을 대상으로 RANKL에 의해 유도된 파골세포 분화에 대한 억제 효능 확인 및 그 기작을 구명하고자 하였다. RAW264.7 파골세포에서 풀무치 추출물의 독성 및 증식 효과를 확인하기 위하여 MTS assay를 진행하였고 $2,000{\mu}g/ml$ 농도까지 세포 독성은 확인되지 않았다. 풀무치 추출물이 파골세포의 분화에 미치는 영향을 확인하기 위해 3일 동안 RAW264.7 세포에 파골세포 분화 촉진제인 RANKL을 단독 처리 및 풀무치 추출물과 함께 처리한 후 TRAP 활성을 비교하였다. 그 결과 RANKL에 의해 증가한 파골세포 분화가 풀무치 추출물 처리에 의해 농도 의존적으로 감소하는 것을 확인하였다. 파골세포 분화와 관련된 유전자(TRAP, RANK, NFATc1 및 CK)의 발현량 변화를 확인한 결과, RANKL 처리 시에 증가한 유전자 발현량이 풀무치 추출물 처리에 의해 현저하게 감소하는 것을 확인하였고, NFATc1, c-Src와 같은 분화 관련 단백질 발현량 또한 풀무치 추출물 처리에 의해 감소하였다. 풀무치 에탄올 추출물은 $NF-{\kappa}B$, ERK 및 JNK 신호전달에 영향을 미쳐 그 결과로 파골세포 분화 억제 효과가 나타나는 것으로 판단된다. 이러한 결과로 보아 풀무치 에탄올 추출물은 골 흡수를 억제하는 역할을 함으로써 골다공증 예방 및 치료를 위한 새로운 기능성 소재로 사용 가능성이 있음을 확인하였다.

• International Journal of Oral Biology
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• 제45권3호
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• pp.126-133
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• 2020

Homer proteins are scaffold proteins that regulate calcium (Ca²⁺) signaling by modulating the activity of multiple Ca²⁺ signaling proteins. In our previous report, Homer2 and Homer3 regulated NFATc1 function through its interaction with calcineurin, which then acted to regulate receptor activator of nuclear factor-kappa B ligand (RANKL)-induced osteoclastogenesis and bone metabolism. However, to date, the role of Homers in osteoclastogenesis remains unknown. In this study, we investigated the roles of Homer2 and Homer3 in aging-dependent bone remodeling. Deletion of Homer2/Homer3 (Homer2/3 DKO) markedly decreased the bone density of the femur. The decrease in bone density was not seen in mice with Homer2 (Homer2^-/-) and Homer3 (Homer3^-/-) deletion. Moreover, RANKL treatment of bone marrow-derived monocytes/macrophages in Homer2/3 DKO mice significantly increased the formation of multinucleated cells and resorption areas. Finally, Homer2/3 DKO mice decreased bone density in an aging-dependent manner. These findings suggest a novel potent mode of bone homeostasis regulation through osteoclasts differentiation during aging by Homer proteins, specifically Homer2 and Homer3.

• 대한의생명과학회지
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• 제23권4호
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• pp.295-302
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• 2017

Mononuclear osteoclast precursors derived from hematopoietic progenitors fuse together and then become multinucleated mature osteoclasts by macrophage-colony stimulating factor (M-CSF) and receptor activator of nuclear factor-${\kappa}B$ ligand (RANKL). Especially, the binding of RANKL to its receptor RANK provides key signals for osteoclast differentiation and bone-resorbing function. RANK transduces intracellular signals by recruiting adaptor molecules such as TNFR-associated factors (TRAFs), which then activate mitogen activated protein kinases (MAPKs), Src/PI3K/Akt pathway, nuclear factor-${\kappa}B$ (NF-${\kappa}B$) and finally amplify NFATc1 activation for the transcription and activation of osteoclast marker genes. This review will briefly describe RANKL-RANK signaling pathways and key molecules critical for osteoclast differentiation.

• 동의생리병리학회지
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• 제24권1호
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• pp.74-79
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• 2010

Osteoclasts are bone-resorbing giant cells that differentiate from hematopoietic cells of the monocyte/macrophages. Excessive osteoclast differentiation leads to gradual loss of bone mass causing fracture of the skeleton. The aim of this study was to develop a drug candidates for the treatment of osteoporosis. RANKL-induced osteoclast differentiation was dose-dependently inhibited by myricetin. Myricetin inhibited the expression of c-Fos, NFATc1, and TRAP in BMMs treated with RANKL. Myricetin disrupted the structure of actin ring and suppressed osteoclastic bone resorption. Also, myricetin induced apoptosis in mature osteoclasts. Myricetin inhibited the phosphorylation of ERK in mature osteoclasts treated with M-CSF. The activation of caspase-9 and caspase-3 was increased by myricetin treatment. Our results suggest that myricetin may be an effective agent to prevent bone diseases such as osteoporosis.

