• 한국자원식물학회지
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• 제28권5호
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• pp.561-567
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• 2015

본 연구에서는 세복수초 추출물에 대한 항암활성을 평가하고자 간암세포주인 SK-Hep1 세포주에서 MTT를 통한 세포독성을 평가하고 자가포식(autophagy) 형성정도를 확인하였다. 또한, 종양형성능 측정(Xenograft assay)를 통하여 세복수초 추출물에 대한 항암활성평가를 수행하였다. 그 결과 in vivo및 in vitro에서 모두 항암활성이 뛰어나게 나타났으며, 세복수초 추출물의 항암작용은 자가포식(autophagy)을 증가시키는 것으로 나타났다. 세복수초 추출물은 in vitro및 in vivo에서 모두 LC3의 발현을 농도의존적으로 증가시켜며 p62의 발현을 억제시키는 것으로 확인되었으며, 따라서 세복수초 추출물은 자가포식(autophagy) 활성을 증가시켜 암세포의 세포사멸을 유도하는 것으로 판단되어 간암치료제 개발 및 간암치료제와의 병용요법 등 새로운 작용기전의 항암신약개발 소재로서의 가능성이 있음을 제시한다.

• 대한한의학회지
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• 제26권4호
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• pp.12-21
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• 2005

Objectives: In the present study, we investigated whether an aqueous extract of Armeniacae semen induces apoptotic neuronal cell death upon mouse N2a neuroblastoma cells. Methods: 1. Cell viability was determined by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTI) assay. 2. For in situ detection of apoptotic cells, terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) assay, 4,6-diamidino-2-phenylindole (DAPI) staining. 3. The fraction of cells was revealed by flow cytometric analysis used that. 4. For detection of apoptotic DNA cleavage, DNA fragmentation assay was performed. 5. For detection of bax and bcl-2, Western blot analysis was performed. 6. Caspase enzyme activity was measured using caspase-3 assay. Results: From the present results, N2a neuroblastoma cells treated with Armeniacae semen extract exhibited several characteristics of apoptosis. A treatment of Armeniacae semen extract was shown to increase the expression of Bax, a proapoptotic protein, and the treatment decreased the expression of Blc2, an anti-apoptotic protein. In addition, Armeniacae semen extract increased the caspase-3 enzyme activity. Conclusions: The present results show that Armeniacae semen extract induces apoptotic cell death in mouse N2a neuroblastoma cells.

• 대한한의학회지
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• 제26권4호
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• pp.130-142
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• 2005

Objectives: Amygdalin is known to be a natural compound which has antitussive and anticancer activities. Amygdalin is abundant in the seeds of bitter almond and apricots of the Prunus genus, and other rosaceous plants. We investigated whether amygdalin induces apoptosis. Materials and Methods : 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)assay, terminal deoxynuclotidyl transferase (TdT)-mediated dUTP nick end-labeling (TUNEL) assay, 4,6-diamidino-2-phenylindole (DAFI) staining, flow cytometric analysis, DNA fragmentation assay, western blot, and caspase-3 enzyme assay were performed on ME-180 cervical cancer cells treated with amygdalin. Results: Through morphological and biochemical analyses, it was demonstrated that ME-180 cells treated with amygdalin exhibit several apoptotic features. It was shown that amygdalin induces increases in levels of Bax and caspase-3 and a decrease in Bcl-2 expression. Conclusions: These results suggest the possibility that amygdalin exerts an anti-tumor effect on human cervical cancer.

• 대한한방내과학회지
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• 제26권1호
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• pp.60-73
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• 2005

Objectives : The aim of this study was to evaluate the effects of Loranthus parasiticus Merr. on cell cycle and expression of related genes in HepG2 cells. Methods : The MTT assay, cell counting assay, $[^3H]-Thymidine$ incorporation assay, flow cytometric analysis, quantitative RT-PCR and western blot assay were studied. Results : In the water extract of Loranthus parasiticus Merr., inhibition of cell proliferation and DNA synthesis in HepG2 cells was seen. These inhibitory effects were due to inhibition of G l-S transition in cell cycle. After treatment with the extract, expression of cyclin D1(G1 check point related gene) was inhibited particularly in dose-dependent and time-dependent manners. Conclusion : These results suggest that the inhibition of cell cycle progression by Loranthus parasiticus Merr. in HepG2 cell is due to suppression of cyclin D1(G1 check point related gene) mRNA expression and protein synthesis.

