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dc.contributor.advisor고광석-
dc.contributor.author이서연-
dc.creator이서연-
dc.date.accessioned2016-08-26T04:08:10Z-
dc.date.available2016-08-26T04:08:10Z-
dc.date.issued2015-
dc.identifier.otherOAK-000000116301-
dc.identifier.urihttps://dspace.ewha.ac.kr/handle/2015.oak/213021-
dc.identifier.urihttp://dcollection.ewha.ac.kr/jsp/common/DcLoOrgPer.jsp?sItemId=000000116301-
dc.description.abstractSeveral mechanisms for the pathogenesis of many liver diseases have been proposed such as oxidative stress, endotoxins, cytokines and compromised immune system. Also, altered methionine metabolism has been shown in the patients with liver disease. In this regard, it is important to maintain normal methionine metabolism in liver diseases. Therefore, we focused on S-adenosylmethionione (SAMe), taurine and betaine which participate in the methionine metabolism pathways. The aims of this study were to examine the effects of SAMe and its combinations with taurine and/or betaine. Because SAMe is a key metabolite in methionine metabolism and a precursor of glutathione (GSH), the combinations included SAMe. First, to examine the effects of SAMe, taurine or betaine treatment on ethanol-induced liver injury, mice were administered SAMe, taurine or betaine pretreatment followed by ethanol treatment and were sacrificed 18 hr afterward. We found that SAMe, taurine or betaine treated mice attenuated the fall in hepatic GSH levels compared to mice treated with ethanol alone. Especially, SAMe treatment was shown to have effect on GSH level by upregulating glutamate-cysteine ligase catalytic subunit (GCLC), which is rate limiting enzyme subunit for GSH synthesis. We evaluated the effects of SAMe and its combinations with taurine and/or betaine on ethanol-treated AML12 cells. SAMe pretreatment and its combinations were shown to preserve intracellular GSH level. And the mRNA levels of GCLC were increased more than 1.5-fold in SAMe and its combination pretreated groups. Furthermore, we evaluated the effects of SAMe and its combinations with taurine and/or betaine on lipopolysaccharide (LPS) or polyinosinic-polycytidylic acid (Poly(I:C)) stimulated RAW 264.7 cells. Intracellular GSH levels fell following LPS or Poly(I:C) treatment. Interestingly, SAMe and its combinations pretreatment increased GSH levels, coinciding with a preservation in the mRNA levels of GCLC in LPS- or Poly(I:C)-treated RAW 264.7 cells. In addition, SAMe, taurine or betaine inhibit the increased inflammatory mediators, especially taurine or betaine pretreatment was shown to inhibit the LPS- or Poly(I:C)-induced increase in mRNA levels of tumor necrosis factor-α (TNF α) and inducible nitric oxide synthase (iNOS) in RAW 264.7 cells. Therefore, we suggest that SAMe combination with taurine and betaine treatment have anti-oxidant functions as well as anti-inflammatory properties.;간질환의 경우 산화적 스트레스(oxidative stress), 내독소혈증 (endotoxemia), 면역기능 이상 등이 많이 발생하게 되며 이것이 발병 원인이 되기도 한다. 최근 연구들에 따르면 간질환에서 나타나는 비정상적인 methionine 대사의 변화가 간질환을 악화시키는 것으로 나타났으며 이에 따른 간 내 glutathionine (GSH)의 농도 저하 또한 관찰되었다. GSH는 간에서 강력한 항산화 기능을 하는 물질로 GSH의 농도를 유지시키는 것이 간 보호에 중요한 역할을 하는 것으로 사료된다. 이에 본 연구에서는 methionine 대사에 관여하는 S-adenosylmethionine (SAMe) 그리고 taurine, betaine 및 그들의 혼합물이 가지는 효과를 알아보고자 하였다. 우선, 생쥐에게 SAMe, taurine, betaine을 선처리한 후, 급성 알코올성 간손상을 유도하였을 때, 이러한 선처리가 간 내 GSH 농도저하를 완화시키고 특히 SAMe의 처리는 GSH 생합성을 조절하는 유전자인 glutamate cysteine ligase catalytic subunit (GCLC)의 mRNA 발현을 증가시키는 것을 동물실험을 통하여 확인하였다. SAMe 는 GSH의 직접적인 전구체일 뿐만 아니라 GSH의 생합성을 조절하는 유전자들의 발현을 조절하는 것으로 알려져 SAMe를 혼합물의 필수 구성 성분으로 혼합물의 효과를 세포실험을 통해 밝히고자 하였다. 생쥐의 일반 간세포주인 AML12 세포를 이용하여 GSH 농도 유지기능에 미치는 영향과 알코올 처리로 인해 일어나는 변화에 대한 효과를 확인한 결과, SAMe 또는 혼합물을 선처리한 경우에 GSH 수준을 유지하는 것으로 보이며 특히 GCLC의 mRNA 발현이 control 그룹에 비해 1.5배 정도 증가하는 것을 확인하였다. 따라서 본 실험의 결과에 비추어 볼 때, SAMe가 GCLC 유전자의 발현량을 증가시킴으로써 GSH의 농도를 유지하는데 있어 가장 중요한 역할을 하는 것으로 사료된다. 대식세포인 RAW 264.7 세포에 SAMe, taurine, betaine과 그들의 혼합물을 처리 후, lipopolysaccharide (LPS)의 처리를 통해 내독소혈증을 유발하거나 바이러스성 감염과 유사한 영향을 미치는 polyinosinic-polycytidylic acid (Poly(I:C))를 처리하여 이에 대한 예방 또는 완화 효과를 확인하고자 하였다. 그 결과, SAMe와 그것이 포함된 혼합물의 선처리는 LPS 또는 Poly(I:C)로 인해 감소되는 GSH를 보존하고 GCLC mRNA 발현량 감소를 저해하는 것으로 확인되었다. 흥미롭게도 taurine과 betaine 또는 그들이 포함된 혼합물을 선처리시에 LPS 또는 Poly(I:C)로 인해 증가하는 염증성 매개 인자인 tumor necrosis factor-α (TNF α)와 inducible nitric oxide synthase (iNOS)의 mRNA 발현이 억제되는데 효과가 탁월했음이 밝혀졌다. 이와 같이 SAMe, taurine과 betaine 혼합물은 항산화 기능 뿐만 아니라 항염증 기능에 대한 가능성을 가져 간질환을 예방 또는 완화시키는데 도움을 줄 수 있을 것이라고 사료된다.-
