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dc.contributor.advisor고광석-
dc.contributor.author조은혜-
dc.creator조은혜-
dc.date.accessioned2021-01-25T16:30:25Z-
dc.date.available2021-01-25T16:30:25Z-
dc.date.issued2021-
dc.identifier.otherOAK-000000173096-
dc.identifier.urihttp://dcollection.ewha.ac.kr/common/orgView/000000173096en_US
dc.identifier.urihttps://dspace.ewha.ac.kr/handle/2015.oak/256133-
dc.description.abstractProhibitin 1 (Phb1) is a pleiotropic protein that has multiple functions in the mammalian cells such as regulation of cell proliferation and stabilization of mitochondrial protein. It mainly presents in the mitochondrial inner membrane and functions as a chaperone protein which stabilizes the protein created in mitochondria and is being suggested as a potential therapeutics target for a variety of diseases, including liver cancer. In normal hepatocytes, Phb1 helps cells to grow and function normally. Acetaminophen (APAP) is one of the world’s most commonly used over-the-counter drugs as an analgesic. At lower dosage, it is effective and safe for soothing light pain, but overdose is well known to cause liver damage because of the accumulation of N-acetyl-p-benzoquinone imine (NAPQI). APAP-induced hepatotoxicity is mainly induced by depletion of glutathione (GSH), resulting in oxidative stress and mitochondrial dysfunction. Because S-adenosylmethionine (SAMe) is a precursor of GSH, it can increase intracellular GSH concentration. Since APAP metabolism and regulation of SAMe mainly occur in the liver, this study was aimed to understand the mRNA expression relates to APAP and GSH metabolism regulated by Phb1 in normal hepatocytes- AML 12 cells. Therefore, we investigated the effects of Phb1 expression levels on hepatotoxicity caused by APAP and the hepatoprotective effects on liver injury. For this experiment we used two different Phb1 silencing level, which are high efficiency (HE, >80%) and low efficiency (LE, >40%, <60%). These siRNA transfected cells were further pre-treated with 0.5 mM of SAMe for 20 h before treatment of APAP at different doses (1-3 mM) for 24 h. Then the cell viability, intracellular GSH, and the mRNA expression level of key enzymes were determined. APAP-mediated hepatotoxicity was significantly increased in Phb1 siRNA-transfected AML 12 cells compared to the control. When APAP was treated at a range of concentration 1-3 mM, APAP treatment of 2 mM concentration resulted in the most apparent hepatotoxicity in all HE group, but there were no significant changes in LE groups. It suggests that Phb1 expression level affects APAP toxicity susceptibility. When SAMe was pretreated, Phb1 deficiency led to significant mRNA expressions level of thioredoxin reductase (ThR), NAD(P)H quinone oxidoreductase 1 (Nqo1), and glutamate-cysteine ligase (Gcl) at 3 mM of APAP to protect the cells from oxidative stress. Overall in this study, Phb1 protects hepatocytes against APAP toxicity even though its expression levels are down to 50% of the wild type expression levels, and SAMe has hepatoprotective effects against hepatotoxicity.;Prohibitin 1 (Phb1)은 주로 미토콘드리아 내막에 위치하는 세포 증식 조절이나 미토콘드리아 단백질의 안정성에 관여하는 단백질이다. 