골흡수 증가시 tartrate-resistant acid phosphatase 양성세포의 동태 : in Initial Bone Resorption after the Cortical Bone Defect

손승원,김익동,채종민,곽정식,손태중 慶北大學校 醫科大學 1994 慶北醫大誌 Vol.35 No.1

목적 : 골흡수에 중심적인 역할을 하는 파골세포의 유래에 대하여 아직도 많은 논란이 있는 바 최근 tartrate 내성 acid phosphatase라는 효소가 파골세포와 그 전구세포를 인지 할 수 있는 표지자로 사용하게 됨에 따라 파골세포의 분화를 촉진시킨 경우와 억제시킨 경우에서 tartrate 내성 acid phosphatase의 양성세포의 동태를 관찰하여 그 세포의 유래와 골 표면에서의 파골세포의 형성과정을 알아보고저 한다. 대상 및 방법 : Sprague-Dawley계 흰쥐의 경골 앞쪽 전반을 실톱으로 골결손을 만들어 골흡수를 유도한 다음 파골세포의 전구세포로부터 다핵의 파골세포로의 융합내지는 분화를 촉진시키기 위하여 부갑상선 조직을 골결손 부위에 이식시킨 군과 반대로 파골세포의 분화을 억제시키기 위하여 국소적 prostaglandin생산 억제제인 indomethacin을 체중 1㎏당 2㎎을 국소에 매일 주사한 군으로 나누어 실험을 하였다. 골결손을 일으킨 후 제 1 일, 제 2 일, 제 3 일, 제 4 일 및 제 5 일에 경골을 절취하여 광학현미경을 위한 tartrate 내성 acid phosphatase (AcP)염색과 투과전자현미경을 위한 AcP의 조직화학적 염색을 하여 관찰하였다. 결과 : 골결손을 일으킨 후 아무런 처치를 하지 않은 대조군에서는 tartrate 내성 AcP양성의 단핵을 가진 파골세포의 전구세포가 골결손 제 1 일 후부터 나타나기 시작하여 제 2 일 째 가장 많이 나타났다. 제 3 일에서는 다핵을 가진 파골세포가 출현하기 시작하여 제 4 일 째 가장 많이 관찰되었으며 5일 후에는 파골세포의 숫자가 적어졌으며 골결손 주위에 신생 유골이 형성되기 시작하였다. 그리고 파골세포의 전구세포가 나타날 때 골내막의 골아세포 아래 세포층에는 tartrate 내성 AcP음성이면서 단핵의 탐식구들도 많이 출현하여 골결손 제 2 일 째 역시 가장 않은 숫적 증가를 보였다. 이 후 점차 소실되었다. 그리고 골결손을 일으킨 후 부갑상선 조직을 이식한 실험군에서는 골결손 제 1 일 후부터 tartrate 내성 AcP양성의 단핵을 가진 파골세포의 전구세포들과 그 주위에 단구 또는 대식구의 탐식구들이 왕성하게 나타나기 시작하여 제 2 일 째부터 다핵을 가진 파골세포의 출현을 보이며 피질골의 골흡수가 많이 일어났다. AcP의 조직 화학적반응을 한 후 투과전자현미경적 관찰에서 파골세포의 전구세포나 다핵의 이미 형성된 파골세포에 새로이 다른 종류의 탐식구로 보이는 세포들과 밀접하게 접촉내지는 융합하는 과정을 시사하는 소견도 관찰할 수가 있었다. 그러나 prostaglandin의 생산 억제제인 indomethacin을 국소에 매일 주사한 군에서는 골아세포들의 변형이 일어나지 않았으며 이어 파골세포들의 형성 및 활성화가 매우 적어 골흡수가 현저히 억제되었다. tartrate 내성 AcP양성의 단핵의 파골세포의 전구세포들이 골결손 제 3 일 후부터 매우 드물게 출현되었다. 결론 : 골조직 표면에서 파골세포의 전구세포들이 혈구세포인 단구 또는 대식구들과 융합이 이루어질 것으로 추측되며, 부갑상선 호르몬이나 prostaglandin 등의 골흡수 촉진 인자들이 파골세포내의 tartrate내성 AcP의 활성도를 증가시키기며, 단구나 대식구와의 융합을 촉진시킨 것으로 생각된다. 그리고 골조직 표면에서의 파골세포의 분화 융합에는 골아세포가 어떤 인자를 분비하여 매개할 것으로 생각된다. An electron microscopic cytochemical study was carried out to investigate the origin of osteoclast and the mechanism for differentiation of osteoclast precursor around the bone tissue during bone resorption after cortical bone defect. In the present study an incomplete cortical defect was made in the tibial shaft of rats using a bone saw, and thereafter tartrate-resistant acid phosphatase cell population examined under two conditions. When transplantation of the parathyroid tissue was done into medullary cavity of the tibia. For the other condition, indomethacin, one of prostaglandin inhibitors, was administered daily at a dose of 2 ㎎/1 ㎏/day. The animals were sacrified at 1, 2, 3, 4 and 5 days after bone defect. The tibial bones were extirpated and examined tartrate-resistant acid phosphatase cells by light microscopy and ultrastructural acid phosphatase-cyto-chemical study. The results were summarized as follow: In the control groups, the first tartrate-resistant acid phosphatase positive nultinucleated osteoclasts appeared along the endosteal surface 3 days after bone defect, and the highest number of these cells was found 4 days after boine defect. Tartrate-resistant acid phosphatase negative mononuclear cells also incerased 1-2 days before the formation of osteoclasts. In the parathyroid transplantation group, the osteoblast converted to stellate shape and the bone matrix was exposed. Formation of tartrate-resistant acid phosphatase positive osteoclast precursors and multinucleated osteoclasts was markedly incresed. There were findings that macrophage has fused with preexisting osteoclast. In the indomethacin group, on day 2, the osteoblasts did not observed shape change, and osteoclastic bone resorption was inconspicuous. The maturation of tartrate-resistant acid phosphatase positive osteoclast precursors was delayed. The results suggested that macrophages fuse with pre-existing osteoclasts and/or osteoclast precursors around the bone tissue, and the bone resorbing agents stimulate fusion of the osteoclast precursors into multinucleated osteoclasts as well as the tartrate-resistant acid phosphatase activity of the preosteoclasts. The osteoblasts seem to mediate formation of multinucleated osteoclasts around the bone surface.

