• 생명과학회지
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• 제22권8호
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• pp.1052-1056
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• 2012

미역(Undaria pinnatifada)은 낮은 칼로리 및 요오드의 원료로써 천연체중조절식품으로 알려져 있다. 미역이 체중조절식품으로 알려져 있음에도 불구하고, 지방세포 분화 및 지방축적에 관한 저해 기작은 연구가 미비하다. 본 연구에서는 3T3-L1에서 지방세포로 분화가 일어나는 단계에서 미역에탄올추출물의 효과 및 기작을 확인하였다. 미역에탄올추출물의 독성과 지방축적저해효과는 MTT assay, Oil red O staining, RT-PCR과 western blot으로 분석하였다. 미역에탄올추출물은 50 ${\mu}g/ml$의 농도에서 독성을 띄지 않았다. 3T3-L1의 분화 및 지방세포에서 triglyceride축적과정동안 50 ${\mu}g/ml$의 미역에탄올추출물을 처리하였으며, 미역에탄올추출물은 지방세포에서 triglyceride의 축적을 40% 감소시켰다. 지방세포 특이적 단백질인 Peroxisome proliferator activated receptor ${\gamma}$ ($PPAR{\gamma}$), leptin과 Hormone sensitive lipase (HSL)의 발현은 RT-PCR과 western blot으로 확인하였다. $PPAR{\gamma}$의 과발현은 지방세포의 분화를 촉진시킨다. 또한 지방세포 크기의 증가와 세포 내 triglyceride의 함량에 따라 leptin은 세포 외로 분비된다. 그러므로 $PPAR{\gamma}$와 leptin은 비만의 지표로 사용된다. 첨가한 미역에탄올추출물의 농도가 높아질수록 $PPAR{\gamma}$와 leptin의 발현이 억제되었다. 이상의 결과를 통하여, 미역의 에탄올 추출물은 지방전구세포의 분화를 억제시키며, 지방세포 내 triglyceride의 축적을 저해하는 것으로 판단된다.

• 미생물학회지
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• 제36권2호
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• pp.142-149
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• 2000

국내에서 유행하는 Plasmodium vivax의 유전자를 조작하여 만든 재조항 Circumsporozrozite(CS) 단백질 (22)을 이용하여 말라리아에 대한 혈청학적 검사의 유용성을 평가하였다. 최초발병일로부터 진단까지 걸린 기간을 기준으로 환자들의 면역반응을 Western blot으로 알아본 결과, 15일 이내에 진단받은 환자들은 43.8%(14/32)가 양성반응을 보였고, 16일 이상 경과한 환자들은 94.4%(17/18)가 양성반응을 보여 전체적으로는 62%(31/50)의 양성률을 보였다. Blood stage 항원에 대한 항체를 갖고 있음에도 불구하고 재조합 CS단백질 항원에 대해 음성인 환자들이 22.6%(7/31)였다. 비유행지 주민(경북 예천군)들은 10.7%(3/28),유행시 주민(인천시 강화군)들은 27.6%(13/47)의 재조합 CS 단백질에 대한 항체 양성을 보였다. 재조합 CS 단백질 항원의 역학조사시 유용성을 알아보기 위하여 말라리아 유행지인 경기도 파주시 조산리, 마정리, 항양리, 뇌조리 거주 주민 422명의 전혈을 채취하여 혈액도말법과 중합효소연쇄 반응법으로 항원검사를 실시하였으며 blood stage 항원을 이용한 간접면역형광법과 재조합 CS 단백질 항원을 이용한 효소면역측정법으로 말라리아 항체보유 여부를 조사하였다. 혈액도말법에서는 422명 모두 음성이었으나, 중합효소연쇄반응법에서는 2명(0.47%)이 양성이었다. 간접면역형광법에서는 42명(9.95%), 재조합 CS 단백질 항원을 이용한 효소변역측정법에서는 71명(16.82%)이 양성으로 나타났다. 두 검사에서 모두 항체 양성을 보인 사람은 8명(11.27%)에 불과하였다.

