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학술연구/단체지원/교육 등 연구자 활동을 지속하도록 DBpia가 지원하고 있어요.
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논문 기본 정보
- 자료유형
- 학위논문
- 저자정보
- 지도교수
- 황혜진
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- 2022
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문모초(Veronica peregrina L.)는 주로 아시아에서 자라는 식물로 논밭 근처와 냇가에서 자라는 흔한 식물이다. 개불알풀 속(Veronica)에 속하는 식물들의 연구는 많이 진행되었지만 문모초에 관한 연구는 현재까지 많이 알려지지 않았다. 이에 본 연구에서는 문모초 에탄올 추출물(Ethanol Extract of Veronica peregrina L.; EEVP)의 항비만 및 항산화 효능을 확인하고자 하였다.
EEVP의 처리가 지방전구세포(preadipocyte)인 3T3-L1의 성장과 중성지방(triglyceride; TG)의 축적에 미치는 영향을 확인하기 위하여, EEVP를 50-200 μg/mL로 처리하였다. Oil Red O 염색 전과 후로 구분하여 지방구(lipid droplet) 염색 정도를 관찰한 결과 EEVP에 의해 lipid droplet의 크기와 양이 감소되는 것을 확인하였고, TG 농도를 측정한 결과 미분화 대조군과 비교하여 농도 의존적으로 감소되어 3T3-L1 preadipocyte의 분화를 현저하게 억제함을 확인하였다.
3T3-L1 세포를 이용하여 지방세포(adipocyte) 분화를 억제하는 성분은 지속적으로 연구가 진행되고 있다. adipocyte 축적의 주요 인자로 peroxisome proliferator-activated receptor gamma (PPARγ), adipocyte protein 2 (aP2), sterol regulatory element binding proteins (SREBP), CCAAT- enhancer-binding proteins (C/EBPs) 등이 있다. 본 연구결과 EEVP 처리 시, western blot 실험을 통하여 PPARγ, C/EBPα, C/EBPβ, SREBP-1C의 발현이 억제되었으며, aP2, leptin과 같은 adipocyte expressed genes에서도 EEVP에 의해 농도 의존적으로 감소하여 adipocyte 전사 인자의 발현 수준을 현저히 감소시키는 것으로 확인되었다. 또한 EEVP의 처리에 따른 AMP-activated protein kinase (AMPK)와 AMPK의 하위단계인 acetyl-CoA carboxylase (ACC)의 인산화에 대한 효과를 확인한 결과, p-AMPK와 p-ACC가 EEVP의 농도가 높아짐에 따라 증가하는 것으로 나타나 EEVP의 preadipocyte 억제 효과에는 AMPK가 관여한다는 것을 알 수 있었다.
EEVP의 항산화 효과를 검증하기 위하여 hydrogen peroxide (H2O2)로 산화적 스트레스(oxidative stress)를 유발시킨 3T3-L1 세포에서 EEVP의 처리 후의 자연세포사(apoptosis)에 대한 실험에서 억제되는 효과를 관찰하였다. EEVP의 처리는 nuclear factor erythroid 2-related factor-2 (Nrf2)의 활성화에 의해 heme oxygenase-1 (HO-1)의 발현을 증가시켰고, Kelch-like ECH-associated protein 1 (Keap1)은 EEVP의 처리에 따라 농도 의존적으로 감소하였다. Comet assay 실험을 진행한 결과 H2O2 단독 처리한 3T3-L1 세포에서의 DNA는 손상되어 나타났으며, 이에 EEVP로 처리한 세포에서는 EEVP의 농도 의존적 으로 DNA 손상이 감소하는 것으로 나타났고, 활성산소종 (reactive oxigen species; ROS) 생성이 감소하는 것을 확인하였다. 또한, H2O2 단독 처리한 3T3-L1 세포에서 p-γH2AX의 활성 은 증가되었고, EEVP 처리 시 활성이 약해지는 것으로 나타나, EEVP가 DNA 손상을 회복시킨다는 것을 알 수 있었다. 뿐만 아니라 미토콘드리아와 세포질의 cytochrome c를 보았을 때 H2O2로 유도된 상태에서 EEVP를 처리했을때 미토콘드리아에 있는 cytochrome c 발현이 농도 의존적으로 증가되어 EEVP가 미토콘드리아 기능 손상을 억제시키는 것을 확인하였다. 또한 EEVP의 처리 시 Bax/Bcl-2 발현이 감소되며 caspase-3, PARP 의 활성화가 대조군만큼 감소되는 결과를 보여 apoptosis 현상으 로부터 세포를 보호하는 것을 확인할 수 있었다. 이 결과로 EEVP의 활성이 AMPK signaling pathway를 통해 항산화 효과가 나타나는 것을 알 수 있었다.