Alkaline Phosphatase, Tartrate-resistant Acid Phosphatase, and BMP6(Vgr-1) Expression during Chondrocyte Differentiation and Osteogenesis in Fetal Mouse Metatarsals

Gu, Y.,Gitelman, S. E.,Martincic, D.,Zernik, J. H.,Minkin, C. Korean Academy of Oral Biology and the UCLA Dental 1997 International Journal of Oral Biology Vol.22 No.2

Changes in the expression of alkaline phosphatase (ALP), tartrate-resistant acid phosphatase (TRAP or TrATPase, tartrate-resistant acid-ATPase), and bone morphogenic protein-6 (BMP6, also known as Vgr-1) were characterized during chondrocyte differentiation and osteogenesis in fetal mouse metatarsals. Histochemical observations demonstrate an increase in ALP expression both in differentiating hypertrophic chondrocytes and in osteogenic cells of the surrounding perichondrium/periosteum during days 15, 16, 17, and 18, of gestation. Expression of TRAP is first localized to a few small mononuclear osteoclast precursors in the periosteum of day 16 limbs and then a marked increase is demonstrated between days 17 and 18 of gestation as osteoclasts/chondroclasts invade periosteal bone surfaces and spicules of mineralized cartilage. Enzyme assays show a 9-fold increase in ALP activity in the developing limbs from day 15 to day 18 of gestation (0.05 to 0.43 mmole p-nitrophenol hydrolyzed/min/mg protein), and a 5.5-fold increase in TrATPase activity for the same time period (0.15 to 0.80% [^32P]g-ATP hydrolyzed/min/mg protein). Steady state levels of RNA for ALP, TRAP, and BMP6/Vgr-1 were evaluated using quantitative PCR amplification and our results demonstrate a 5-fold increase in ALP, a 500-fold increase in TRAP/TrATPase, and a 6-fold increase in BMP6/Vgr-1 mRNA levels between days 15 and 18 of gestation.

