• Molecules and Cells
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• 제41권3호
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• pp.234-243
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• 2018

In recent years, the interest towards the relationship between incretins and bone has been increasing. Previous studies have suggested that glucagon-like peptide-1 (GLP-1) and its receptor agonists exert beneficial anabolic influence on skeletal metabolism, such as promoting proliferation and differentiation of osteoblasts via entero-osseous-axis. However, little is known regarding the effects of GLP-1 on osteoblast apoptosis and the underlying mechanisms involved. Thus, in the present study, we investigated the effects of liraglutide, a glucagon-like peptide-1 receptor agonist, on apoptosis of murine MC3T3-E1 osteoblastic cells. We confirmed the presence of GLP-1 receptor (GLP-1R) in MC3T3-E1 cells. Our data demonstrated that liraglutide inhibited the apoptosis of osteoblastic MC3T3-E1 cells induced by serum deprivation, as detected by Annexin V/PI and Hoechst 33258 staining and ELISA assays. Moreover, liraglutide upregulated Bcl-2 expression and downregulated Bax expression and caspase-3 activity at intermediate concentration (100 nM) for maximum effect. Further study suggested that liraglutide stimulated the phosphorylation of AKT and enhanced cAMP level, along with decreased phosphorylation of $GSK3{\beta}$, increased ${\beta}-catenin$ phosphorylation at Ser675 site and upregulated nuclear ${\beta}-catenin$ content and transcriptional activity. Pretreatment of cells with the PI3K inhibitor LY294002, PKA inhibitor H89, and siRNAs GLP-1R, ${\beta}-catenin$ abrogated the liraglutide-induced activation of cAMP, AKT, ${\beta}-catenin$, respectively. In conclusion, these findings illustrate that activation of GLP-1 receptor by liraglutide inhibits the apoptosis of osteoblastic MC3T3-E1 cells induced by serum deprivation through $cAMP/PKA/{\beta}-catenin$ and $PI3K/Akt/GSK3{\beta}$ signaling pathways.

• 한국자원식물학회:학술대회논문집
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• 한국자원식물학회 2020년도 춘계학술대회
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• pp.90-90
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• 2020

Panax ginseng C.A. Meyer (P. ginseng) is known to exert a wide range of pharmacological effects both in vitro and in vivo. Although studies on ginsenoside, antioxidant activity, and anticancer effect of wild simulated ginseng (WSG) have been conducted, there is little research on the effect of WSG on bone metabolism. In this study, we investigated the potential anti-osteoporotic properties of WSG on the growth and differentiation of MC3T3-E1 cells. WSG significantly increased the viability and proliferation of MC3T3-E1 cells. WSG activated intracellular alkaline phosphatase (ALP) activity in MC3T3-E1 cells. In addition, WSG increased the mineralized nodules in MC3T3-E1 cells. Furthermore, WSG increased the expression of genes such as Runx2, ALP, OPN and OCN associated with osteoblast growth and differentiation in a dose-dependent manner.

• 대한한방부인과학회지
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• 제31권4호
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• pp.1-15
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• 2018

목 적: 본 연구는 길경(Platycodon grandiflorum root)를 함유한 복합 한약재 추출물(ExMH-PGR)의 골다공증 예방 및 치료효과를 알아보기 위해 MC3T3-E1 조골세포주와 RAW 264.7 파골세포주를 이용하여 in vitro 수준에서 검증하였다. 방 법: 배양액에 다양한 농도의 ExMH-PGR를 첨가하여 MC3T3-E1 세포와 RAW 264.7 세포의 세포증식을 비교하였다. MC3T3-E1 세포에서 Alkaline phosphatase (ALP) 활성, 콜라겐 합성, 오스테올칼신 생성, 무기질 축적을 분석하였다. RAW 264.7 세포에서 Tartrate-resistant acid phosphatase (TRAP) 활성과 actin ring 형성 정도를 분석하였다. 결 과: ExMH-PGR는 $25{\mu}g/mL$ 농도까지 유의한 수준으로 ALP 활성, 콜라겐 합성, 그리고 오스테올칼신 형성을 증가시켰다. $50{\sim}200{\mu}g/mL$의 ExMH-PGR은 TRAP 활성과 actin ring 형성을 유의하게 억제했다. 결 론: ExMH-PGR은 조골세포의 활성을 촉진하고 파골세포의 활성을 억제하는 효과가 있어 골다공증 치료에 유용할 가능성이 있다.