정상 간세포에서 Phb1은 세포가 정상적으로 자라고 기능하도록 돕는 역할을 한다. Acetaminophen (APAP)는 전 세계적으로 많이 사용되고 있는 진통∙해열제이다. 치료용량에서는 통증 완화에 효과적으로 안전한 약품이지만 과다복용은 중간독성 대사체인 N-acetyl-p-benzoquinone imine (NAPQI)가 간 내에 축적되면서 간 손상을 유발할 수 있다. 또한, NAPQI는 glutathione (GSH)의 고갈을 유발하여 산화스트레스 및 미토콘드리아 기능장애를 일으킬 수 있다. S-adenosylmethionine (SAMe)는 GSH의 precursor로 세포 내의 GSH 농도로 높일 수 있다. APAP 및 SAMe 대사 조절은 주로 간에서 이루어지기 때문에 정상 간 세포인 AML 12 세포에서 Phb1 발현과 APAP 독성용량으로 인한 간 손상 및 SAMe로 인한 간 독성 완화효과를 알아보고자 연구를 진행하였다. 본 연구에서는 마우스 간세포주인 AML 12를 Phb1 siRNA를 사용하여 유전자 Phb1을 knockdown (KD)시켰다. 이 때 두가지 타입의 siRNA를 사용하였는데, 하나는 Phb1의 발현량이 50-60%가 되는 low efficiency (LE) siRNA이고, 다른 하나는 10%가 되는 high efficiency (HE) siRNA이다. KD시킨 세포에 첫번째 그룹은 APAP를 dose-dependent하게 1-3mM 처리하여 24시간동안 배양하였고, 두번째 그룹은 0.5 mM 농도의 SAMe를 먼저 처리하여 20시간동안 배양한 후, APAP를 1-3mM 처리한 다음 24시간동안 배양하였다. 세포 독성 및 유전자발현 변화를 보기 위해 cell viability, mRNA 발현 수준 및 세포 내 GSH 농도를 측정하였다. APAP로 인한 독성은 siRNA를 처리하지 않은 control group과 비교하였을 때 전반적으로 유의적인 차이를 보였다. 특히 Phb1 HE group에서 APAP를 2mM 처리하였을 때 독성 관련 marker들이 증가하였다. 반면, SAMe를 함께 처리한 group은 Phb1 HE group에서 APAP를 3mM 처리하였을 때 산화스트레스에 대응하기 위한 항산화 유전자들의 발현이 증가하였다. 이를 통해 Phb1이 절반 이상의 발현량을 보이면 APAP로 인한 간 독성으로부터 세포를 보호할 수 있고, SAMe 또한 세포 내 GSH 농도를 높임으로써 간 독성을 예방하는 효과를 보이는 것으로 해석할 수 있다.-
dc.description.tableofcontentsI. Introduction 1 A. Literature review 1 1. Drug metabolism in the liver and drug-induced hepatotoxicity 1 2. Hepatotoxicity induced by Acetaminophen 3 3. Function of Prohibitin 1 in liver disease 6 4. Hepatoprotective function of S-adenosylmethionine 8 B. Hypothesis 11 C. Objectives 12 Ⅱ. Materials and Methods 13 A. Cell culture 13 B. Small interfering RNA transfection 14 C. Cell viability assay 15 D. S-adenosylmethionine and Acetaminophen treatment 16 E. RNA isolation, Reverse transcription, and Real-time PCR 17 F. Cellular Glutathione (GSH) concentration 21 G. Statistical analysis 22 Ⅲ. Results 23 1. The silencing efficiency of Phb1 by siRNA transfection 23 2. Cell viability 26 3. mRNA expression of inflammatory cytokines 29 4. mRNA expression of acetaminophen metabolism 32 5. mRNA expression of oxidative stress 35 6. Intracellular glutathione concentration 38 7. mRNA expression of glutathione synthesis 41 Ⅳ. Discussion 44 Ⅴ. Conclusion 51-
dc.formatapplication/pdf-
dc.format.extent1580472 bytes-
dc.languageeng-
dc.publisher이화여자대학교 대학원-
dc.subject.ddc600-
dc.titleThe Relationship between Prohibitin1 Expression Levels and Hepatotoxicity Induced by Acetaminophen and Hepatoprotective Effects of S-adenosylmethionine in AML 12 cells-
dc.typeMaster's Thesis-
dc.creator.othernameCho, Eun Hye-
dc.format.pageviii, 59 p.-
dc.identifier.thesisdegreeMaster-
dc.identifier.major대학원 식품영양학과-
dc.date.awarded2021. 2-
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