The Time Course Changes in Bone Metabolic Markers after Administering the Anti-Receptor Activator of Nuclear Factor-Kappa B Ligand Antibody and Drug Compliance among Patients with Osteoporosis

Kazuhide Inage,Sumihisa Orita,Kazuyo Yamauchi,Yoshihiro Sakuma,Go Kubota,Yasuhiro Oikawa,Takeshi Sainoh,Jun Sato,Kazuki Fujimoto,Yasuhiro Shiga,Kazuhisa Takahashi,Seiji Ohtori 대한척추외과학회 2015 Asian Spine Journal Vol.9 No.3

Study Design: Retrospective study. Purpose: We conducted a study to investigate the time course changes in bone metabolic markers after the administration of the anti-receptor activator of nuclear factor-kappa B ligand (RANKL) antibody and to assess drug compliance among osteoporotic patients. Overview of Literature: The anti-RANKL antibody is expected to provide an improvement in those with a bone metabolism disorder. However there are only a few clinical reports available on the effect of treatment. Methods: We included 40 post-menopausal osteoporotic patients who received the anti-RANKL antibody. To determine the time course changes in the bone metabolic markers, we measured the serum tartrate-resistant acid phosphatase 5b (TRACP 5b; a bone resorption marker) and the serum N-terminal propeptide of type 1 collagen (P1NP; a bone formation marker) levels prior to and 1 month after administrating the anti-RANKL antibody. To evaluable drug compliance, we assessed the dropout rate during treatment and at 6 months after treatment. Results: The average TRACP 5b level significantly decreased from 574.8 mU/dL before treatment to 153.2 mU/dL 1 month after treatment (p <0.05). There was no significant difference in the average P1NP level, which was 56.9 μG/L and 35.1 μG/L before and 1 month after treatment, respectively (p >0.05). As for drug compliance, we did not have any dropouts during the treatment or after 6 months (dropout rate: 0%). Conclusions: Our study suggests that anti-RANKL antibody treatment suppresses bone resorption and maintains bone formation.

Soy Isoflavones Mitigate Long-Term Femoral and Lumbar Vertebral Bone Loss in Middle-Aged Ovariectomized Mice

Kim, Dae-Won,Yoo, Ki-Yeon,Lee, Yoon-Bok,Lee, Keun-Ha,Sohn, Heon-Soo,Lee, Sung-Joon,Cho, Kyung-Hwan,Shin, Yong-Kook,Hwang, In-Koo,Won, Moo-Ho,Kim, Dong-Woo The Korean Society of Food Science and Nutrition 2009 Journal of medicinal food Vol.12 No.3

We evaluated the protective effects of soy isoflavones (SIF) against osteoporosis in middle-aged ovariectomized (OVX) mice. SIF (30 mg/kg or 60 mg/kg) or $17{\beta}$-estradiol ($E_2$) was administered to OVX mice for 4 months after bilateral ovariectomy. We observed the biochemical markers of bone turnover, e.g., alkaline phosphatase (ALP) and tartrate-resistant acid phosphatase (TRAP), in serum. We also observed the bone mineral density (BMD) in femurs and lumbar vertebrae. In addition, we examined trabecular bone and interstitial cells in the femur using hematoxylin and eosin staining. The decrease in ALP levels and the increase in TRAP levels normally resulting from ovariectomy were suppressed by administration of 60 mg/kg SIF or $E_2$. Administration of 60 mg/kg SIF or $E_2$ also maintained the BMD, trabecular bone, and interstitial cells in OVX mice compared to those in pre-OVX mice. These results suggest that 60 mg/kg SIF effectively mitigates ovariectomy-induced osteoporosis in middle-aged mice.

