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양파와 유자 추출물의 허혈/저산소성 뇌혈관장벽 손상에 대한 보호 효과 및 기전 연구

The Effects of Onion and Yuzu Extracts against Ischemia/Hypoxia-Induced Dysfunction of Blood-Brain Barrier

초록/요약

This study investigated the potential beneficial effects of natural products and their major component on redistribution of ZO-1 and FoxO3a-mediated degradation of claudin-5 during ischemia/hypoxia using the mouse middle cerebral artery occlusion (MCAO) model and hypoxia model. The possible underlying mechanisms are also investigated, especially those linked to the oxidative stresses. Onion extract (OE) and its major component, quercetin (Quer), as well as yuzu extract (YE) and hesperidin (HSP) prevented brain ischemia-induced brain edema in dose-dependent manners. Evans blue extravasation in the ischemic hemisphere of the mouse brain was also significantly reduced by treatment with the four materials. In addition, they inhibited the immunoreactivity of tight junction proteins (TJs; ZO-1 (Zonula occludens-1) and claudin-5). In in vitro model, pre-treatment of OE, Quer, YE, and HSP significantly attenuated hypoxia-induced Blood-Brain Barrier (BBB) hyperpermeability. The four materials also inhibited hypoxia-induced redistribution of ZO-1, degradation of claudin-5, increase of matrix metalloproteinase (MMP)-3/9 mRNA levels, and translocation of Forkhead box O 3a (FoxO3a) into nucleus. The effects of antioxidant, trolox, and N-acetyl-cysteine (NAC) as well as siFoxO3a transfection also mimicked those effects of OE, Quer, YE, and HSP. However, transfection of siFoxO3a did not inhibit transcription of MMP-9 and redistribution of ZO-1 induced by hypoxia. In addition, OE, Quer, YE, and HSP attenuated the generation of oxidative stress induced by hypoxia, indicating that they may have antioxidant effects against hypoxia induced-dysfunction of BBB. The results from this study demonstrate that OE, Quer, YE, and HSP prevent BBB hyperpermeability and tight junction proteins (TJs) disruption in MCAO and hypoxia model. In addition, these findings suggest that BBB protection by the four materials involves reduction of MMPs transcription, the inhibition of ZO-1 redistribution, and FoxO3a inhibition-mediated suppression of claudin-5 degradation, possibly through its antioxidant effects in hypoxia model.

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TABLE OF CONTENTS

ABSTRACT i
TABLE OF CONTENTS iii
LIST OF FIGURES vi
ABBREVIATION viii
I. INTRODUCTION 1
II. MATERIALS AND METHODS 4
A. Transient focal cerebral ischemia 4
B. Measurement of brain water content 5
C. Measurement of Evans blue extravasation 5
D. Immunohistochemistry for TJs 5
E. Cell culture 6
F. Endothelial cell monolayer permeability assay 6
G. Immunocytochemistry for TJs and FoxO3a 7
H. Western blot analysis 7
I. Preparation of cell fractions 8
J. Polymerase chain reaction (PCR) 8
K. siRNA transfection 9
L. Measurement of DCF-DA fluorescence 9
M. Preparation of the OE, Quer, YE, and HSP 9
N. Statistical analysis 10


III. RESULTS 11
A. The effects of OE, Quer, YE, and HSP on water content in brain ischemia model 11
B. The effects of OE, Quer, YE, and HSP on Evans blue extravasation in brain ischemia model 14
C. The effects of OE, Quer, YE, and HSP on TJs disruption in brain ischemia model 16
D. The effects of OE, Quer, YE, and HSP on BBB permeability in hypoxia model 18
E. The effects of OE, Quer, YE, and HSP on disruption of ZO-1 and claudin-5 in hypoxia model 22
F. The effects of OE, Quer, YE, and HSP on the expression of MMP-3 and MMP-9 in hypoxia model 26
G. The effects of OE, Quer, YE, and HSP on translocation of FoxO3a into nucleus in hypoxia model 28
H. FoxO3a modulates expression of MMP-3 and MMP-9 in hypoxia model 30
I. FoxO3a affects the degradation of claudin-5 but redistribution of ZO-1 in hypoxia model 32
J. FoxO3a regulates hypoxia-induced BBB hyperpermeability 34
K. The effects of oxidative stress scavengers on hypoxia-induced BBB dysfunction in hypoxia model 36
IV. DISCUSSION 40
V. CONCLUSION 46
VI. REFERENCES 47
국문요약 52

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