Lysophosphatidylcholine의 혈관평활근세포에 대한 세포 독성

강영희 ( Young Hee Kang ),이영주 ( Young Joo Lee ),이동윤 ( Dong Yun Lee ),유미라 ( Mee Ra Rhyu ),최두석 ( Doo Seok Choi ),윤병구 ( Byung Koo Yoon ) 대한폐경학회 2012 대한폐경학회지 Vol.18 No.3

Objectives: To investigate the cytotoxic effects of lysophosphatidylcholine (lysoPC), an active component of oxidized low-density lipoproteins (LDL), on vascular smooth muscle cells (VSMCs). Methods: VSMCs were derived from rat aorta. Cell death was assessed by 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay, lactic dehydrogenase (LDH) assay, and DNA fragmentation assay. Apoptosis was quantified by propidium iodide staining and fluorescent activated cell sorting (FACS) analysis, and intracellular free radical production was determined using 2``, 7``-dichlorofluorescin diacetate (DCF-DA). In addition, the changes in caspases, bcl-2 and bax proteins were evaluated by western blot analysis. Results: LysoPC over 25 μM induced more than 50% of the cell death at 10 hours on MTT assay with no change in the level of LDH. The DNA ladder pattern showed that cell death induced by lysoPC was caused by apoptosis, which was associated with increased free radical production. Vitamin E, a potent antioxidant and caffeic acid phenylethyl ester (CAPE), an inhibitor of nuclear factor-kappaB (NF-kB), blocked apoptosis. The casepase-3 precursor decreased and the active form of caspase-8 increased. Total bcl-2 and bax proteins did not change with lysoPC treatment, but translocation of bax from cytosole to the mitochondria membrane was observed. Conclusion: LysoPC induces apoptosis in VSMCs via an oxidant mechanism, dependent on NF-kB. (J Korean Soc Menopause 2012;18:139-146)

혈관 평활근세포 증식에 대한 국내 자생종 승마의 효과

강영희 ( Young Hee Kang ),이동윤 ( Dong Yun Lee ),최두석 ( Doo Seok Choi ),윤병구 ( Byung Koo Yoon ),이영주 ( Young Joo Lee ),류미라 ( Mee Ra Rhyu ) 대한폐경학회 2012 대한폐경학회지 Vol.18 No.2

연구목적: 혈관 평활근세포 증식에 대한 국내 자생종 승마의 효과를 밝히기 위해 시행되었다. 연구재료 및 방법: 3개월된 암컷 Sprague-Dawley 쥐의 대동맥으로부터 혈관 평활근세포를 얻었다. 눈빛승마, 왜승마, 세잎승마, 촛대승마 등 4종의 자생 승마를 80% ethanol, absolute methanol 그리고 40% 2-propanol을 사용하여 추출하였다. 10% fetal bovine serum (FBS)에서 배양한 혈관 평활근세포를 승마 추출물 (0.3mg/mL)로 24시간 동안 처리한 후 bromodeoxyuridine incorporation assay를 사용하여 세포증식을 조사하였다. dichlorofluorecin diacetate을 첨가한 후 flow cytometry를 이용하여 세포내 ROS 생성을 정량화하였다. 결과: 추출법이 다른 국내 자생종 승마 추출물을 처리한 결과, 혈관 평활근세포의 증식이 유의하게 23-30% 억제되었다. 또한 강력한 항산화제인 비타민 E는 10% FBS로 유도된 세포증식을 감소시켰다. 승마 추출물의 작용기전을 밝히기 위한 추가 연구 상 10% FBS에 의한 ROS 생성이 승마에 의해 감소되었다. 세포증식이나 ROS 생성에 대한 효과는 추출 방법과 승마 종에 따라 차이가 없었다. 결론: 국내 자생종 승마는 산화 스트레스 감소를 통하여 혈관 평활근세포 증식을 억제한다. 향후 다양한 심혈관계 영향에 대한 추가 연구가 필요할 것으로 사료된다. Objectives: To examine the effect of extracts of Korean native Cimicifuge species on cell proliferation in vascular smooth muscle cells (VSMC). Methods: VSMC were isolated from rat aorta. Cell proliferation was assessed by measure of bromodeoxyuridine incorporation into the cells. Differences in Reactive oxygen species (ROS) levels were examined after exposure to the extracts of Korean native Cimicifuge species using the detection reagents dichlorofluorecin diacetate. The rhizomes/roots were air-dried and milled with a commercial food mixer. Milled rhizomes/roots of each Cimicifuga species were separately extracted by 80% ethanol, absolute methanol, and 40% 2-propanol using homogenizer and evaporated under reduced pressure at low temperatures. Effects of extracts dissolved in phosphate-buffered saline (0.3 mg/mL) were examined. Results: Ethanolic, methanolic or propanolic extracts of 4 Korean native Cimicifuge species (Cimicifuga [C] davurica, C. japonica, C. heracleifolia var. bifida Nakai, C. simplex) were screened. The addition of extracts of each Korean native Cimicifuge species to cells in the presence of 10% fetal bovine serum (FBS) potently inhibited cell proliferation. Significant decrease of 23%-30% was observed. Vitamin E, a potent antioxidant, inhibited 10% FBS-stimulated cell proliferation of VSMC. We also demonstrated that extracts of each Korean native Cimicifuge species decreased intracellular ROS generation induced with 10% FBS. The effect of Korean native Cimicifuge species was not species-specific and solvent-specific. Conclusion: TExtracts of Korean native Cimicifuge species inhibit VSMC proliferation via inhibition of intracellular ROS. These findings suggest that Cimicifuge species used for reducing menopause symptoms might be cardioprotective in women.