본 연구 결과 EEVP는 preadipocyte 분화억제 및 항산화 효능을 갖는 것으로 나타나 향후 항비만 및 항산화 기능성 소재로서의 활용이 가능할 것으로 사료된다.
EEVP의 처리가 지방전구세포(preadipocyte)인 3T3-L1의 성장과 중성지방(triglyceride; TG)의 축적에 미치는 영향을 확인하기 위하여, EEVP를 50-200 μg/mL로 처리하였다. Oil Red O 염색 전과 후로 구분하여 지방구(lipid droplet) 염색 정도를 관찰한 결과 EEVP에 의해 lipid droplet의 크기와 양이 감소되는 것을 확인하였고, TG 농도를 측정한 결과 미분화 대조군과 비교하여 농도 의존적으로 감소되어 3T3-L1 preadipocyte의 분화를 현저하게 억제함을 확인하였다.
3T3-L1 세포를 이용하여 지방세포(adipocyte) 분화를 억제하는 성분은 지속적으로 연구가 진행되고 있다. adipocyte 축적의 주요 인자로 peroxisome proliferator-activated receptor gamma (PPARγ), adipocyte protein 2 (aP2), sterol regulatory element binding proteins (SREBP), CCAAT- enhancer-binding proteins (C/EBPs) 등이 있다. 본 연구결과 EEVP 처리 시, western blot 실험을 통하여 PPARγ, C/EBPα, C/EBPβ, SREBP-1C의 발현이 억제되었으며, aP2, leptin과 같은 adipocyte expressed genes에서도 EEVP에 의해 농도 의존적으로 감소하여 adipocyte 전사 인자의 발현 수준을 현저히 감소시키는 것으로 확인되었다. 또한 EEVP의 처리에 따른 AMP-activated protein kinase (AMPK)와 AMPK의 하위단계인 acetyl-CoA carboxylase (ACC)의 인산화에 대한 효과를 확인한 결과, p-AMPK와 p-ACC가 EEVP의 농도가 높아짐에 따라 증가하는 것으로 나타나 EEVP의 preadipocyte 억제 효과에는 AMPK가 관여한다는 것을 알 수 있었다.