Soy Isoflavones Mitigate Long-Term Femoral and Lumbar Vertebral Bone Loss in Middle-Aged Ovariectomized Mice

김대원,유기연,Yoon-Bok Lee,Keun-Ha Lee,Heon-Soo Sohn,이성준,조경환,Yong Kook Shin,황인구,원무호,Dong-Woo Kim 한국식품영양과학회 2009 Journal of medicinal food Vol.12 No.3

We evaluated the protective effects of soy isoflavones (SIF) against osteoporosis in middle-aged ovariectomized (OVX) mice. SIF (30mg/kg or 60mg/kg) or 17β-estradiol (E2) was administered to OVX mice for 4 months after bilateral ovariectomy. We observed the biochemical markers of bone turnover, e.g., alkaline phosphatase (ALP) and tartrate-resistant acid phosphatase (TRAP), in serum. We also observed the bone mineral density (BMD) in femurs and lumbar vertebrae. In addition, we examined trabecular bone and interstitial cells in the femur using hematoxylin and eosin staining. The decrease in ALP levels and the increase in TRAP levels normally resulting from ovariectomy were suppressed by administration of 60mg/kg SIF or E2. Administration of 60mg/kg SIF or E2 also maintained the BMD, trabecular bone, and interstitial cells in OVX mice compared to those in pre-OVX mice. These results suggest that 60mg/kg SIF effectively mitigates ovariectomy-induced osteoporosis in middle-aged mice.

아보카도 과육, 과피 및 씨 추출물이 조골세포 분화 및 파골세포 형성에 미치는 영향

김미진(Mi-Jin Kim),임남경(Nam-Kyung Im),유미희(Mi-Hee Yu),김현정(Hyun-Jeong Kim),이인선(In-Seon Lee) 한국식품영양과학회 2011 한국식품영양과학회지 Vol.40 No.7

본 연구에서는 아보카도가 골 형성에 미치는 영향을 검토하고자 아보카도 과육, 과피 및 씨로 나누어 각각 메탄올 추출물을 제조하여 osteoblastic MC3T3-E1 cells을 이용한 골 형성능과 마우스 골수 세포로부터 유래된 파골세포를 이용한 골 흡수능을 측정하였다. 아보카도 과육 추출물을 제외한 과피 및 씨 추출물은 조골세포의 증식 및 ALP 활성을 증가시켰으며, 파골세포에 대해서는 아보카도 과육 및 과피 추출물에서 세포독성 없이 TRAP 활성을 억제하는 것을 확인하였다. 또한 아보카도 과피의 핵산 분획은 조골세포의 증식 및 ALP 활성을 크게 증가시켰으며, 아보카도 과피 에틸아세테이트 분획은 파골세포의 분화지표인 TRAP 활성을 크게 억제하였다. 따라서 아보카도 과피는 조골세포의 증식과 파골세포의 억제에 관여할 수 있는 우수한 소재로 향후 골다공증의 치료제로서의 개발 가능성을 가진 천연물소재로 생각된다. Avocado (Persea americana Mill., Family Lauraceae) is an important subtropical crop in the Americas where it has been cultivated for several thousand years. To investigate the bioactivities of avocado, which acts on bone formation, we prepared methanol extracts from the sarcocarp, peels, and seeds of avocado. The methanol extracts of peels and seeds showed higher bone-forming activity than avocado sarcocarp extracts accompanied by MC3T3-E1 osteoblast proliferation and alkaline phosphatase (ALP) activity. Additionally, the extracts of sarcocarp and peel from avocado also decreased tartrate-resistant acid phosphatase (TRAP) activity against differentiation of osteoclasts, derived from mouse bone marrow macrophages. The hexane fraction from avocado peels showed strong bone-forming activity accompanied by osteoblast proliferation and ALP activity (1 7 0 .7 ± 8.4%), and the ethyl acetate fraction from avocado peel decreased TRAP activity (5.2±0.3%) and differentiated osteoclasts at 50 ㎍/㎖. Therefore, avocado is expected to be a natural source for developing medicinal agents to prevent bone-related diseases, such as osteoporosis, by increasing osteoblast differentiation and reducing osteoclast activity.

RANKL에 의해 유도되는 파골세포 분화에 대한 시금치 추출물의 영향

김동규(Dong-Gyu Kim),김미혜(Mi-Hye Kim),강민정(Min Jung Kang),신정혜(Jung Hye Shin) 한국식품영양과학회 2015 한국식품영양과학회지 Vol.44 No.4