생체생장에 관한 세포생물학적 연구 - Polyamine 과 auxin 이 β - 1 , 4 - endoglucanase 활성에 미치는 영향

조영동(Young Dong Cho),강정훈(Jung Hoon Kang),이영미(Young Mee Lee),이순희(Sun Hi Lee),이준승(June Seung Lee),강영희(Young Hee Kang) 한국식물학회 1988 Journal of Plant Biology Vol.31 No.3

Spermidine, spermine and IAA promoted an increase in β-1,4-endoglucanase activity in hypocotyls of Clycine max. The optimal concentration for the increase of the enzyme activity was 10^-6 M for spermidine, 10^-8 M for spermine and 10^-6 M for IAA. However, IAA had innocuous effect on arginine decarboxylase and ornithine decarboxylase activities, and the content of polyamine. Such cumulative results suggest that the increase in β-1,4-endoglucanase activity by IAA is not attributed by the effect on the biosynthesis of polyamine by IAA but spermidine, spermine and IAA induce cell wall loosening and therefore extension growth of cells.

생체발생 및 분화기구의 세포생물학적 연구 8 - 2. 옥수수 배젓에서 Peptide 가 cAMP Level 에 미치는 영향

조영동(Young Dong Cho),강영희(Young Hee Kang),이순희(Sun Hi Lee),김성기(Seong Ki Kim) 한국식물학회 1984 Journal of Plant Biology Vol.27 No.1

Activities of corn endospern adenylate cyclase and phosphodiesteraes were found right after germination, and phosphodiesterase activity was shown to increase steadily. Protease activity was also found. Corn peptide fraction purified by using Sephadex G-25 column was shown to enhance corn phosphodiesterase activity but inhibit bovine phosphodiesterase activity. And the fraction inhibits corn adenylate cyclase activity. Trypsintreated peptide fraction was shown to enhance phosphodiesterase activity 80% compared to that of native peptide fraction. However, in case of DNase phosphodiesteraes was shown to be innocuous. According to cumulative results, it is more likely that peptide fraction produced by protease inhibits adenylate cyclaes activity and enhance phosphodiesterase, decreasing cAMP level.

생체 생장에 관한 세포 생물학적 연구 - 당근 세포의 D - glucose - 6 - phosphate cyclohydrolase 활성에 미치는 polyamine 의 영향 -

조영동(Young Dong Cho),이순희(Sun Hi Lee),강영희(Young Hee Kang),김명원(Myeong Won Kim),김성호(Seong Ho Kim),진언선(Eon Seon Jin) 한국식물학회 1986 Journal of Plant Biology Vol.29 No.4

Effects of putrescine, spermidine and spermine on the activity of D-glucose-6-phosphate cyclohydrolase in the Daucus carota L. protoplast cultured for 4 days and effects of polyamines on the incorporation of D-[u^-14C]-glucose treated to protoplasts in culturemedium were investigated. The activity of D-glucose-6-phosphate cyclohydrolase was increased by polyamines and among them spermine was the most effective. Polyamines increased protein synthesis and this due to the increasing effect of the polyamines on the synthesis of glycoprotein which is one of cell wall components. The synthesis of cell polysaccharides, such as pectic substances, hemicelluloses and celluloses was increased by polyamines, which stimulated synthesis of pectin substances and hemicellulose more greatly than that of cellulose, and spermidine was the most effective. In the light of the above results it seems that the polyamines increase cell wall regeneration by the stimulation of enzyme activities which synthesize cell wall components.