EEVP의 항산화 효과를 검증하기 위하여 hydrogen peroxide (H2O2)로 산화적 스트레스(oxidative stress)를 유발시킨 3T3-L1 세포에서 EEVP의 처리 후의 자연세포사(apoptosis)에 대한 실험에서 억제되는 효과를 관찰하였다. EEVP의 처리는 nuclear factor erythroid 2-related factor-2 (Nrf2)의 활성화에 의해 heme oxygenase-1 (HO-1)의 발현을 증가시켰고, Kelch-like ECH-associated protein 1 (Keap1)은 EEVP의 처리에 따라 농도 의존적으로 감소하였다. Comet assay 실험을 진행한 결과 H2O2 단독 처리한 3T3-L1 세포에서의 DNA는 손상되어 나타났으며, 이에 EEVP로 처리한 세포에서는 EEVP의 농도 의존적 으로 DNA 손상이 감소하는 것으로 나타났고, 활성산소종 (reactive oxigen species; ROS) 생성이 감소하는 것을 확인하였다. 또한, H2O2 단독 처리한 3T3-L1 세포에서 p-γH2AX의 활성 은 증가되었고, EEVP 처리 시 활성이 약해지는 것으로 나타나, EEVP가 DNA 손상을 회복시킨다는 것을 알 수 있었다. 뿐만 아니라 미토콘드리아와 세포질의 cytochrome c를 보았을 때 H2O2로 유도된 상태에서 EEVP를 처리했을때 미토콘드리아에 있는 cytochrome c 발현이 농도 의존적으로 증가되어 EEVP가 미토콘드리아 기능 손상을 억제시키는 것을 확인하였다. 또한 EEVP의 처리 시 Bax/Bcl-2 발현이 감소되며 caspase-3, PARP 의 활성화가 대조군만큼 감소되는 결과를 보여 apoptosis 현상으 로부터 세포를 보호하는 것을 확인할 수 있었다. 이 결과로 EEVP의 활성이 AMPK signaling pathway를 통해 항산화 효과가 나타나는 것을 알 수 있었다.
본 연구 결과 EEVP는 preadipocyte 분화억제 및 항산화 효능을 갖는 것으로 나타나 향후 항비만 및 항산화 기능성 소재로서의 활용이 가능할 것으로 사료된다.
목차
Ⅰ. 서론·······························································································································1Ⅱ. 재료 및 방법···············································································································71. 실험재료 ·······················································································································71) 문모초 에탄올 추출물 제조······························································································72. 항비만 활성 ·················································································································81) 3T3-L1 preadipocytes 세포 배양 ··············································································82) MTT assay를 이용한 세포 생존율 측정 ······································································83) 3T3-L1 preadipocytes 분화 유도 ··············································································94) Oil Red O 염색 및 triglyceride 정량 ···········································································105) 3T3-L1의 단백질 발현을 위한 western blot ·····························································103. 항산화 활성 ···············································································································121) 세포 생존력 측정········································································································122) ROS 수치 측정············································································································123) DNA 손상에 대한 comet assay ····················································································134) H2O2 처리한 3T3-L1의 western blot··········································································145) Annexin V 염색에 의한 apoptosis 검출 ·····································································146) 미토콘드리아 막 전위차 측정························································································157) 형광현미경을 통한 apoptosis 분석················································································164. 통계분석 ···································································································································16Ⅲ. 결과·········································································································································171. 항비만 활성 ······························································································································171) EEVP가 3T3-L1 preadipocytes의 생존율에 미치는 영향······························································172) EEVP가 lipid droplet 생성과 triglyceride에 미치는 영향····························································193) EEVP가 adipogenic transcription factor 및 adipocyte expressed genes의 발현에 미치는 영향·····224) EEVP로 인한 adiopcytes에서 AMPK 및 ACC의 효과 ·····································································245) Compound C가 lipid droplet 생성과 triglyceride에 미치는 영향·····················································266) Compound C가 AMPK와 adipocytes genes에 미치는 영향 ·····························································292. 항산화 활성 ···································································································································321) H2O2 유도된 3T3-L1 세포에서 EEVP의 독성 억제효과 ·································································322) H2O2 유도된 3T3-L1 세포에서 EEVP에 대한 Nrf2/HO-1 와 AMPK의 유전자 발현 효과 ················343) H2O2 유도된 3T3-L1 세포에서 EEVP의 ROS 생성 억제 효과···························································364) H2O2 유도된 3T3-L1 세포에서 EEVP의 DNA 손상 억제 효과····························································375) H2O2 유도된 3T3-L1 세포에서 EEVP의 미토콘드리아 기 능에 대한 영향·········································406) H2O2 유도된 3T3-L1 세포에서 EEVP의 apoptosis 억제 효 과··························································43Ⅳ. 고찰 및 결론·································································································································46Ⅴ. 참고문헌 ······································································································································52Abstract ··········································································································································64감사의 글···········································································································································67
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