BV-2 microglia에서 F1,6DP가 LPS로 유도된 세포내 산화적 스트레스에 미치는 영향
Fructose-1,6-diphosphate Regulates Cellular Redox through Up-regulation of Pentose Phosphate Pathway in BV-2 Microglia
- 주제(키워드) Fructose-1 , 6-diphosphate + F1 , 6DP + ROS + iNOS + NO + MAPKs + PPP + G6PD + BV-2 + microglia
- 발행기관 아주대학교
- 지도교수 이수환
- 발행년도 2006
- 학위수여년월 2006. 8
- 학위명 박사
- 학과 및 전공 일반대학원 신경과학기술과정
- 본문언어 영어
초록/요약
Fructose-1,6-disphosphate (F1,6DP), a glycolytic intermediate, is known to have protective activities against ischemia / reperfusion injury and septic shock induced by bacterial lipopolysaccharide (LPS), a Gram-negative cell wall component. In this study, it was shown that F1,6DP could effectively reduce LPS-induced iNOS expression and NO production in BV-2 mouse microglia. F1,6DP attenuated iNOS expression through down-regulation of AP-1 DNA binding activity and interception of MAPKs, JAK / STAT and Akt / GSK-3β pathways. In consideration of ROS-contribution to iNOS expression and NO production, it was tested whether F1,6DP affects LPS-induced ROS production in BV-2 cells. F1,6DP significantly reduced LPS-induced ROS production in BV-2 cells. It was also found that F1,6DP enhanced LPS-induced activity of glucose-6-phopsphate dehydrogenase (G6PD), a key enzyme of PPP, which paralleled the enhancement of LPS-stimulated G6PD expression in the levels of mRNA and protein. Ectopic expression of G6PD resulted in the diminution of iNOS expression and NO production as well as ROS production. Considering PPP is one of the most important ROS scavenging mechanism, our data obtained hitherto suggest that F1,6DP can reduce the cellular ROS generation by increasing metabolic flux through PPP, that is, by providing substrate for G6PD, and up-regulation of G6PD. And also, it was suggested that ROS regulation by F1,6DP might be functionally linked to the down-regulation of LPS-induced iNOS expression and NO production in BV-2 microglia.
more목차
ABSTRACT i
TABLE OF CONTENTS iii
LIST OF FIGURE v
ABBREVIATIONS vii
I. Introduction 1
1. Neuronal injury by NO and ROS 1
2. The neuroprotective effects of F1,6DP 4
II. Aims of Study 7
1. Effects of F1,6DP on iNOS expression and NO production 7
2. Effects of F1,6DP on PPP 7
3. Possible linkage between its effects on PPP and iNOS expression 7
III. Materials and Methods 8
1. Materials 8
2. Cell culture 8
3. BV-2 microglial cell viability 9
4. ROS assay 9
5. GSSG / GSH assay 10
6. Western blot analysis 11
7. RNA preparation and RT-PCR 12
8. NO assay 13
9. G6PD assay 13
10. Transfection study 14
11. Statistical analysis 15
IV. Results 16
1. Effects of F1,6DP on BV-2 microglial cell viability 16
2. Effects of F1,6PD on NO production and iNOS expression 16
3. Effects of F1,6DP on iNOS mRNA stabilization 20
4. Effects of F1,6DP on DNA binding activity of transcription factors 20
5. Effects of F1,6DP on MAPKs phosphorylation 23
6. Effects of F1,6DP on JAK / STAT and Akt / GSK-3β pathway 27
7. Effects of F1,6DP on ROS regulation 27
8. Regulation of pentose phosphate pathway by F1,6DP 34
9. Short-term effects of F1,6DP on G6PD activity 40
10. Effects of G6PD overexpression on NO and ROS production 44
V. Discussion 52
VI. References 59
국문 요약 65