dc.description.tableofcontentsI. Introduction 1 A. Literature review 1 1. The hepatic methionine metabolism 1 2. Metabolism of alcohol in liver 4 3. Lipopolysaccharide (LPS) and liver disease 4 4. Polyinosinic-polycytidylic acid (Poly(I:C)) and liver disease 5 5. S-adenosylmethionine (SAMe) role in liver disease 6 6. Taurine role in liver disease 7 7. Betaine role in liver disease 8 B. Hypothesis 10 II. Materials and Methods 12 A. Materials 12 B. Animal experiments 12 C. Plasma aspartate aminotransferase (AST) and alanine aminotransferase (ALT) 13 D. Cell culture 14 E. Cell treatment 14 F. Cell viability 15 G. Glutathione (GSH) concentration 16 H. RNA isolation and quantitative real-time PCR 17 I. Western blot analysis 18 J. Nitric Oxide assay 19 K. Multiplex bead-based cytokine assay 20 L. Statistical analysis 20 III. Results 21 A. Effects of SAMe, taurine and betaine on acute alcohol-induced hepatotoxicity in mice 21 1. Body weight, liver weight and ratio of liver weight to body weight 21 2. Plasma aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels 23 3. Hepatic glutathione (GSH) level 25 4. Hepatic mRNA expression levels of GSH synthetic enzymes 27 B. Effects of SAMe and its combinations with taurine and/or betaine in AML12 cells 29 1. Cell viability 29 2. Intracellular GSH levels in AML12 cells 31 3. mRNA levels of GSH synthetic enzymes in AML12 cells 33 4. Intracellular GCLC and GS protein levels in AML12 cells 35 C. Effects of SAMe and its combinations with taurine and/or betaine in ethanol-treated AML12 cells 37 1. Cell viability 37 2. Intracellular GSH levels in ethanol-treated AML12 cells 39 3. mRNA levels of GSH synthetic enzymes in ethanol-treated AML12 cells 41 4. Intracellular GCLC and GS protein levels in AML12 cells 43 D. Effects of SAMe and its combinations with taurine and/or betaine in LPS-stimulated RAW 264.7 cells 45 1. Cell viability 45 2. Intracellular GSH levels in LPS-stimulated RAW 264.7 cells 47 3. mRNA levels of GSH synthetic enzymes in LPS-stimulated RAW 264.7 cells 49 4. Intracellular GCLC and GS protein levels in LPS-stimulated RAW 264.7 cells 51 5. NO production in LPS-stimulated RAW 264.7 cells 53 6. mRNA levels of TNFα and iNOS in LPS-stimulated RAW 264.7 cells 55 7. IL-1β production in LPS-stimulated RAW 264.7 cells 57 E. Effects of SAMe and its combinations with taurine and/or betaine in Poly(I:C)-stimulated RAW 264.7 cells 59 1. Cell viability 59 2. Intracellular GSH levels in Poly(I:C)-stimulated RAW 264.7 cells 61 3. mRNA levels of GSH synthetic enzymes in Poly(I:C)-stimulated RAW 264.7 cells 63 4. Intracellular GCLC and GS protein levels in Poly(I:C)-stimulated RAW 264.7 cells 65 5. NO production in Poly(I:C)-stimulated RAW 264.7 cells 67 6. mRNA levels of TNFα and iNOS in Poly(I:C)-stimulated RAW 264.7 cells 69 7. IL-1β production in Poly(I:C)-stimulated RAW 264.7 cells 71 IV. Discussion 73 V. Conclusion 79 References 80 국문초록 88-
dc.formatapplication/pdf-
dc.format.extent1235228 bytes-
dc.languageeng-
dc.publisher이화여자대학교 대학원-
dc.subject.ddc600-
dc.titleEffects of sulfur-containing amino acids metabolites on various acute hepatotoxicity models in vitro-
dc.typeMaster's Thesis-
dc.title.translated급성 간독성이 유발된 세포모델들에 대한 황함유아미노산 대사물들의 효과 연구-
dc.creator.othernameLee, Seo Yeon-
dc.format.pageviii, 90 p.-
dc.contributor.examiner손용석-
dc.contributor.examiner황광연-
dc.contributor.examiner고광석-
dc.identifier.thesisdegreeMaster-
dc.identifier.major대학원 식품영양학과-
dc.date.awarded2015. 8-
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