• 생명과학회지
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• 제23권12호
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• pp.1428-1435
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• 2013

본 연구의 목적은 melanocyte (B16F1)에서 quercetin과 kaempferol을 포함하는 미나리 에탄올 추출물(OJE)의 멜라닌 합성효과에 미치는 영향을 조사한 것이다. OJE가 세포수준에서 멜라닌 합성을 억제하는지를 조사하기 위하여 여러 농도의 OJE 존재 하에서 B16F1세포를 배양하였다. 현재 연구에서 DPPH radical scavenging, reducing power, lipid peroxidation 및 DNA oxidation에 미치는 항산화 효과는 cell free system에서 평가되었다. 더욱이 멜라닌 생성에 대한 OJE 효과는 dopaquinone (DOPA) assay 및 tyrosinase 활성으로 결정되었다. 뿐만 아니라 superoxide dismutase (SOD)-1, -2, glutathione reductase (GSH)와 같은 항산화 효소 및 tyrosinase의 단백질발현이 western blot 분석을 이용하여 평가되었다. 본 연구에서 OJE는 지질과산화 억제효과를 나타내었고 fenton 반응에 의해서 생성되는 hydroxyl radical에 의하여 유발되는 DNA 산화를 보호하였다. OJE는 50 ${\mu}g/ml$ 이상에서 멜라닌 합성을 증가시켰고 tyrosinase 활성도 50 ${\mu}g/ml$에서 검출되었다. Western blot 분석에서는 OJE가 농도에 비례하여 tyrosinase SOD-1, -2 및 GSH의 발현 수준을 증가시켰다. 이러한 발견들은 항산화 효과를 가진 OJE가 melanocyte에서 tyrosinase 활성과 melanin 생성을 조절할 수 있어 피부를 산화스트레스로부터 보호할 수 있다는 것을 암시하고 있다.

• Journal of Acupuncture Research
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• 제19권4호
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• pp.74-88
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• 2002

Objectives : This study is aimed to investigate the effects of bee venom and melittin on cell death in synovial cell line. Methods : It was evaluated by using MTT assay, morphologic method, DNA fragmenation, NO generation, flow cytometry, immunocytochemistry analysis, RT-PCR, Western blot. Results : The obtained results are summarized as follows: 1. The MTT assay demonstrated that synovial cell viability was significantly inhibitted dose-dependently by treatment with bee venom and melittin in comparison with control. 2. The morphologic study demonstrated that synovial cell showed apoptosis after treatment with bee venom and melittin for 6 hours using microscope. 3. In case of NO generation bee venom group and melittin group showed significant inhibition in comparison with control. 4. The Flow cytometry demonstrated that apoptosis of synovial cell treated with bee venom and melittin was related with stop of cell cycle in stage of $G_0/G_1$. 5. DNA fragmenation demonstrated that synovial cell treated with bee venom and melittin showed DNA ladder below l Kbp. 6. Immunocytochemistry assay demonstrated that COX-II and PLA2 were strongly down-regulated by treatment with bee venom and melittin whereas iNOS was almostly not expressed by bee venom treatment and slightly expressed by melittin treatment. 7. RT-PCR analysis demonstrated that iNOS were strongly down-regulated by treatment with bee venom and melittin whereas COX-II was almostly not expressed by bee venom treatment and slightly expressed by melittin treatment. 8. Western blot demonstrated that iNOS were strongly down-regulated by treatment with $15{\mu}g/ml$ bee venom whereas COX-II was strongly down-regulated from $5{\mu}g/ml$ bee venom. Conclusions : These results suggest that bee venom and melittin have significant effect on cell death in synovial cell line and further study is needed in vivo.

• 대한한방내과학회지
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• 제31권2호
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• pp.331-345
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• 2010

Objectives : This study was designed to investigate the effects of Saenggantanggami-bang (SG) on nonalcoholic fatty liver disease. Methods : HepG2 cells were used in an in vitro model. HepG2 cells were divided into three groups. The Normal group was incubated with no fatty acid. The Control group was incubated with 1mM palmitic acid to introduce fat overloading. The PA-SG group was incubated with 1mM palmitic acid and various concentrations of Saenggantanggami-bang (SG). Cell viability and cytotoxicity were analyzed by MTT assay and LDH assay. Intracellular triglyceride (TG) levels, reactive oxygen species (ROS) levels, ATP amount, and GST activity were measured. Cell death pattern and protective effect of SG on cell death were studied by DNA fragmentation and caspase-3 intensity (western blot). Results : Compared with the Control group, cell viability of the PA-SG group significantly increased (P<0.01), cytotoxicity of the PA-SG group decreased (P<0.01), and intracellular TG levels and ROS levels of the PA-SG group decreased (P<0.05). In DNA fragmentation assay, necrotic pattern was observed and DNA fragment decreased in the PA-SG group. In western blot, apoptotic pattern was observed, caspase-3 intensity of the PA-SG group was reduced significantly, but there were no significant differences in intracellular ATP amount and GST activity between the control group and the PA-SG group. Conclusion : The results suggest that Saenggantanggami-bang can be a potential candidate for the clinical treatment of nonalcoholic fatty liver disease.

