The function of sestrin2 on the progress of atherosclerosis
Sestrin2가 동맥경화증의 기저 메커니즘에 미치는 영향 분석
- 주제(키워드) Sestrin2 , Atherosclerosis , Inflammation
- 발행기관 고려대학교 대학원
- 지도교수 류혜진
- 발행년도 2017
- 학위수여년월 2017. 8
- 학위구분 박사
- 학과 대학원 의과학과
- 세부전공 의생명과학전공
- 원문페이지 79 p
- 실제URI http://www.dcollection.net/handler/korea/000000077094
- 본문언어 영어
- 제출원본 000045920284
초록/요약
Background & Objectives: Sestrin2 (sesn2) is recently regarded as a novel regulator in metabolic disorders such as obesity and non-alcoholic fatty liver disease (NAFLD). Sesn2 is strongly linked to adenosine monophosphate-activated protein kinase (AMPK) activation, which plays a role in maintaining cell homeostasis and protects against inflammatory and oxidative stresses in various cell types. AMPK signaling can regulate the development and advancement of atherosclerosis, however, the function of sesn2 in the vascular endothelium has not yet been clarified. Methods: To determine the role of sesn2 in the development of atherosclerosis, scramble or sesn2-silencing small interfering RNA (siRNA) oligomers were inserted into human umbilical vein endothelial cells (HUVECs), human monocytic cell line (THP-1), and aortic tissues of C57BL/6 mice. The efficiency of sesn2-knockdown was evaluated by Western blotting. Lipopolysaccharide (LPS) was administrated to siRNA-treated cells and mice to induce atherosclerotic events such as inflammation and decrease in cell viability. 5-Aminoimidazole-4-carboxamide ribonucleotide (AICAR) was used to induce AMPK activation, and parthenolide was used for nuclear factor kappa B (NF-κB) inhibition. Results; Sesn2-knockdown was strongly associated with LPS-mediated atherosclerotic reactions. In HUVECs and THP-1 cells, sesn2-knockdown significantly reduced AMPK phosphorylation and increased LPS-mediated NF-κB phosphorylation, leading to up-regulation of several pro-inflammatory cytokines. LPS-induced expression of endothelial adhesion molecules and attachment of THP-1 cells to HUVECs were both increased by sesn2-knockdown, and these elevations were blocked after treatment with either the AMPK activator or NF-κB inhibitor. Furthermore, several cell death-related events such as reactive oxygen species (ROS) production, nuclear condensation and degradation, and endoplasmic reticulum (ER)-mediated stress were significantly increased in sesn2-knockdown HUVECs, but not after AICAR treatment. In aortic tissues obtained from C57BL/6 mice, LPS-mediated NF-κB phosphorylation was increased in sesn2-knockdown aortas. LPS-mediated expression of endothelial adhesion molecules, serum level of pro-inflammatory cytokines, and attachment of CD11b-positive cells to arterial walls were all increased by sesn2-knockdown, but not after treatment with the NF-κB inhibitor. In addition, the administration of the AMPK activator significantly decreased ER-stress-related signaling in sesn2-knockdown aortic tissues. Conclusion: Sesn2-knockdown increased LPS-mediated atherosclerotic events such as increase in expression of endothelial adhesion molecules, pro-inflammatory cytokines, monocyte attachment to arterial walls, and ER-mediated stress, through inactivation of AMPK. These results suggest that sesn2 might be a novel therapeutic target to prevent the development and advancement of atherosclerosis.
more목차
ABSTRACT I
CONTENTS IV
LIST OF FIGURES VII
I. INTRODUCTION 1
1. Atherosclerosis: A Chronic Inflammatory Disease 1
2. Inflammation, Oxidative stress and Atherosclerosis 1
3. AMPK: An Anti-Atherosclertic Molecule 2
4. What is Sestrin2? 2
II. METERIALS & METHODS 6
1. Cell culture and transfection 6
2. Compounds 6
3. Western blotting 7
4. Chromatin Immunoprecipitation (ChIP) assay 8
5. Quantitative Real-time PCR (qPCR) 8
7. Adhesion assay 9
8. Enzyme-linked immunosorbent assay (ELISA) 9
9. Measurement of cell viability 10
10. Dihydroethidium (DHE), Hoechst Staining, and Terminal deoxy-nucleotidyl transferase dUTP nick end labeling (TUNEL) assay 10
11. Animals and material treatments 10
12. Immunohistochemistry (IHC) 13
13. Statistical analysis 13
III. RESULTS 14
1. Sesn2 is a stress-inducible gene. 14
2. AMPK phosphorylation is decreased in sesn2 knockdown cells. 19
3. Sesn2-knockdown elevates LPS-mediated NF-κB activation through AMPK dephosphorylation. 22
4. LPS-mediated NF-κB phosphorylation is decreased by sesn2 over-expression via an AMPK-dependent mechanism 26
5. LPS-mediated expression of endothelial adhesion molecules and adhesion of THP-1 cells to HUVECs are increased by sesn2-knockdown. 29
6. LPS-mediated expression of proinflammatory cytokines is increased in sesn2-knockdown HUVECs and THP-1 cells 33
7. LPS-mediated ER stress-mediated signaling is increased in sesn2-knockdown HUVECs. 35
8. LPS-mediated cell death-related events are increased in sesn2-knockdown HUVECs. 38
9. Acute down-regulation of sesn2 gene is induced in aortic tissues of C57BL/6 mice. 41
10. LPS-mediated aortic atherosclerotic events are increased in sesn2-knockdown C57BL/6 mice. 43
11. ER stress-related signaling by LPS is increased in sesn2-knockdownC57BL/6 mice. 48
12. Atherosclerosis-related proteins induced by sesn2-knockdown are mainly found in endothelial cells. 51
IV. DISCUSION 53
Limitation 56
Conclusion 57
V. REFERENCE 60
KOREAN ABSTRACT 65
ACKNOWLEDGEMENT 67
