Exogenous Thioredoxin Reduces Lipid Accumulation under Inflammatory-Induced Conditions
- 주제(키워드) thioredoxin , antioxidant , inflammation
- 발행기관 고려대학교 대학원
- 지도교수 김찬화
- 발행년도 2014
- 학위수여년월 2014. 2
- 학위구분 박사
- 학과 일반대학원 생명공학과
- 원문페이지 114 p
- 실제URI http://www.dcollection.net/handler/korea/000000048748
- 본문언어 영어
- 제출원본 000045793068
초록/요약
Redox-regulating molecule, recombinant human thioredoxin (rhTRX) which shows anti-inflammatory, and anti-oxidative effects against lipopolysaccharide (LPS)-stimulated inflammation and regulate protein expression levels. LPS-induced reactive oxygen intermediates (ROI) and NO production were inhibited by exogenous rhTRX. In this study identified up/down-regulated intracellular proteins under the LPS-treated condition in exogenous rhTRX-treated A375 cells compared with non-LPS-treated cells via 2-DE proteomic analysis. Also, cytokines were quantitatively measured in in vivo mouse inflammation models using cytometry bead array. Exogenous rhTRX inhibited LPS-stimulated production of ROI and NO levels. TIP47 and ATP synthase may influence the inflammation-related lipid accumulation by affecting lipid metabolism. The modulation of skin redox environments during inflammation is most likely to prevent alterations in lipid metabolism through up-regulation of TIP47 and ATP synthase and down-regulation of inflammatory cytokines. Taken together, these results demonstrate that exogenous rhTRX has anti-inflammatory properties and intracellular regulatory activity in vivo and in vitro. Monitoring of LPS-stimulated pro-inflammatory conditions treated with rhTRX in A375 cells could be useful for diagnosis and follow-up of inflammation reduction related with candidate proteins. These results have a therapeutic role in skin inflammation therapy.
more목차
List of Tables i
List of Figures iii
List of Abbreviations vi
Abstract vii
Ⅰ. Introduction 1
Ⅱ. Literature Review 4
1. Cellular antioxidant enzymes in Thioredoxin system 4
1.1. Thioredoxin 4
1.2. TRX as a biomarker for oxidative stress 6
2. Protective effects of TRX against inflammation 7
2.1. Skin inflammation 7
2.2. Regulatory effects on cytokine production 9
2.3. Molecular mechanism of TRX action against inflammation 11
Ⅲ. Materials and Method 16
1. Cell culture 16
2. Plasmid construction and rhTRX purification 18
3. rhTRX labeling assay 21
3.1. Fluorescence labeling of rhTRX 21
3.2. Confocal Microscopy 23
3.3. Exogenous rhTRX internalization assay 24
4. Protein precipitation and interaction study 25
4.1. Subcellular fractionation of A375 cells 25
4.2. Pull down and Co-IP assay 27
5. Measurement of intracellular ROIs and NO levels 30
6. Protein identification by ESI-Q-TOF MS/MS 31
7. In vivo mouse model and Cytometric Bead Array 35
8. Oil Red O staining 37
9. Statically analysis 38
Ⅳ. Results 39
1. Early steps of exogenous rhTRX bind to plasma membrane and internalization into cytosol 39
2. Fractionation of plasma membrane with exogenous His-rhTRX interaction 42
3. Identification of rhTRX-associated proteins 44
4. rhTRX inhibits LPS-stimulated production of ROI and NO 53
5. rhTRX attenuates LPS-stimulated changes in protein expression 57
5.1. Total protein expression of A375 melanoma cells stimulated with bacterial LPS and rhTRX, as compared with those without treatment of LPS and rhTRX 57
5.2. Identification of differentially expressed proteins in bacterial LPS-stimulation 59
5.3. Direct effects of exogenous rhTRX equilibrate to the expression bacterial LPS-stimulated down-regulation proteins to normal condition 61
6. rhTRX reduces inflammation-induced lipid accumulation 71
7. rhTRX decreases inflammatory cytokines 73
Ⅴ. Discussion 77
Ⅵ. Conclusions 83
Ⅶ. References 84
Abstract in Korean 99