생체발생 및 분화기구의 (分化機構) 세포생물학적 연구 8 - 3. Polyamine 의 옥수수 배의 (胚) Glucose - 6 - Phosphate Dehydrogenase , 6 - Phosphogluconate Dehydrogenase 및 Protein Kinase 활성에 미치는 영향

조영동(Young Dong Cho),강영희(Young Hee Kang),이순희(Sun Hi Lee),조명제(Myung Je Cho) 한국식물학회 1984 Journal of Plant Biology Vol.27 No.2

Palmitoyl CoA was found to inhibit corn embryo axis glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase, which were also inhibited by polyamines. However, reversal of inhibition of both enzymes by palmitoyl CoA was made by spermine. Activity of corn embryo axis protein kinase was found to increase steadily after germination. Activation and inhibition of protein kinase were made by MgCl_2 and all polyamines, respectively. Such results suggest that fatty acid biosynthesis and lypolysis could be regulated to some extent by polyamines in corn embryo axis.

Cell Biological Studies on the Mechanism of Development and Differentitation VI Study on Polyamines and Related Enzymes during Organ Development

조영동,강영희,조명제,김성기,이순희,Cho, Young-Dong,Kang, Young-Hee,Cho, Myung-Je,Kim, Seong-Ki,Lee, Sun-Hi 생화학분자생물학회 1983 한국생화학회지 Vol.16 No.2

1) Corn embryo가 성장함에 따라 putrescine 함량은 계속 증가하나 spermidine과 spermine의 함량은 점점감소하여 일정수준에 도달한다. 2) Corn에서는 L-agmatine으로 부터 putrescine 합성과정에는 separate enzymes가 관여 한다. 3) Corn endogenous putrescine level의 증가와 embryo protein 함량의 증가는 병행한다. 4) Exogenous polyamine 중 putrescine이 가장 Corn exlpant의 성장을 촉진시키고 spermidine와 spermine은 약간씩 촉진시킨다. 5) 발아 후 급격 한 corn에 서 c-AMP 함량의 감소는 주로 phosphodiesterase activity에 기인되는 것 같다. 6) Corn endosperm peptide fraction 이 phosphodiesterase activity를 현저히 높인다. 7) Corn embryo glucose-6-phosphate dehydrogenase는 lipid 합성에 크게 기여하지 않는 것 같다. 1) The amount of putrescine increases continuously but that of spermidine and spermine decreases steadily, and reaches plateau as corn embryo grows. 2) Separate enzymes are involved in formation of putrescine from L-agmating. in corn 3) Increase in level of endogenous putrescine in corn parallels with that of protein. 4) Putrescine of exogenous polyamines stimulate growth, of corn explant, and spermidine and spermine stimulate less than putrescine. 5) After germination, drastic decrease in amount of c-AMP in corn may mainly be due to phosphodiesterase activity. 6) Corn endosperm peptide fraction enhances phosphodiesterse activity apparantly. 7) Corn embryo glucose-6-phosphate dehydrogenase seems to be not essential for lipid biosynthesis.

고혈당으로 유도된 신장 mesangial cell에서 nobiletin의 matrix accumulation과 TGF-β1-Src-caveolin-1 signaling에 의한 사구체 경화증 억제효과

김동연(Dong Yeon Kim),강영희(Young-Hee Kang),강민경(Min-Kyung Kang) 한국영양학회 2020 Journal of Nutrition and Health Vol.53 No.1