• Tuberculosis and Respiratory Diseases
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• 제56권2호
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• pp.187-197
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• 2004

연구배경 : Nitric Oxide(NO)는 매우 다양한 생물학적 조절기능을 수행하는 분자로서 심혈관계, 신경계, 면역기능 등에 관여함은 물론 최근 세포 사에도 직 간접적으로 영향을 미치고 있음이 알려져 있다. NO의 이렇게 복잡한 생물학적 기능 수행은 reactive oxygen species(ROS), metal ions 및 단백질 등과 복잡한 상호작용에 의한 것이며 NO가 나타내는 생물학적 효과는 용량-의존적이며, 세포-특이적이라고 밝혀져 있다. NO는 간세포 및 현관내피세포에서는 아포프토시스를 억제하지만 종양세포 및 신경세포 등에서는 아포프토시스를 유도하는 것으로 보고되고 있다. NO는 여러 호흡기질환의 병태생리에도 관여하는 것으로 알려져 있는바 천식과 같은 염증성 기도 질환에서 호기 NO가 증가되어있는 반면 흡연자나 일차성 폐 고혈압 환자에서는 감소되어 있다고 보고되고 있다. 이러한 배경에서 NO가 폐 상피 세포의 세포 사에 미치는 영향과 신호전달 경로를 밝히기 위하여 본 연구를 시행하였다. 방 법 : 폐 상피 세포로는 A549 세포 주를, NO donor로서는 SNAP (S-nitroso-N-acetyl-penicillamine)과 SNP(sodium nitroprusside)를 사용하였다. 세포 독성 검사는 crystal violet assay를 이용하였고 아포프토시스 assay는 Hoechst 33342와 propium iodide(PI) 이중 염색 후 형광현미경을 이용하여 핵의 형태학적변화를 관찰함으로써 괴사(necrosis)와 감별하였다. 철에 의한 NO 유도성 세포 사 억제 효과를 관찰하기 위하여 RBC와 FeSO4를 이용하였다. NO 유도성세포사의 신호전달 경로에 bcl-2와 p53이 미치는 영향을 평가하기 위하여 bcl-2 과 발현 세포 주 (A549-bcl-2)와 p53 knock out 세포 주 (A549-E6)를 대상으로 세포독성을 비교하였고 p53 활성화는 Western blot을 이용하여 확인하였다. 결 과 : A549 세포 주에서 SNAP과 SNP 모두 농도-의존적 세포독성을 관찰할 수 있었다. 아포프토시스 assay에서 SNAP은 저 농도에서는 아포프토시스를, 고농도에서는 괴사를 유도함을 관찰하였고 SNP는 농도에 상관없이 세포사가 괴사의 형태를 나타냄을 확인하였다. 이는 SNP가 순순한 NO donor가 아니라 cyanide에 의한 세포독성의 결과라고 생각되며 고농도의 SNAP에 의한 괴사 유도는 peroxynitrite 생성에 의한 결과임을 시사한다. SNAP에 의한 세포 사는 RBC와 FeSO4등 철에 의해 억제됨을 확인하였고 bcl-2에 의해서 억제되었으며 p53을 활성화시키고 p53 knock out에 의해 차단되었다. 결 론 : 폐상피세포에서 NO는 저 농도에서는 아포프토시스를 고농도에서는 괴사에 의한 세포 사를 유도하며 철이 중요한 억제제이며 bcl-2 및 p53이 신호전달 경로에 있어서 중요한 역할을 담당하는 것으로 생각된다.

• Biomolecules & Therapeutics
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• 제13권3호
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• pp.190-197
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• 2005