• Nutrition Research and Practice
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• 제10권2호
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• pp.148-153
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• 2016

BACKGROUND/OBJECTIVES: Bone formation and bone resorption continuously occur in bone tissue to prevent the accumulation of old bone, this being called bone remodeling. Osteoblasts especially play a crucial role in bone formation through the differentiation and proliferation. Therefore, in this study, we investigated the effects of Scytosiphon lomentaria extract (SLE) on osteoblastic proliferation and differentiation in MC3T3-E1 cells. MATERIALS/METHODS: A cell proliferation assay, alkaline phosphatase (ALP) activity assay, alizarin red staining and protein expression analysis of osteoblastic genes were carried out to assess the osteoblastic proliferation and differentiation. RESULTS: The results indicated that treatment of SLE promoted the proliferation of MC3T3-E1 cells and improved ALP activity. And, SLE treatment significantly promoted mineralized nodule formation compared with control. In addition, cells treated with SLE significantly upregulated protein expression of ALP, type 1 collagen, bone morphogenetic protein 2, runt-related transcription factor 2, osterix, and osteoprotegerin. CONCLUSIONS: The results demonstrate that SLE promote differentiation inducement and proliferation of osteoblasts and, therefore may help to elucidate the transcriptional mechanism of bone formation and possibly lead to the development of bone-forming drugs.

• Journal of Acupuncture Research
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• 제29권6호
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• pp.11-21
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• 2012

Objectives : BHH10 is traditional medicine herb used for enhancing body resistance against various diseases. The aim of this study was to identify BHH10 extract induces osteogenic activity in human osteoblast-like MC3T3-E1 cells. Methods : MC3T3-E1, pre-osteoblast cell line, were treated with BHH10 of various concentrations($0.1{\mu}g/mL$, $1{\mu}g/mL$, $10{\mu}g/mL$). And then, the effect of BHH10 on osteoblast differentiation was examined by alkaline phosphatase(ALP) activity, von Kossa staining and RT-PCR for osteoblast differentiation markers such as osteocalcin(OCN), osteopontin(OPN). Results : BHH10 had dose-dependent effect on the viability of osteoblastic cells, and dose-dependently increased alkaline phosphatase(ALP) activity. BHH10 markedly increased mRNA expression for OCN, OPN in MC3T3-E1 cells. Also, BHH10 significantly induced mineralization in the culture of MC3T3-E1 cells. Conclusions : In conclusion, these results propose that BHH10 can play an important role in osteoblastic bone formation, osteogenesis, and may possibly lead to the development of bone-forming drugs.

• International Journal of Oral Biology
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• 제34권1호
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• pp.37-42
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• 2009

The attachment and adhesion of RAW 264.7 and MC3T3-E1 cells to titanium (Ti) discs with various degrees of roughness was investigated. The attachment, adhesion, and proliferation of these cells were evaluated after 4 hr, 24 hr and 7 day incubations. Both RAW 264.7 and MC3T3-E1 cells showed a time-dependant correlation between attachment and adhesion on the surface of the titanium discs. Both types of cells tended to have higher survival rate on these discs as the surface roughness increased. The percentage of adherent inflammatory RAW 264.7 cells was greater than MC3T3-E1 cells at 24 hr, but this was reversed at 7 days in culture. The morphology of osteoblastic MC3T3-E1 cells at 24 hr, determined using a surface emission microscope (SEM), appeared flattened and spread out while inflammatory RAW 264.7 cells were predominantly spherical in shape. The adhesion of both cell types on the titanium discs was dependant on the levels of fibronectin adsorbed on the disc surface, indicating that serum constituents modulate the efficient adhesion of these cells. Our data indicate that the cellular response to the titanium surface is dependent on the types of cells, surface roughness and serum constituents.

• Nutrition Research and Practice
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• 제4권5호
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• pp.356-361
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• 2010

Zinc is an essential trace element required for bone formation, however not much has been clarified yet for its role in osteoblast. We hypothesized that zinc would increase osteogenetic function in osteoblasts. To test this, we investigated whether zinc treatment enhances bone formation by stimulating osteoblast proliferation, bone marker protein alkaline phosphatase activity and collagen synthesis in osteoblastic MC3T3-E1 cells. MC3T3-E1 cells were cultured and treated with various concentrations of zinc (0, 1, 3, 15, 25 uM) along with a normal osteogenic medium (OSM) as control for 1, 5, 10 days. As measured by MTT assay for mitochondrial metabolic activity, cell proliferation was stimulated even at low zinc treatment (1-3 ${\mu}M$) compared to OSM, and it was stimulated in a zinc concentration-dependent manner during 5 and 10 days, with the most pronounced effect at 15 and 25 uM Zn. Cellular (synthesized) alkaline phosphatase (ALP) activity was increased in a zinc concentration-dependent manner, so did medium (secreted) ALP activity. Cellular collagen concentration was increased by zinc as time went by, therefore with the maximum zinc stimulatory effect in 10 days, and medium collagen concentration showed the same pattern even on 1 and 5 day. This zinc stimulatory effect of collagen synthesis was observed in cell matrix collagen staining. The study results imply that zinc can increase osteogenic effect by stimulating cell proliferation, ALP activity and collagen synthesis in osteoblastic cells.

