Phytic acid와 myo-inositol이 비알코올성지방간 세포 모델에서 지질 축적에 미치는 영향 :Effects of Phytic acid and myo -inositol on lipid accumulation in non-alcoholic fatty liver cell model

심은아

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자료유형: 학위논문

저자정보: 심은아 (가톨릭대학교, 가톨릭대학교 대학원)

지도교수: 김혜경

발행연도: 2023

저작권: 가톨릭대학교 논문은 저작권에 의해 보호받습니다.

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비알코올성 지방간 질환은 간 조직에 중성지방이 5% 이상 축적되는 것으로 정의되며 지질대사에 장애가 생기는 경우 나타난다. Phytic acid(PA)와 myo-inositol(MI)은 곡류, 견과류, 콩류 등에 존재하는 천연물질로 항암효과, 혈당저하, 혈중지질저하, 지질 함량 감소 등 건강에 유익한 영향이 보고되었으나 간 세포의 지질 축적에 미치는 직접적인 효과에 대해서는 아직까지 연구된 바 없었다. 따라서 본 연구에서는 지방산 처리에 의한 HepG2 세포의 지방간 유도 모델에서 PA와 MI가 지질 축적억제에 직접적인 영향을 미치는지 알아보았다. PA와 MI는 간 세포에서 지방산에 의한 중성지방 축적을 직접적으로 감소시켰다. 내인성 지방 합성 억제효과를 알아보기 위해 지방산 합성효소인 acetyl-CoA carboxylase (ACC) 및 fatty acid synthase(FAS)의 발현을 측정하였고 PA와 MI에 의해 부분적으로 발현이 감소하는 것을 확인하였다. 또한, 장쇄 지방산의 막 수용체인 cluster of differentiation 36(CD36)의 유전자 발현량이 감소하는 것으로 간 세포 내 지방산 유입이 억제됨을 확인하였다. 지방산 β-산화에 관여하는 조절인자인 carnitine palmitoyl transferase 1(CPT-1)과 peroxisome proliferator-activated receptor α(PPARα)유전자 발현이 PA와 MI의 처리에 의해 증가한 것을 통해 지방산 산화가 촉진됨으로써 지질 축적량을 감소시킬 수 있음을 확인하였다. PA와 MI가 콜레스테롤 합성에 미치는 영향을 알아보기 위해 관련 효소인 HMG CoA reductase (HMGCR)의 발현을 측정한 결과 PA와 MI에서 모두 유의적인 감소가 나타나 간 내 콜레스테롤 합성 억제에 영향을 줄 수 있음을 확인하였다. 또한, PA와 MI의 지질의 합성 억제 및 β-산화 증가의 효과가 AMP-activated protein kinase (AMPK)의 활성화에 의한 것인지 알아보기 위해 AMPK 활성 형태인 인산화된 AMPK와 AMPK에 의해 매개되는 지질 대사 관련 조절인자의 단백질 발현을 측정하였다. PA와 MI 처리 시 모두 AMPK의 활성화가 증가하였다. AMPK 활성화에 따라 매개되는 전사인자인 SREBP1c는 활성이 억제되었고, ACC는 비활성화 되었다. 따라서 PA와 MI는 AMPK 활성화 경로를 통해 지질 합성억제 및 산화 촉진이 유발되어 간 지질축적을 억제할 수 있음을 확인할 수 있었다. 결론적으로 PA와 MI는 유리지방산으로 유도한 비 알코올성 지방간 세포모델에서 간세포 내 지질 축적을 완화하는 효과가 있음을 확인하였고, 이러한 결과는 선행 동물실험에서 나타난 간 조직의 지질 함량 감소를 세포수준에서의 효과로 뒷받침할 수 있는 근거로서 의미가 있다.