Advanced glycation end products (AGE) have been implicated in the pathogenesis of diabetic complications including nephropathy. However, the role of AGE in the activation of mesangial cells cultured under high glucose has not been elucidated. The effects of aminoguanidine, which prevents formation of AGE and protein cross-linking, on the synthesis of $TGF-{\beta}1$ and fibronectin by rat mesangial cells cultured under high glucose for 2 weeks were examined and compared with the effects of $N^G$-nitro-L-arginine methyl ester (NAME), a selective nitric oxide synthase inhibitor, because aminoguanidine also inhibits the inducible nitric oxide synthase. Culture of mesangial cells in 30 mM (high) glucose for 2 weeks induced 1.5-fold (ELISA) and 1.9-fold (Western blot analysis) increase in AGE in the culture media compared to 5.6 mM (control) glucose. Northern blot analysis revealed 1.5-fold increase in $TGF-{\beta}1$ and 1.7-fold increase in fibronectin mRNA expression in cells cultured under high glucose compared to control glucose. Increases in mRNA expression were followed by increased protein synthesis. Mink lung epithelial cell growth inhibition assay revealed 1.4-fold increase in $TGF-{\beta}1$ protein in high glucose media compared to control. Fibronectin protein also increased 2.1-fold that of control glucose by Western blot analysis. Administration of aminoguanidine suppressed AGE formation in a dose dependent manner and at the same time suppressed $TGF-{\beta}1$ and fibronectin synthesis by mesangial cells cultured in both control and high glucose. In contrast, NAME did not affect high glucose-induced changes. These findings support a role for AGE in high glucose-induced upregulation of $TGF-{\beta}1$ and fibronectin synthesis by mesangial cells.

• 한국미생물·생명공학회지
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• 제45권2호
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• pp.178-183
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• 2017

본 연구는 새로운 기능성 화장품 소재를 개발하기 위해 천연물 재료인 말오줌나무 추출물 활용 가능성 연구하였다. 이 목적을 이루기 위하여, 말오줌나무의 세포독성효과를 MTT assay를 통해 확인한 결과, $500{\mu}g/ml$ 농도에서 100% 이상의 세포 생존율을 나타내었다. 항염증 활성을 효과적으로 확인하기 위하여, LPS로 유도된 대식세포 내 NO 생산을 억제하는 효과를 griess의 방법으로 조사하였다. 그 결과 NO의 생성이 말오줌나무 추출물의 농도 의존적으로 저해되었음을 확인하였다. 말오줌나무 추출물을 LPS로 유도된 RAW 264.7 대식세포에서 전염증성 인자(iNOS, COX-2)들을 생성하여 측정하였다. 그 후, iNOS와 COX-2의 단백질 발현 억제 효과를 측정하기 위해 50, 100, $500{\mu}g/ml$ 농도에서 western blot을 수행하였고, ${\beta}$-actin를 양성대조군으로 사용하였다. iNOS와 COX-2의 mRNA 발현 억제 효과를 측정하기 위해 50, 100, $500{\mu}g/ml$ 농도에서 RT-PCR을 수행하였고, 양성 대조군으로 GAPDH를 사용하였다. 결과적으로, western blot으로 iNOS와 COX-2의 단백질 발현 억제 효과를 측정한결과 $500{\mu}g/ml$ 농도에서 각각 31.2%, 54.7%의 감소 효과를 보였으며, iNOS, COX-2의 mRNA 발현 억제 효과를 RT-PCR로 측정한 결과 $500{\mu}g/ml$ 농도에서 각각 72.2%, 89% 정도로 감소하였다. 이러한 결과들을 통해 말오줌나무 추출물은 항염증 효과를 가진 천연물 소재로 활용 가능할 것으로 생각된다.

• The Korean Journal of Physiology and Pharmacology
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• 제18권2호
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• pp.183-190
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• 2014

This project's aim was to determine the reserpine-induced gastric ulcer preventive effect of polysaccharide of Larimichthys crocea swim bladder (PLCSB) in ICR mice. The anti-gastric ulcer effects of polysaccharide of Larimichthys crocea swim bladder was evaluated in mice model using morphological test, serum levels assay, cytokine levels assay, tissue contents analysis, reverse transcription-polymerase chain reaction (RT-PCR) analysis and western bolt assay. High concentration (50 mg/kg dose) of PLCSB reduced IFN-${\gamma}$ as compared to low concentration (25 mg/kg dose) and control mice. SS and VIP serum levels of PLCSB treated mice were higher than those of control mice, and MOT and SP serum levels were lower than control mice. Gastric ulcer inhibitory index of PLCSB treatment groups mice were much lower than control mice, and the high concentration treated mice were similar to the ranitidine treated mice. The SOD and GSH-Px activities of PLCSB treated mice were higher than control mice, close to normal mice and ranitidine treated mice. PLCSB treated mice also showed the similar contents of NO and MDA to normal group. By RT-PCR and western blot assay, PLCSB significantly induced inflammation in tissues of mice by downregulating NF-${\kappa}B$, iNOS, and COX-2, and upregulating $I{\kappa}B-{\alpha}$. These results suggest that PLCSB showed a good gastric ulcer preventive effect as the gastric ulcer drug of ranitidine. Polysaccharide of Larimichthys crocea swim bladder may be used as a drug material from marine products.