본 연구에서는 고혈당으로 인해 유발되는 당뇨병성 신장병증의 대표적인 증상인 사구체 경화증을 완화시키는 nobiletin의 효능에 대해 알아보고자 하였다. 신장 세포인 HRMC를 이용하여 고혈당에서의 세포외 기질 축적 단백질의 발현과 경화에 관여하는 신호 전달 억제 효능을 확인한 결과 nobiletin은 고혈당의 자극에 의해 증가하는 섬유화 단백질인 collagen IV, fibronectin 그리고 CTGF의 발현을 억제하였으며, 여기에 관여하는 TGF-β1-Src-caveolin-1 신호 전달 경로를 통해 사구체 경화증을 억제하는 것을 확인하였다. 따라서 nobiletin은 고혈당으로 유도된 당뇨병성 신장병증에 있어 사구체 경화증을 예방하는 기능성 성분으로서의 활용 가능성을 확인하였다. Purpose: Diabetic nephropathy is one of the most important diabetic complications prompted by chronic hyperglycemia, characterized by glomerulosclerosis, tubular fibrosis, and it eventually causes kidney failure. Nobiletin is a polymethoxyflavone present in tangerine and other citrus peels, and has anti-cancer and anti-inflammatory effects. This study investigated the effects of nobiletin on glomerular fibrosis through inhibition of the transforming growth factor (TGF)-β1-Src-caveolin-1 pathway. Methods: Human renal mesangial cells (HRMC) were incubated in media containing 33 mM glucose with or without 1–20 uM nobiletin for 3 day. The cellular expression levels of fibrogenic collagen IV, fibronectin, connective tissue growth factor (CTGF), TGF-β1, Src and caveolin-1 were all examined. In addition, TGF-β1, Src and caveolin-1 proteins were screened to reveal the relationship among TGF-β1-Src-caveolin-1 signaling in glomerular fibrosis. Results: High glucose promoted the production of collagen IV, fibronectin and CTGF in HRMC, which was inhibited in a dose dependent manner by 1–20 uM nobiletin. The Western blot data showed that high glucose elevated the expression of TGF-β1, Src, caveolin-1 and Rho GTPase. When nobiletin was treated to the HRMC exposed to high glucose, the expression of TGF-β1-Src-caveolin-1 was dampened. Finally, TGF-β1-Src-caveolin-1 signaling pathway was activated in high glucose-exposed HRMC, and such activation was encumbered by nobiletin. Conclusion: These result demonstrated that nobiletin blunted high glucose-induced extracellular matrix accumulation via inhibition of the TGF-β1-Src-caveolin-1 related intracellular signaling pathway. Nobiletin may be a potent renoprotective agent to counteract diabetes-associated glomerular fibrosis that leads to kidney failure.

생체발생 및 분화기구의 세포학적 연구 ( Ⅵ ) 기관발달에 따른 Polyamine 및 관련된 효소에 관한 연구

조영동,강영희,조명제,김성기,이순희 ( Young Dong Cho,Young Hee Kang,Myung Je Cho,Seong Ki Kim,Sun Hi Lee ) 생화학분자생물학회 1983 BMB Reports Vol.16 No.2

1) The amount of putrescine increases continuously but that of spermidine and spermine decreases steadily, and reaches plateau as corn embryo grows. 2) Separate enzymes are involved in formation of putrescine from L-agmating. in corn 3) Increase in level of endogenous putrescine in corn parallels with that of protein. 4) Putrescine of exogenous polyamines stimulate growth, of corn explant, and spermidine and spermine stimulate less than putrescine. 5) After germination, drastic decrease in amount of c-AMP in corn may mainly be due to phosphodiesterase activity. 6) Corn endosperm peptide fraction enhances phosphodiesterse activity apparantly. 7) Corn embryo glucose-6-phosphate dehydrogenase seems to be not essential for lipid biosynthesis.

생체생장에 관한 세포생물학적 연구 - 당근 뿌리의 원형질체에서 polyamine 과 Ca2+ 이 β - glucan synthetase 활성도에 미치는 영향

이순희(Sun Hi Lee),강영희(Young Hee Kang),표병식(Byoung Sik Pyo),조영동(Young Dong Cho),김명원(Myeong Won Kim),이준승(June Seung Lee) 한국식물학회 1987 Journal of Plant Biology Vol.30 No.3

The effect of polyamine, Ca^2+ and calmodulin on GS(β-glucan synthetase) activity was studied in Daucus carota root. The Ca^2+ is shown to have no effect on the GS activity wherease the GS Ⅱ activity is increase in response to increase in concentration of the Ca^2+. When the protoplasts are cultured, for 4 days, the GS Ⅱ activity increases as a tunction of time and reaches a maximum after 3 dyays at a time when the network of cellulose microfibrils is known to be synthesized. The effect of the Ca^2+ and 1 mM spermine on the GS Ⅱ activity turns out to be synergistic, especially more synergistic at lower concentration of the Ca^2+. The GS Ⅱ activity seems to be enhanced by the Ca^2+. The GS Ⅱ activity in the protoplast treated by the calcium channel blocker, verapamil, turns out to be lower than that of the control. Cumulative results suggest that the Ca^2+ stimulates the cell wall regeneration via enhancement of the GS Ⅱ activity responsible for synthesizing the cell wall component throught synergistic effect with spermine.