F1,6DP에 의한 뇌혈관장벽 기능 조절 기전
Protective Effects of Fructose-1,6-diphosphate on LPS-induced Blood-Brain Barrier (BBB) Dysfunction
- 주제(키워드) Blood-Brain Barrier (BBB) , Murine brain cerebrovascular endothelial cells (bEnd.3) , Fructose-1 , 6-diphosphate (F1 , 6DP) , Toll-like receptor 4 (TLR4) , Intercellular adhesion molecular-1 (ICAM-1)
- 발행기관 아주대학교
- 지도교수 이수환
- 발행년도 2009
- 학위수여년월 2009. 8
- 학위명 박사
- 학과 및 전공 일반대학원 신경과학기술과정
- 실제URI http://www.dcollection.net/handler/ajou/000000010099
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
The blood-brain barrier (BBB) impedes the influx of un-wanted substances from blood milieu to brain. Dysfunction of BBB has been implicated in the pathogenesis of a variety of CNS diseases such as stroke and Alzheimer’s disease. Therefore, the control of the barrier properties has been regarded as one of the critical issues for the treatment of various cerebral diseases. In this study, it was examined whether F1,6DP, a putative neuroprotectant, could attenuate LPS-induced BBB dysfunction. Firstly, new in vitro BBB model was constructed using brain microvascular cell line (bEnd.3) and primary cultured mouse astrocyte (PA). This co-culture model was comparable to well-known HUVEC-C6 glioma co-culture system in terms of permeability criteria such as transendothelial electrical resistance (TEER) and transcellular-/ paracellular permeability ratio. Inflammatory stimuli such as LPS and TNFα decreased TEER and increased the ratio of the permeability coefficient of propranolol and sucrose. F1,6DP restored the changes in TEER and permeability coefficients induced by inflammatory stimuli. And also, F1,6DP reversed the alterations in the distribution of endothelial tight junction (TJ) proteins, such as occludin and zonula occludin-1 (ZO-1), which were spreaded throughout the cytoplasm in the presence of LPS. In addition, F1,6DP significantly reduced the LPS-induced expression of intercellular adhesion molecule-1 (ICAM-1) in bEnd.3 cells. NF-κB inhibitors such as MG132 and Bay 11-7082 reduced LPS-induced ICAM-1 expression at the levels of mRNA and protein in bEnd.3 cells, suggesting strong involvement of NF-κB. These results were supported by EMSA and NF-κB reporter assay. F1,6DP also reduced LPS-induced ICAM-1 expression through the inhibition of NF-кB activation. F1,6DP blocked NF-κB activation by ectopic expression of the key molecules in TLR4 signaling pathway such as MyD88 , TRAF6, and TBK1. Interestingly, F1,6DP significantly diminished IRF3 activity, in which TBK1 appeared to be the target molecule. These results suggest that F1,6DP could restore the function of BBB altered by inflammatory stimuli maintaining the functional integrity of BBB and suppressing ICAM-1 expression via inhibiting TLR4 signal pathways in bEnd.3 cells, thereby contribute to the protection from brain injury.
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TABLE OF CONTENTS
ABSTRACT i
TABLE OF CONTENTS iii
LIST OF FIGURE vi
LIST OF TABLE viii
LIST OF TABLE viii
ABBREVIATIONS ix
I. INTRODUCTION 1
A. The role of blood-brain barrier in brain 1
B. The importance of cell adhesion molecules in brain inflammation 2
C. The role of F1,6DP for BBB maintenance 3
D. Toll-like receptor 4
II. AIMS OF STUDY 8
A. Effects of F1,6DP on BBB permeability using in vitro BBB model 8
B. Effects of F1,6DP on ICAM-1 expression in brain microvascular endothelial cells 8
III. MATERIALS AND METHODS 9
A. Materials 9
B. Cell culture 9
C. Cell viability 10
D. Bioelectric and permeability assessment 11
E. Western blot analysis 12
F. Immunostaining of cell adhesion molecules 13
G. Cell surface enzyme-linked immunosorbent assay 14
H. RNA preparation and RT-PCR 15
I. Monocyte-endothelial cell adhesion assay 16
J. Electrophoretic mobility shift assay (EMSA) 17
K. ROS assay 18
L. Plasmid 18
M. Luciferase assay 18
N. Statistical analysis 19
IV. RESULTS 20
PART I. Effects of F1,6DP on BBB permeability in vitro BBB model 20
A. Establishment of an in vitro BBB model 20
B. Effects of F1,6DP on the alterations in the distribution of the endothelial tight junction proteins 27
C. Effects of ROS and COX inhibitors on BBB disruption 29
PART II. Effects of F1,6DP on ICAM-1 expression 33
A. Effects of F1,6DP on bEnd.3 cell viability 33
B. Effects of F1,6DP on adherence of leukocyte to bEnd.3 cell 33
C. Effects of F1,6DP on ICAM-1 expression 36
D. Effects of F1,6DP on NF-κB activation 36
E. Effects of LPS on TLR4-related protein expression 40
F. Effects of dominant-negative mutants of TLR4-related proteins on NF-κB activation 40
G. Regulation of MyD88-dependent pathway by F1,6DP 43
H. Effects of MAPK inhibitors on NF-κB activation 43
I. Effects of F1,6DP on NF-κB activation through TRIF-dependent pathway 49
J. Effects of F1,6DP on IFNβ expression through TRIF-dependent pathway 51
V. DISCUSSION 56
REFERENCES 63
국문 요약 77