• 한국치위생학회지
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• 제23권4호
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• pp.219-226
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• 2023

연구목적: 이 연구는 대황 추출물이 골수 유래 대식세포(BMM)에서 파골세포 분화에 미치는 영향을 조사하는 것을 목적으로 한다. 파골 세포는 골 재흡수 및 재형성에 중요한 역할을 하며, 파골 세포의 조절 장애는 다양한 골 관련 질환을 유발할 수 있다. 잠재적인 항염증 특성을 가진 약용 식물인 대황은 뼈 대사를 조절하는 것으로 제안되었다. 연구방법: 생후 5주령의 C57BL/6 마우스의 대퇴골과 경골에서 BMM을 분리하고 M-CSF(mouse macrophage colony-stimulating factor) 존재하에 3일간 배양한 후 M-CSF와 파골 세포 분화를 유도하기 위한 핵 인자-κB 리간드(RANKL)의 활성화제를 처리하였다. 연구결과: 대황 추출물로 처리하면 BMM에서 파골 세포 분화가 현저하게 억제되었다. 또한 대황 추출물은 파골세포 형성에 필수적인 유전자인 TRAP(tartrate-resistant acid phosphatase) 및 CTSK(cathepsin K)의 mRNA 발현을 억제하였다. 또한 파골세포 분화에 중요한 전사 인자인 활성화된 T 세포 c1(NFATc1)의 핵 인자의 RANKL 유도 발현을 억제하였다. 결론: 이러한 결과는 대황 추출물이 BMMs에서 파골 세포 형성에 억제 효과가 있음을 나타낸다. 따라서 대황 추출물은 비정상적인 파골 세포 활동과 관련된 뼈 관련 질환의 치료를 위한 유망한 치료제이다. 잠재적인 임상 적용을 완전히 이해하기 위해서는 메커니즘에 대한 추가 연구와 탐색이 필요하다.

• International Journal of Oral Biology
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• 제44권3호
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• pp.89-95
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• 2019

Piperlongumine (PL) is a natural product found in long pepper (Piper longum). The pharmacological effects of PL are well known, and it has been used for pain, hepatoprotection, and asthma in Oriental medicine. No studies have examined the effects of PL on bone tissue or bone-related diseases, including osteoporosis. The current study investigated for the first time the inhibitory effects of PL on osteoclast differentiation, bone resorption, and osteoclastogenesis-related factors in RAW264.7 macrophages stimulated by the receptor activator for nuclear factor-${\kappa}B$ ligand (RANKL). Cytotoxicity was examined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and osteoclast differentiation and bone resorption were confirmed by tartrate-resistant acid phosphatase (TRAP) staining and pit formation analysis. Osteoclast differentiation factors were confirmed by western blotting. PL exhibited toxicity in RAW264.7 macrophages, inhibiting osteoclast formation and bone resorption, in addition to inhibiting the expression of osteoclastogenesis-related factors, such as tumor necrosis factor receptor-associated factor 6 (TRAF6), c-Fos, and NFATc1, in RANKL-stimulated RAW264.7 macrophages. These findings suggest that PL is suitable for the treatment of osteoporosis, and it serves as a potential therapeutic agent for various bone diseases.

• Anatomy and Cell Biology
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• 제56권1호
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• pp.94-108
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• 2023

Cancer cell heterogeneity is a serious problem in the control of tumor progression because it can cause chemoresistance and metastasis. Heterogeneity can be generated by various mechanisms, including genetic evolution of cancer cells, cancer stem cells (CSCs), and niche heterogeneity. Because the genetic heterogeneity of CSCs has been poorly characterized, the genetic mutation status of CSCs was examined using Exome-Seq and RNA-Seq data of liver cancer. Here we show that different surface markers for liver cancer stem cells (LCSCs) showed a unique propensity for genetic mutations. Cluster of differentiation 133 (CD133)-positive cells showed frequent mutations in the IRF2, BAP1, and ERBB3 genes. However, leucine-rich repeat-containing G protein-coupled receptor 5-positive cells showed frequent mutations in the CTNNB1, RELN, and ROBO1 genes. In addition, some genetic mutations were frequently observed irrespective of the surface markers for LCSCs. BAP1 mutations was frequently observed in CD133-, CD24-, CD13-, CD90-, epithelial cell adhesion molecule-, or keratin 19-positive LCSCs. ASXL2, ERBB3, IRF2, TLX3, CPS1, and NFATC2 mutations were observed in more than three types of LCSCs, suggesting that common mechanisms for the development of these LCSCs. The present study provides genetic heterogeneity depending on the surface markers for LCSCs. The genetic heterogeneity of LCSCs should be considered in the development of LCSC-targeting therapeutics.