파골세포의 분화에 대한 시금치 추출물의 영향을 확인하고자 RANKL을 처리한 RAW264.7 세포에서 세포독성, TRAP(+) 다핵세포의 형성, 파골세포 분화 관련 유전자의 발현, 그리고 단백질 발현을 확인하였다. 물과 25, 50, 75 및 100% 에탄올 시금치 추출물의 세포독성을 측정한 결과 모든 추출물들이 100 μg/mL 이하의 농도에서 RAW264.7 세포에 독성을 유발하지 않았다. TRAP 염색을 통해 TRAP(+) 다핵세포의 수와 효소 활성을 측정한 결과 물 추출물을 제외한 모든 추출물이 대조군에 비해 분화 억제 및 효소 활성저해 효과가 있었다. 특히 100 μg/mL 농도의 100% 에탄올 추출물은 RANKL만 처리한 대조군과 비교해 80%의 유의한 TRAP(+) 다핵세포 숫자 감소와 44%의 TRAP 효소 활성 저해율을 보였다. 시금치 에탄올 추출물은 RANKL에 의한 파골세포 분화의 지표가 되는 관련유전자인 NFAT, c-FOS, cathepsin K 및 TRAP의 발현을 억제하였다. 또한 단백질 수준에서 시금치 에탄올 추출물은 RANKL에 의해 증가된 NFATc1의 발현을 현저히 감소시키는 것으로 확인되었고, 또한 c-FOS의 활성화 형태인 인산화된 c-FOS의 발현뿐만 아니라 인산화되지 않은 비활성의 c-FOS 발현도 감소시켰다. 반면 파골세포의 분화에 직간접적인 영향을 미친다고 알려진 MAPK 중 ERK의 활성에는 거의 영향을 미치지 않는 것으로 보아 시금치 에탄올 추출물은 c-FOS의 활성, 비활성형 전체를 감소시킴으로 파골세포 분화를 감소시키는 것으로 확인되었다. Inhibition of osteoclast differentiation is the most important target for prevention of inflammatory bone resorption and bone diseases. Here, we investigated the effect of spinach ethanol extract on osteoclast differentiation in RAW264.7 cells. Spinach was extracted with ethanol at a concentration ranging from 0 to 100% (0, 25, 50, 75, and 100% ethanol). Inhibitory effects of receptor activator of NF-κB ligan (RANKL)-induced osteoclast differentiation were evaluated using tartrate-resistant acid phosphatase (TRAP) stain assay. The most effective eanol concentration for osteoclast differentiation was 100%. Spinach extract (100% ethanol) suppressed RANKL-induced osteoclast differentiation and TRAP activity. Spinach extract (100% ethanol) also suppressed expression of osteoclast differentiationrelated marker genes (NFATc1, c-FOS, cathepsin K, and TRAP) and down-regulated RANKL-induced NF-κB and ERK phosphorylation during osteoclast differentiation. Taken together, our results suggest that spinach extract is effective against reducing osteoclast differentiation through the NF-κB-mediated pathway.

저칼슘식이 생쥐 하악골에서 파골세포의 Tartrate저항성 산성인산분해효소 활성에 대한 세포화학적 연구

박은주,임도선,김현만,고재승 대한구강해부학회 1994 대한구강해부학회지 Vol.18 No.1

The present study was carried out to exanine the morphological changes and TRACP (Tartrate resistant acid phosphatase) activity in the resorption of mandibular bones of mouses resulted, established by low calcium diet. Mice were divided into two groups; one was the calcein group for fluorescent microscopy, the other was the non-calcein group for light and electron microscopy. After calcein injection, mice(ICR) were fed either a low calcium diet (0.01% Ca, 0.75% P) or control diet (0.1% Ca. 0.75% P). Non-calcein group were also fed a low calcium diet or control diet. Mice were sacrificed on 3, 7 and 14 days. For fluorescent microscopy study, fixed undecalcified alveolar bones were embedded in Epon 812, and ground sectioned to study the new bone formation after calcein injection. For the TRACP histochemical study, fixed decalcified alveolar bones were incubated in p-NPP (p-nitrophenyl phosphate) media. For the transmission electron microscopic study, incubated alveolar bone were post fixed in OsO_(4), embedded with Epon 812 for the study of TRACP localzation and activity. The observed results were as follows. 1. Low calcium diet induced bone resorption in buccal and lingual sides of alveolar bone, while the lingual side of alveolar bone was not resorbed in the control group. Bone resorption increased over the period of low calcium diet. 2. The alveolar bone proper of the lingual alveolar bone continued to deposit new bone of the periodontal side even after the low calcium diet. 3. After low calcium diet TRACP activity increased in the cytoplasm of osteoclast, extracellular ruffled border. TRACP activity was also found at the eroded bone matrix after bone resorption, and in the cytoplasm of detached osteoclast. In the immature osteoclast enzyme activity was confined in rER. No enzyme activity was found in the osteoblast.

