Identification of natural product autophagy regulator by high content screening
- 발행기관 강릉원주대학교 일반대학원
- 지도교수 도움말 Prof. Heesu Lee and Prof. Jae Wook Lee
- 발행년도 2024
- 학위수여년월 2024. 2
- 학위명 석사
- 학과 및 전공 도움말 일반대학원 KIST강릉분원학.연협동과정
- 세부분야 해당없음
- 실제URI http://www.dcollection.net/handler/kangnung/000000011609
- UCI I804:42001-000000011609
- 본문언어 영어
초록/요약 도움말
Autophagy is a crucial autolytic process for balancing energy sources during critical developmental periods and disruptions in nutrient absorption. The extent of this process is directly correlated with its benefits, due to its role in eliminating toxic factors and promoting cell viability. Consequently, autophagy has emerged as a novel and essential approach for regulating disease progression, offering scientific intrigue and clinical relevance. Autophagy also plays a pivotal role in the removal of misfolded and misassembled proteins, the removal of damaged organs such as mitochondria, and endoplasmic reticulum, and the elimination of intracellular pathogens in peroxisomes(4). Autophagy is impaired in many neurodegenerative diseases, diabetes, sepsis, etc (28,29). Therefore, autophagy activation by small molecules has become an attractive therapeutic strategy. Recent literature shows that natural products are the prominent source for novel small molecular drugs. In my thesis, we screened a library of natural products extracts comprising from various medicinal plants and discovered many plant extracts that activate autophagy. Here, we report a novel plant extract, BE0161F1, which significantly activates the autophagy-lysosomal pathways and its underlying molecular mechanisms. The induction of autophagy by BE0161F1 was investigated by immunoblotting and GFP-LC3B imaging. The data showed a significant increase in the autophagy marker LC3BII and autophagosome vesicle formation as a function of concentration and time in HeLa cell. In addition, acridine orange image analysis indicated the development of acidic lysosome after BE0161F1 (12.5 µg/mL, 25 µg/mL, 50 µg/mL) treatment. These data confirm that BE0161F1 induces the formation of autophagosomes and autolysosomes. Furthermore, BE0161F1 treatment of HeLa-TFEB-GFP cells significantly increased the level of TFEB-GFP translocation in the nucleus compared to the control. Additionally, the results of qPCR analysis revealed the upregulation of TFEB target genes such as CSTD, LAMP1, ATC16L, and p62/SQSTM. These data strongly support the idea that BE0161F1 induces the autophagy-lysosomal pathway and that it should be further explored to uncover its therapeutic potential.
more목차 도움말
Content
List of Abbreviations 3
List of figures 6
Abstract 8
1. Introduction 10
1.1. Autophagy mechanism. 10
1.2. Autophagy-associated diseases 16
2. Materials and methods 22
2.1. Chemicals and reagents. 22
2.2. Autophagy screening assay 22
2.3 Western blot. 22
2.4. qPCR of TFEB target genes 22
2.5. Acridine orange staining. 23
2.6. Statistical analysis. 24
3. Results and discussion. 25
3.1. Screening BE0161F1 - a novel extract activator of autophagy 25
3.2. BEO161F1 induces autophagy in a dose-dependent manner. 33
3.3. BE0161F1 induces Lysosome generation. 41
3.4 BEO161F1 induces the autophagy-lysosome pathway by activating transcription factor EB (TFEB). 45
Conclusion 50
Reference 56
ACKNOWLEDGMENT 60
