• The Korean Journal of Physiology and Pharmacology
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• 제23권1호
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• pp.47-54
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• 2019

Estrogen withdrawal in post-menopausal women leads to overactivation of osteoclasts, which contributes to the development of osteoporosis. Inflammatory cytokines are known as one of mechanisms of osteoclast activation after estrogen deficiency. SPA0355 is a thiourea derivative that has been investigated for its antioxidant and anti-inflammatory activities. However, its efficacy in bone resorption has not been previously investigated. The aim of this study was to investigate the impact of SPA0355 on the development of osteoporosis and to explore its mode of action. In vitro experiments showed that SPA0355 inhibited receptor activator of $NF-{\kappa}B$ ligand (RANKL)-induced osteoclastogenesis in primary bone marrow-derived macrophages. This effect appears to be independent of estrogen receptor activation as ICI 180,782 failed to abrogate its effects on osteoclasts. Further signaling studies revealed that SPA0355 suppressed activation of the MAPKs, Akt, and $NF-{\kappa}B$ pathways. SPA0355 also increased osteoblastic differentiation, as evidenced by its effects on alkaline phosphatase activity and mineralization nodule formation. Intraperitoneal administration of SPA0355 to ovariectomized mice prevented bone loss, as verified by three-dimensional images and bone morphometric parameters derived from ${\mu}CT$ analysis. Noticeably, SPA0355 did not show hepatotoxicity and nephrotoxicity and also had little effect on hematological parameters. Taken together, the results indicate that SPA0355 may protect against bone loss in ovariectomized mice by stimulation of osteoblast differentiation and by inhibition of osteoclast resorption. Therefore, SPA0355 is a safe and potential candidate for management of postmenopausal osteoporosis.

• 치위생과학회지
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• 제19권3호
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• pp.198-204
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• 2019

Background: Oxidative stress is a known to be associated with in the pathogenesis of many inflammatory diseases, including periodontitis. Ursolic acid is a pentacyclic triterpenoid with has antimicrobial, antioxidative, and anticancer properties. However, the role of ursolic acid in the regulating of osteogenesis remains undetermined. This study was aimed to elucidate the crucial osteogenic effects of ursolic acid and its ability to inhibit oxidative stress by targeting the immediate early response 3 (IER3)/nuclear factor erythroid 2-related factor 2 (Nrf2) pathway. Methods: Cell proliferation was determined using water-soluble tetrazolium salt assay, cell differentiation was evaluated by alkaline phosphatase (ALP) activity, and formation of calcium nodules was detected using alizarin red S stain. Generation of reactive oxygen species (ROS) was determined using by DCFH-DA fluorescence dye in hydrogen peroxide ($H_2O_2$)-treated MG-63 cells. Expression levels of IER3, Nrf2, and heme oxygenase-1 (HO-1) were analyzed using western blot analysis. Results: Our results showed that ursolic acid up-regulated the proliferation of osteoblasts without any cytotoxic effects, and promoted ALP activity and mineralization. $H_2O_2$-induced ROS generation was found to be significantly inhibited on treatment with ursolic acid. Furthermore, in $H_2O_2$-treated cells, the expression of the early response genes: IER3, Nrf2, and Nrf2-related phase II enzyme (HO-1) was enhanced in the presence of ursolic acid. Conclusion: The key findings of the present study elucidate the protective effects of ursolic acid against oxidative stress conditions in osteoblasts via the IER3/Nrf2 pathway. Thus, ursolic acid may be developed as a preventative and therapeutic agent for mineral homeostasis and inflammatory diseases caused due to oxidative injury.

• 생명과학회지
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• 제14권6호
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• pp.967-974
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• 2004