• Natural Product Sciences
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• 제14권1호
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• pp.21-26
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• 2008

The leaves of Acanthopanax species have traditionally been used as a tonic and a sedative as well as in the treatment of rheumatism and diabetes. Chiisanoside is the major active lupane triterpenoid of Acanthopanax leaves. To investigate the bioactivities of chiisanoside, which act on bone metabolism, the effects of chiisanoside on the function of osteoblastic MC3T3-E1 cells were studied. Chiisanoside $(0.02{\sim}20\;{\mu}M)$ significantly increased the growth of MC3T3-E1 cells and caused a significant elevation of alkaline phosphatase (ALP) activity, collagen content, and nodules mineralization in the cells (P < 0.05). The effect of chiisanoside (2 ${\mu}M$) in increasing ALP activity was completely prevented by the presence of tamoxifen, suggesting that the effect of chiisanoside might be partly estrogen receptor mediated. Moreover, cotreatment of p38 inhibitor SB203580 or JNK inhibitor SP600125 inhibited chiisanoside-mediated ALP upregulation, suggesting that the induction of differentiation by chiisanoside is associated with increased activation of p38 and JNK mitogen-activated protein kinases. Our data indicate that the enhancement of osteoblast function by chiisanoside may result in the prevention for osteoporosis.

• Imaging Science in Dentistry
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• 제33권3호
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• pp.179-185
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• 2003

Purpose: To investigate the effects of irradiation on the phenotypic expression of the MC3T3-El osteoblastic cell line, particularly on the expression of osteocalcin and osteopontin. Materials and Methods: Cells were irradiated with a single dose of 0.5, 1,4, and 8 Gy at a dose rate of 5.38 Gy/min using a cesium 137 irradiator. After the specimens were harvested, RNA was extracted on the 3rd, 7th, 14th, and 21st day after irradiation. The RNA strands were reverse-transcribed and the resulting cDNAs were subjected to amplification by PCR. Results: The irradiated cells demonstrated a dose-dependent increase in osteocalcin and a dose-dependent decrease in osteopontin mRNA expression compared with the non-irradiated control group, The amount of osteocalcin mRNA expression decreased significantly at the 3rd day after irradiation of 0,5, 1,4, and 8 Gy, and also decreased significantly at the 3rd, 14th, and 21 st day after irradiation in the 8 Gy exposed group compared with the control group, The degree of osteopontin mRNA expression increased significantly at the 7th day after irradiation of 0,5, 1,4, and 8Gy, Conclusion: These results showed that each single dose of 0,5, 1, 4, and 8 Gy influenced the mRNA expression of osteocalcin and osteopontin associated with the calcification stage of osteoblastic cells, suggesting that each single dose affected bone formation at the cell level.

• International Journal of Oral Biology
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• 제39권2호
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• pp.121-128
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• 2014

Sambucus sieboldiana (SS) is a member of the family Caprifoliaceae and has been recommended as a functional material because of its several bioactivities. Although numerous literatures are available on the pharmacological and biological activities, the biological activity of SS in bone regeneration process has not yet been well-defined. Therefore, in this study, the effect of SS was investigated in the proliferation and differentiation of MC3T3-E1 osteoblastic cell line. The treatment of SS did not significantly affect the cell proliferation in MC3T3-E1 cells. SS significantly accelerated the mineralization and significantly increased the expression of alkaline phosphatase (ALP) and osteocalcin (OC) mRNAs, compared to the control, in the differentiation of MC3T3-E1 cells. SS significantly accelerated the decrease of osteonectin (ON) mRNA expression as compared with the control in a time-dependent manner in the differentiation of MC3T3-E1 cells. These results suggest that the SS facilitate the osteoblast differentiation and mineralization in MC3T3-E1 osteoblastic cells. Therefore, there may be potential properties for development and clinical application of bone regeneration materials.