• 대한치과보철학회지
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• 제42권2호
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• pp.175-191
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• 2004

Statement of problem: Platelet-rich plasma(PRP) is well known to be very effective method to stimulate and accelerate the healing of bone and soft tissue. However, there are few reports which deal with the mechanisms of the PRP on the activation of the osteoblasts. Purpose: This study was aimed to investigate the effect of growth factors in PRP on the activity of osteoblasts. Material and method: To evaluate the effect on human, human osteoblast cell line was cultured. PRP was extracted from the blood of a healthy volunteer. Using the recombinant growth factors of PDGF, $TGFT-\beta$, IGF-1, bFGF which are mainly found at bone matrix and their neutralizing antibody, the effect of PRP on the attachment and proliferation of osteoblasts was evaluated. To evaluate the autocrine and paracrine effects, conditioned media(CM) of PRP was made and compared with PRP. By the western blot analysis, the expression of growth factors in PRP, CM was examined. Cell morphology was compared by the light microscope. Results : 1) The effects of CM on osteoblast were similar to the effects of PRP. 2) PRP, CM, recombinant $TGF-\beta$, bFGF, IGF-1 showed significantly higher cellular attachment than control(p<0.05) in the cell attachment assay. In the cell proliferation assay, PRP, CM, recombinant $TGF-\beta$, IGF-1, bFGF, PDGF increased significantly cell proliferation(p<0.01). Among the recombinant growth factors, IGF-1 showed the highest cellular attachment and proliferation. 3) In the western blot assay, bFGF, IGF-1, PDGF weve equally expressed in PRP and CM. 4) The attachment of osteoblast cell decreased significantly after the addition of neutralizing antibody against $TGF-\beta$, IGF-1(p<0.05). In the cell proliferation assay, the addition of neutralizing antibody against $TGF-\beta$, bFGF, PDGF, IGF-1 decreased significantly the cellular proliferation(p<0.05). The amount of decreasing in the cell attachment and proliferation is the highest in at-lGF-1. 5) The cells in control group were flattened and elongated with a few cellular processes in the a light microscope. But, the cells appeared as spherical, plump cells with well developed cellular processes in experimental groups. The cells in PRP and CM had more prominent developed features than recombinant growth factor groups. Conclusions : These findings imply that PRP maximize the cellular activity in early healing period using the synergistic effect, autocrine, paracrine effects of growth factors and increase the rate and degree of bone formation.

• 대한한의학방제학회지
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• 제22권1호
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• pp.151-165
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• 2014

Objectives : The aim of this study was to evaluate the efficacy of Aconiti Lateralis Preparata Radix (AP) and Acanthopanacis Cortex (AT) extracts in bone-derived adipocyte OP9 cell, osteoclast and osteoblast-like MG63 cells. Methods : MTT assay was used to evaluate the cytotoxicity of AP and AT extracts on OP9, osteoclast and MG63 cells. OP9 cells were treated with AP and AT, and the alterations in fat storage in the cells were determined by the Oil red O. To explain effects of RANKL-induced osteoclast differentiation in bone marrow macrophages, we performed the TRAP staining. The protein level of CAAAT/enhancer binding protein alpha ($C/EBP{\alpha}$) and peroxisome proliferator-activated receptor ${\gamma}$ ($PPAR{\gamma}$) as a adipocyte differentiation marker, and adiponectin was examined using western blot in differentiated OP9 cells. Effects of related genes were confirmed by luciferase assay using reporter assay. Results : AP and AT was not toxic on OP9 and MG63 cells, but AT was a little cytotoxic to osteoclast at the dose of $100{\mu}g/m{\ell}$. They could inhibit differentiation of OP9 cells and osteoclast with results of oil red O staining and TRAP staining. By western blot, AP and AT decreased the expression of $PPAR{\gamma}$ and $C/EBP{\alpha}$ which is the key transcription factor in adipogenesis and adiponectin secretion. AT also inhibited the BMP-4 activity in luciferase assay. AP also inhibited BMP-4 and Wnt3a activity, stimulated ER-${\beta}$ activity but inhibited androgen receptor activity. Conclusions : These results show AP and AT can be useful in osteoporosis and obesity via inhibition of osteoclast and adipocyte differentiation.