골조직은 골아세포, 파골세포, 골세포 등으로 구성되며, 골개조시 여러 인자가 세포증식, 분화, 활성화 및 골대사 조절에 관여한다. 이때 조골세포의 활성은 골형성에 중요하므로, 본 연구에서는 MC3T3-E1 조골세포주를 이용하여 식용자원인 복분자의 조골세포의 증식과 분화활성에 미치는 영향을 조사하였다. 복분자 추출물이 조골세포의 성장에 미치는 영향을 MTT 검색법으로 조사한 결과, 복분자 추출물 $10\;{\mu}g/mL$ 처리시 대조군과 비교하여 $142\%$ 증가하여 조골세포에 대해 높은 성장률을 보였다. 복분자 추출물이 ALP 효소 활성에 미치는 영향을 3일 간격으로 배지교환 및 시료처리를 하면서 27일 동안 배양시간에 따른 변화를 측정하여 조사하였다. 그 결과, 농도 1, 10, $100\;{\mu}g/mL$ 처리시 $100\;{\mu}g/mL$을 제외한 나머지 분획물들이 시간이 지남에 따라 ALP 활성을 증가시켰고, $10\;{\mu}g/mL$ 농도의 추출물은 27일째에 대조군에 비해 약 2.6배 이상, 양성대조군에 비해 약 1.5배 이상 ALP 활성을 증가시켰다. 복분자 추출물은 다시 ALP 효소 염색법과 Alizarin Red 염색으로 조골세포의 ALP활성유도, 분화와 석회화 형성능을 재확인하였으며 골기질 유전자의 발현의 변화도 확인하였다. 따라서 종래의 골질환에 좋다고 알려진 식품인 복분자 추출물이 양성대조군에 비해 빠르게 세포 증식과 분화를 유도하고 있어 앞으로 복분자에 대한 좀 더 깊은 분자생물학 수준 등의 구체적인 연구들과 기작연구가 지속적으로 이루어져야 할 것이라 추측된다.

• Journal of Acupuncture Research
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• 제23권5호
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• pp.69-77
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• 2006

Background : Mesenchymal stem cells (MSCs) are present in most of the tissue matrix, taking part in their regeneration when injury or damage occurs. The aim of this study was to investigate the presence of cells with pluripotential characteristics in human subchondral bone and the capacity of these cells to differentiate to osteoblast. Methods : Human subchondral bone were digested with collagenase. Isolated cells were cultured with a-MEM, 15% FBS, 10-8M dexamethasone and 50 ng/mL ascoric acid. Cells from 0 day(isolated cells), 7 day (first subculture) and 14 days (third subculture) were used to carry out phenotypic characterization experiments flowcytometry analysis with 11 monoclonal antibodies) and osteogenic differentiation experiments. Osteogenic differentiation of cells was assessment by quantification of bone extracellular matrix components by following analysis: alkaline phosphatase(ALP) stains to detect ALP activity, RT-PCR and western blot to detect osteocalcin (OCN), osteopontin (OPN) and type I collagen(Col I), and Alizarin red stains to detect calcium deposition. Results : Flowcytometry analyses showed that in our population more than 98% of cells were positive for MSC markers: SH-2(CD105, 99%), CD29 (95%), CD73 (95%). Cells were negative for hematopoietic markers (CD11b, CD34, and CD45). Furthermore, cells showed positive stain to multipotent markers such as CDl17 (c-kit) (15.1%), and CD166 (74.9%), and cell adhesion molecules such as CD54 (78.1%) and CD106 (63.5%). The osteogenic specific marker analyses showed that the culture of these cells for 7 and 14 days stimulates ALP, OCN, OPN and Col I synthesis by RT-PCR and Western blot analysis. Also, after 14 days in the culture of MSCs induces mineralization by Arizarin red stain. Conclusion : In this work, we demonstrated a new and efficient method for osteoblastic differentiation of human subchondral bone stem cells. As MSCs takes part in reparative processes of adult tissues, these cells could play an important role in osteogenesis.

• 한국자원식물학회지
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• 제33권4호
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• pp.227-236
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• 2020