• Journal of Ginseng Research
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• 제39권2호
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• pp.125-134
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• 2015

Background: Black ginseng (Ginseng Radix nigra, BG) refers to the ginseng steamed for nine times and fine roots (hairy roots) of that is called fine black ginseng (FBG). It is known that the content of saponin of FBG is higher than that of BG. Therefore, in this study, we examined antitumor effects against MCF-7 breast cancer cells to target the FBG extract and its main component, ginsenoside Rg5 (Rg5). Methods: Action mechanism was determined by MTT assay, cell cycle assay and western blot analysis. Results: The results from MTT assay showed that MCF-7 cell proliferation was inhibited by Rg5 treatment for 24, 48 and 72 h in a dose-dependent manner. Rg5 at different concentrations (0, 25, 50 and $100{\mu}M$), induced cell cycle arrest in G0/G1 phase through regulation of cell cycle-related proteins in MCF-7 cells. As shown in the results from western blot analysis, Rg5 increased expression of p53, $p21^{WAF1/CIP1}$ and $p15^{INK4B}$ and decreased expression of Cyclin D1, Cyclin E2 and CDK4. Expression of apoptosiserelated proteins including Bax, PARP and Cytochrome c was also regulated by Rg5. These results indicate that Rg5 stimulated cell apoptosis and cell cycle arrest at G0/G1 phase via regulation of cell cycle-associated proteins in MCF-7 cells. Conclusion: Rg5 promotes breast cancer cell apoptosis in a multi-path manner with higher potency compared to 20(S)-ginsenoside Rg3 (Rg3) in MCF-7 (HER2/ER+) and MDA-MB-453 (HER2+/ER) human breast cancer cell lines, and this suggests that Rg5 might be an effective natural new material in improving breast cancer.

• Journal of Acupuncture Research
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• 제19권1호
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• pp.189-202
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• 2002

Objective : This study is designed to investigate the effects of bee venom and melittin on cell death in neuroblastoma cell line after pretreatment with NG(nerve growth inhibitory substance) Methods : It was evaluated by using MTT assay, morphological method, DNA fragmenation, flow cytometry, immunocytochemistry analysis, RT-PCR and Western blot. Results : The MTT assay demonstrated that neuroblastoma cell viability was significantly inhibited dose-dependently by treatment with bee venom and melittin after pretreatment with NG in comparison awith control. The morphological study and fow cytometry demonstrated that neuroblastoma cell showed apoptosis. DNA fragmenation showed DNA ladder below 1 Kbp. Immunocytochemistry assay demonstrated that Fos and MAPK were down-regulated. RT-PCR analysis demonstrated that Fos and MAPK was down-regulated. Western blot demonstrated that Fos and MAPK were down-regulated from $1{\mu}g/ml$ bee venom in neuroblastoma cell pretreated with NG. Conclusion : These result suggests that bee venom and melittin after NG treatment have significant anti-cancer effect and further study is needed in vivo.

• Ocean and Polar Research
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• 제41권2호
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• pp.79-88
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• 2019

In this study, the inhibitory effect of Atriplex gmelinii C. A. Mey. against the activity of MMP-2 and MMP-9 secreted from phorbol-12-myristate-13-acetate (PMA)-stimulated HT-1080 cells was evaluated by gelatin zymography and enzyme-linked immunosorbent assay (ELISA), reverse transcription polymerase-chain reaction (RT-PCR), and Western blot assay. Specimens of the halophyte A. gmelinii were extracted twice for 24 hr with methylene chloride ($CH_2Cl_2$), and then twice with methanol (MeOH), in turn. Each extract significantly inhibited the enzymatic activities in gelatin zymography and MMP ELISA kit, and expression of MMP-2 and 9 in mRNA and protein levels. Two crude extracts were combined and then the combined crude extracts were fractionated into n-hexane, 85% aqueous methanol (85% aq.MeOH), n-butanol (n-BuOH), and water ($H_2O$) fractions, according to solvent polarity. Among solvent-partitioned fractions, the 85% aq.MeOH fraction showed the strongest inhibitory effect against MMP-2 and -9 in gelatin zymography and MMP ELISA kit. In RT-PCR, all solvent-partitioned fractions significantly suppressed mRNA expression of MMP-2 and -9. On the other hand, in Western blot assay, all solvent-partitioned fractions except $H_2O$ significantly reduced expression levels of protein. HT 1080 cell migration was most significantly inhibited by the n-BuOH fraction followed by the 85% aq.MeOH and $H_2O$ fractions. These results suggest that A. gmelinii could be used as a potential source to inhibit tumor cell metastasis.