Aster saponin A2 inhibits osteoclastogenesis through mitogen-activated protein kinase-c-Fos-NFATc1 signaling pathway

Xiang-Dong Su,Seo Y Yang,Saroj K Shrestha,Yunjo Soh 대한수의학회 2022 Journal of Veterinary Science Vol.23 No.4

Background: In lipopolysaccharide-induced RAW264.7 cells, Aster tataricus (AT) inhibits the nuclear factor kappa-light-chain-enhancer of activated B cells and MAPKs pathways and critical pathways of osteoclast development and bone resorption. Objectives: This study examined how aster saponin A2 (AS-A2) isolated from AT affects the processes and function of osteoclastogenesis induced by receptor activator of nuclear factor kappa-B ligand (RANKL) in RAW264.7 cells and bone marrow macrophages (BMMs). Methods: The cell viability, tartrate-resistant acid phosphatase staining, pit formation assay, polymerase chain reaction, and western blot were carried out to determine the effects of AS-A2 on osteoclastogenesis. Results: In RAW264.7 and BMMs, AS-A2 decreased RANKL-initiated osteoclast differentiation in a concentration-dependent manner. In AS-A2-treated cells, the phosphorylation of ERK1/2, JNK, and p38 protein expression were reduced considerably compared to the control cells. In RAW264.7 cells, AS-A2 suppressed the RANKL-induced activation of osteoclast-related genes. During osteoclast differentiation, AS-A2 suppressed the transcriptional and translational expression of NFATc1 and c-Fos. AS-A2 inhibited osteoclast development, reducing the size of the bone resorption pit area. Conclusion: AS-A2 isolated from AT appears to be a viable therapeutic therapy for osteolytic illnesses, such as osteoporosis, Paget’s disease, and osteogenesis imperfecta.

애엽 추출물이 골 대사에 미치는 영향

이승민(Seung Min Lee),김명규(Myung Gyou Kim),이승연(Seung Youn Lee),강태훈(Tae Hoon Kang) 한국식품영양과학회 2010 한국식품영양과학회지 Vol.39 No.3

본 연구에서는 마우스 골수유래 파골세포와 난소절제 동물모델을 이용하여 애엽 추출물이 골대사에 미치는 영향을 살펴보았다. 애엽 추출물이 파골세포의 분화 및 활성에 미치는 영향을 알아보기 위하여 마우스 골수유래 전구세포에 M-CSF와 RANKL을 처리하여 파골세포로의 분화를 유도 한 후, TRAP 염색법을 실시하고 TRAP 활성도를 측정하였다. 그 결과 애엽 추출물은 TRAP 양성 다핵세포의 형성과 TRAP 활성을 농도 의존적으로 감소시켰으며, 파골세포의 골흡수 또한 애엽 추출물의 최고농도(30 μg/mL) 처리 시 약 90%까지 억제되는 것을 확인하였다. 난소절제 동물모델 을 이용한 실험에서도 애엽 추출물이 난소절제에 따른 급격한 골밀도 감소를 막아준다는 사실을 확인할 수 있었다. 이상의 결과로 미루어 볼 때, 애엽 추출물이 에스트로겐 분비 감소로 인하여 급격한 골 손실이 나타나는 폐경기 여성의 골다공증 예방에 효과가 있을 것으로 사료된다. Artemisia princeps has been utilized as a traditional medicine for a variety of diseases in Korea. In this study, we investigated the effects of Artemisia princeps extract (APE) on bone metabolism both in vitro using primary mouse bone marrow-derived macrophage and in vivo using ovariectomized rats. APE decreased the number of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells and TRAP activity. Also, APE inhibited bone resorptive activity of differentiated osteoclasts. In ovariectomized rats, APE alleviated the decrease in the trabecular bone mineral density. These results showed that APE might be useful for the prevention of postmenopausal bone loss.