고령사회에서 노년기 건강의 큰 문제로 대두되고 있는 골다공증은 특히 폐경 후 여성들에게서 가장 그 발생빈도가 높게 나타났으며, 현재 골다공증 예방 및 치료에 사용되고 있는 약제는 대부분 골흡수 억제제로써 진행된 골소실을 회복 시킬 수는 없기 때문에 골형성 증가를 통한 골다공증 예방과 치료에 관한 연구가 활발히 이루어지고 있다. 산양삼(cultivated wild Panax ginseng, CWP)에 대한 연구는 다수가 원기회복, 자양강장 및 면역증강 효과 등에 대한 것이나 골대사에 미치는 영향에 대한 연구는 거의 없는 실정이다. 이에 본 연구에서는 산양삼 추출물이 조골세포에서 골관련 유전자 발현에 미치는 영향을 확인함으로써 골다공증 예방 및 치료 효과를 갖는 천연 소재로의 활용 가능성을 검토하고자 하였다. 산양삼 추출물 처리가 조골 세포의 증식에 미치는 영향을 알아보기 위해 MTT assay를 실시하였고, MC3T3-E1 세포생존률은 FBS가 첨가되지 않은 배양액만 처리한 대조군과 산양삼 추출물을 처리한 실험군 모두에서 동일한 수준으로 나타났으며 이로써 산양삼 추출물의 안전성을 확인할 수 있었다. 또한 산양삼 추출물을 처리한 실험군과 대조군과의 세포증식률을 비교하였을 때 산양삼 추출물 50 ㎍/mL 농도 처리군에서 유의적으로 세포증식이 촉진되었으며 25 ㎍/mL과 100 ㎍/mL 농도 처리군에서도 대조군보다 높은 경향을 나타내었다. 산양삼 추출물이 조골 세포의 활성에 미치는 영향을 알아보기 위해 조골세포의 분화초기 표지인자인 ALP활성을 측정하였으며 그 결과 모든 산양삼 추출물 처리군이 대조군과 비교하여 유의적으로 높은 활성을 나타내었으며 특히 산양삼 추출물 50 ㎍/mL 농도 처리군에서 가장 높은 활성을 나타내었다. 산양삼 추출물의 농도에 따른 석회화 형성도를 확인하기 위해 무기질화된 세포의 기질을 alizarin red로 염색하였고 산양삼 추출물을 처리한 실험군과 대조군과의 석회화 형성도를 비교하였을 때 산양삼 추출물 50 ㎍/mL 농도 처리군에서 유의적으로 석회화 형성이 촉진되었으며 25 ㎍/mL과 100 ㎍/mL 농도 처리군에서도 대조군보다 높은 경향을 나타내었다. 산양삼 추출물이 MC3T3-E1 조골세포에서 골 형성 관련 유전자 발현에 미치는 영향을 확인하기 위해 Runx2, ALP, OPN, OCN등의 유전자를 정량 real-time PCR을 통해 분석하였으며 대조군과 비교하여 모든 산양삼 추출물 처리군에서 농도 의존적이고 유의적으로 골 형성 관련 유전자발현이 증가되었다. 따라서 산양삼추출물이 골 형성 관련 유전자인 Runx2, ALP, OPN, OCN 발현을 증가시켜MC3T3-E1 조골세포의 분화를 촉진하고, 골 석회화 형성 촉진에 기여하였을 것으로 사료된다. 그러나 산양삼 추출물이 골형성과 관련하여 어떠한 기전으로 유전자의 발현을 조절하였는지에 대한 유전자 및 단백질 수준의 추가적인 연구와 산양삼 추출물의 분화 촉진과 석회화 형성능이 산양삼의 사포닌계 진세노사이드 성분의 영향인지에 대한 후속 연구가 필요할 것으로 사료된다.

• 한국산림과학회지
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• 제113권1호
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• pp.73-87
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• 2024

복령(Wolfiporia hoelen (Fr.) Y.C.Dai & V. Papp)은 아시아 및 유럽 등 많은 나라에서 약용 및 식용으로 사용하고 있는 중요한 약재이다. 복령의 주요 성분들의 약리 활성이 많은 연구를 통해 밝혀지고 있으며, 골대사에도 영향을 주는 것으로 나타났다. 이에 본 연구는 복령 균핵 50% 에탄올 추출물이 골질환 예방 및 천연 치료제의 소재로 활용 가능성이 있는지 알아보고자 하였다. 복령 균핵의 에탄올 추출물을 계통분획법에 따라 n-헥산, 디클로로메탄, 에틸아세테이트순으로 분획하여 C2C12세포에 처리하였으며, 디클로로메탄 분획물에서 C2C12 세포의 ALP 분화 활성이 대조구보다 약 29%가 증가함을 알 수 있었다. 활성을 보인 디클로로메탄 분획물로부터 총 4개의 화합물을 분리하였으며, 기기분석 결과와 문헌을 참고하여 3α-dehydrotrametenolic acid, ergosterol, pachymic acid와 dehydrotumulosic acid로 동정할 수 있었다. 골대사 효능이 있는 지표물질을 찾기 위하여 4개의 화합물을 C2C12 세포의 ALP 분화 활성과 BMMs의 RANKL 유도 억제 활성을 농도별(1, 3, 10, 30, 100 μM)로 평가하였으며, 세포독성 검정도 동일한 농도에서 수행하였다. 그 결과 분리된 화합물 중 dehydrotumulosic acid의 3 μM 농도에서 ALP 활성이 160%로 나타났으며, BMP-2 대조구보다 약 24% 유의미하게 증가하였다. 또한 BMMs의 RANKL 유도 억제 활성은 dehydrotumulosic acid를 10-100 μM 농도로 처리할 때 대조구보다 약 15-86%로 유의미하게 감소하였으며, 10 μM 농도에서 나타난 RANKL억제 활성은 세포독성과 다른 패턴으로 나타나는 것을 확인하였다. 본 연구결과를 토대로 복령의 균핵 에탄올 추출물이 골모세포 분화를 증진시키거나 억제할 수 있는 가능성을 확인하였으며, 복령의 디클로로메탄 분획물로부터 분리 정제된 dehydrotumulosic acid가 골다공증 예방 및 치료에 도움을 줄 수 있을 것으로 사료된다.