• Ocean and Polar Research
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• 제40권4호
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• pp.249-257
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• 2018

Matrix metalloproteinases (MMPs) are associated with the invasion and metastasis of malignant tumors composed of cancer cells in an increased state of expression. This study evaluates the inhibitory effect of Carex pumila on MMP-2 and MMP-9 activity in phorbol-12-myristate-13-acetate (PMA)-stimulated HT-1080 human fibrosarcoma cells using gelatin zymography, MMPs enzyme-linked immunosorbent assay (ELISA), reverse transcription-polymerase chain reaction (RT-PCR) and Western blot assay. C. pumila was extracted twice with dichloromethane ($CH_2Cl_2$) and methanol (MeOH). Treatment with $CH_2Cl_2$ extract and MeOH extract in PMA-stimulated HT-1080 cells effectively reduced the production of MMP-2 and 9. Also, the combined crude extracts ($CH_2Cl_2$ and MeOH) significantly inhibited the enzymatic activities and the expression of MMP-2 and MMP-9 in mRNA and protein levels. The combined crude extracts were partitioned between $CH_2Cl_2$ and water. The organic layer was further fractionated with n-hexane, 85% aqueous methanol (85% aq.MeOH) and the aqueous layer was separated into n-butanol and water, successively. Of the fractions, 85% aq.MeOH fraction showed the highest inhibitory activity of MMP-2 and MMP-9 in gelatin zymography and MMP ELISA kit. Furthermore, 85% aq.MeOH fraction most significantly suppressed cell migration. In RT-PCR and Western blot assay, n-butanol and 85% aq.MeOH fractions exerted the greatest inhibition on mRNA and protein expression of MMP-2 and MMP-9, respectively. As a result, C. pumila can be used as a good anti-invasive agent source.

• Journal of Acupuncture Research
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• 제36권2호
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• pp.80-87
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• 2019

Background: This study investigated the anti-inflammatory effects of bee venom (BV) through the inhibition of nuclear factor kappa beta ($NF-{\kappa}B$) expression in macrophages and keratinocytes. Methods: Cell viability assays were performed to investigate the cytotoxicity of BV in activated macrophages [lipopolysaccharide (LPS)] and keratinocytes [interferon-gamma/tumor necrosis factor-alpha ($IFN-{\gamma}/TNF-{\alpha}$)]. A luciferase assay was performed to investigate the cellular expression of $NF-{\kappa}B$ in relation to BV dose. The expression of $NF-{\kappa}B$ inhibitors ($p-I{\kappa}B{\alpha}$, $I{\kappa}B{\alpha}$, and p50 and p65) were determined by Western Blot analysis, and the electromobility shift assay. A nitrite quantification assay was performed to investigate the effect of BV, and $NF-{\kappa}B$ inhibitor on nitric oxide (NO) production in macrophages. In addition, Western Blot analysis was performed to investigate the effect of BV on the expression of mitogen-activated protein kinases (MAPK) in activated macrophages and keratinocytes. Results: BV was not cytotoxic to activated macrophages and keratinocytes. Transcriptional activity of $NF-{\kappa}B$, and p50, p65, and $p-I{\kappa}B{\alpha}$ expression was reduced by treatment with BV in activated macrophages and keratinocytes. Treatment with BV and an $NF-{\kappa}B$ inhibitor, reduced the production of NO by activated macrophages, and also reduced $NF-{\kappa}B$ transcriptional activity in activated keratinocytes (compared with either BV, or $NF-{\kappa}B$ inhibitor treatment). Furthermore, BV decreased p38, p-p38, JNK, and p-JNK expression in LPS-activated macrophages and $IFN-{\gamma}/TNF-{\alpha}$-activated keratinocytes. Conclusion: BV blocked the signaling pathway of $NF-{\kappa}B$, which plays an important role in the inflammatory response in macrophages and keratinocytes. These findings provided the possibility of BV in the treatment of atopic dermatitis.