Non-alcoholic fatty liver disease is defined as the accumulation of 5% or more of triglycerides in liver tissue and occurs when lipid metabolism is impaired. Phytic acid (PA) and myo-inositol (MI) are natural substances present in grains, nuts, and beans, and have been reported to have beneficial health effects such as blood sugar, blood lipids, lipid content reduction, and anticancer effects, but the direct effect on lipid accumulation of liver cells has not been studied. Therefore, in this study, we investigated whether PA and MI directly affect lipid accumulation inhibition in the fatty liver induction model of HepG2 cells by fatty acid treatment. PA and MI directly reduced triglyceride accumulated by fatty acids in liver cells. To find out the inhibitory effect of endogenous fat synthesis, the expression of acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS) was measured, and it was confirmed that the expression was partially reduced by PA and MI. In addition, it was confirmed that the inflow of free-fatty acids into the liver was suppressed by a decrease in the amount of gene expression of cluster of differentiation 36 (CD36), a membrane receptor of long-chain fatty acids. It was confirmed that the expression of the carnitine palmitoyl transferase 1 (CPT-1) and peroxisome promoter-activated receptor α (PPARα) gene, which are regulators involved in fatty acid β-oxidation, increased by the treatment of PA and MI, thereby reducing lipid accumulation. To find out the effect of PA and MI on cholesterol synthesis, the expression of HMG CoA reductase(HMGCR), a related enzyme, was measured, and it was confirmed that both PA and MI showed a significant decrease, which could affect inhibition of cholesterol synthesis in the liver. In addition, in order to determine whether the effect of PA and MI on lipid synthesis inhibition and β-oxidation increase was due to the activation of AMP-activated protein kinase (AMPK), protein expression of phosphorylated AMPK, and AMPK-mediated activity were investigated. Protein expression of lipid metabolism-related regulators was measured. AMPK activation was increased during PA and MI treatment, and accordingly, SREBP1c, a transcription factor mediated by AMPK activation, was suppressed and phosphorylated ACC was inactivated. Therefore, it was confirmed that PA and MI can inhibit lipid accumulation in the liver by inhibiting lipid synthesis and promoting oxidation through the AMPK activation pathway.
In conclusion, it was confirmed that PA and MI had the effect of alleviating lipid accumulation in hepatocytes in a non-alcoholic fatty liver cell model induced by free fatty acids. It is meaningful in that it can be produced as evidence to support it.

#비알코올성지방간질환 #phytic acid #myo-inositol

국문 초록 ···················································································································viii
Ⅰ. 서론 ··························································································································1
Ⅱ. 재료 및 방법 ··········································································································7
1. 재료 ···················································································································7
2. 세포배양 ···········································································································8
3. 유리지방산 유도 비알코올성 지방간 세포모델 ·······································8
4. HepG2 세포의 생존도 분석 ·······································································9
5. HepG2 세포 내 지질 축적량 분석 ···························································9
5.1. 총 지질 함량 측정 ············································································9
5.2. 중성지방 함량 측정 ·······································································10
6. 지질축적과 관련된 유전자 발현 분석 ····················································11
7. 지질축적과 관련된 단백질 발현 분석 ····················································13
8. 통계분석 ········································································································14
Ⅲ. 결과 ························································································································15
1. 농도 별 지방산 처리가 HepG2 세포의 생존도 및 지질축적에 미치는 영향 ·················································································································15
2. Phytic acid와 myo-inositol이 비알코올성 지방간 세포모델에서 지질축적 억제에 미치는 영향 ···································································17
2.1. Phytic acid와 myo -inositol 처리 시 비알코올성 지방간
세포모델의 생존도 ··············································································17
2.2. 세포 내 총 지질 함량 ·······································································19
2.3. 세포 내 중성지방 함량 ·····································································19
3. Phytic acid와 myo-inositol이 비알코올성 지방간 세포모델에서 지질축적 관련 조절인자에 미치는 영향 ·················································21
3.1. 지질 합성 관련 조절인자 발현 ·······················································21
3.2. 지방산의 β-산화 관련 조절인자 발현 ········································23
3.3. 세포 내 지질 유입 수용체 유전자 발현 ·······································25
3.4. 콜레스테롤 합성 관련 조절인자 발현 ···········································25
3.5. AMP-activated protein kinase (AMPK) 활성화 및 AMPK 매개 조절인자의 발현 ················································································27
Ⅳ. 고찰 ························································································································30
Ⅴ. 참고 문헌 ··············································································································36
영문 논문제출서 ·········································································································40
영문 인준서 ·················································································································41
영문 초록 ·····················································································································42

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