• Journal of Korean Dental Science
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• 제4권1호
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• pp.6-13
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• 2011

Purpose: Craniosynostosis (CS), one of the most common congenital craniofacial deformities, is the premature closure of cranial sutures. NELL-1 is a novel molecule overexpressed during premature cranial suture closure in human CS. From a functional perspective, NELL-1 has been reported to accelerate chondrocyte maturation and modulate calvarial osteoblast differentiation and apoptosis pathways. The mechanism through which NELL-1 induces these phenomena, however, remains unclear. The purpose of this study is to identify the NELL-1 binding protein(s) through which the biologic mechanism of NELL-1 can be further investigated. Materials and Methods: Far-Western and Immunoprecipitation (IP) assays were performed, independently and in sequence, followed by mass spectrometry to identify the NELL-1 binding proteins. Reverse IP was used to verify and confirm candidate binding protein. Results: The only confirmative protein from current experimentation was vimentin. Vimentin is the major structural component of the intermediate filaments. Conclusion: The present study identified and confirmed vimentin as a NELL-1 binding protein, which opened up a new window to mechanistically facilitate studies on this CS-associated molecule.

• BMB Reports
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• 제49권6호
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• pp.343-348
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• 2016

GATA4 has been reported to act as a negative regulator in osteoblast differentiation by inhibiting the Dlx5 transactivation of Runx2 via the attenuation of the binding ability of Dlx5 to the Runx2 promoter region. Here, we determine the role of GATA4 in the regulation of bone sialoprotein (Bsp) in osteoblasts. We observed that the overexpression of Runx2 or Sox9 induced the Bsp expression in osteoblastic cells. Silencing GATA4 further enhanced the Runx2- and Sox9-mediated Bsp promoter activity, whereas GATA4 overexpression down-regulated Bsp promoter activity mediated by Runx2 and Sox9. GATA4 also interacted with Runx2 and Sox9, by attenuating the binding ability of Runx2 and Sox9 to the Bsp promoter region. Our data suggest that GATA4 acts as a negative regulator of Bsp expression in osteoblasts.

• BMB Reports
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• 제43권8호
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• pp.513-523
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• 2010

Cytokines that bind to and signal through the gp130 co-receptor subunit include interleukin (IL)-6, IL-11, oncostatin M (OSM), leukemia inhibitory factor (LIF), cardiotrophin-1 (CT-1), and ciliary neutrophic factor (CNTF). Apart from contributing to inflammation, gp130 signalling cytokines also function in the maintenance of bone homeostasis. Expression of each of these cytokines and their ligand-specific receptors is observed in bone and joint cells, and bone-active hormones and inflammatory cytokines regulate their expression. gp130 signalling cytokines have been shown to regulate the differentiation and activity of osteoblasts, osteoclasts and chondrocytes. Furthermore, cytokine and receptor specific gene-knockout mouse models have identified distinct roles for each of these cytokines in regulating bone resorption, bone formation and bone growth. This review will discuss the current models of paracrine and endocrine actions of gp130-signalling cytokines in bone remodelling and growth, as well as their impact in pathologic bone remodelling evident in periodontal disease, rheumatoid arthritis, spondylarthropathies and osteoarthritis.

• Asian Pacific Journal of Cancer Prevention
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• 제13권10호
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• pp.5219-5223
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• 2012

Osteopontin (OPN) is an integrin-binding protein, believed to be involved in a variety of physiological cellular functions. The physiology of OPN is best documented in the bone where this secreted adhesive glycoprotein appears to be involved in osteoblast differentiation and bone formation. In our study, we used semi-quantitative RT-PCR of osteopontin in calcification tissue of breast to detect breast cancer metastasis. The obtained data indicate that the expression of osteopontin is related to calcification tissue of breast, and possibly with the incidence of breast cancer. The expression strength of OPN by RT-PCR detection was related to the degree of malignancy of breast lesions, suggesting a close relationship between OPN and breast calcification tissue. The results revealed that expression of OPN mRNA is related to calcification of breast cancer tissue and to the development of breast cancer. Determination of OPN mRNA expression can be expected to be a guide to clinical therapy and prediction of the prognosis of breast cancer patients.