Glycoprotein isolated from Dioscorea japonica activates mouse macrophages
- Issued Date
- 2007
- Abstract
- 국문초록
Discorea japonica에서 분리한 당단백질에 의한 생쥐 대식세포의 활성화에 관한 연구
Discorea japonica는 동양의학에서 조절되지 않는 배뇨, 당뇨 및 만성 설사 등에 전통적으로 사용되어 왔다. 본 연구에서 D. japonica에서 분리한 당단백질 (DJ)의 TNF-α, IL-1β, COX-2 및 iNOS 발현에 미치는 영향을 생쥐 대식세포 Raw 264.7 세포에서 관찰하였다. DJ은 단독으로 TNF-α 및 COX-2 유전자의 발현을 유도하였으나, NO 생성 및 iNOS 유전자발현은 유도하지 못했다. DJ는 높은 농도(25 µg/ml)에서만 IL-1β 유전자의 발현을 유도하였다. Western blot, RT-PCR 및 ELISA 분석으로 DJ와 INF-γ의 병용투여가 iNOS와 IL-1β 유전자 발현의 유도에 강한 상승효과를 보인다는 것을 밝혔다. Electrophoretic mobility shift assay는 DJ가 단독으로 NF-kB/Rel을 활성화시켰고, INF-γ와 동시 투여시 약간 상승시킨다는 것을 보여주었다. 뿐만 아니라, DJ는 단독으로 p44/42 MAPK 및 SAPK/JNK 인산화를 강력하게 유도하였으나, p38 MAPK는 유도시키지 않았다. 반대로, DJ와 INF-γ를 병용투여한 경우 p38 MAPK을 상승적으로 활성화시켰으나, p44/42 MAPK 및 SAPK/JNK은 단지 약간만 활성화시켰다. p38의 특이 억제제인 SB203580은 DJ와 INF-γ의 병용 투여에 의해 유도되는 NO의 생성과 iNOS 유전자 발현을 부분적으로 억제하였다. 이상의 결과는 DJ가 TNF-α, IL-1β, COX-2 유전자 발현을 증가시킴으로써 Raw 264.7 세포를 활성화시키며, 이러한 효과는 p44/42 MAPK 및 SAPK/JNK 경로에 의해 조절될 수 있고, DJ와 INF-γ의 병용투여는 NO의 생성과 iNOS 유전자 발현을 강력하게 상승적으로 유도하며, 이러한 효과는 p38 MAPK에 의해 매개될 수 있다는 것을 의미한다. 그러므로 DJ는 면역조절제로 사용될 수 있을 것으로 생각된다.|Abstract
Dioscorea japonica has been used not only in traditional Chinese medicine, but also in modern medicine to treat for uncontrollable urination, diabetes and chronic diarrhea. In this study, the effect of glycoprotein isolated from D. japonica (DJ) on the expression of TNF-a, IL-1b, COX-2 and iNOS was assessed in RAW 264.7 cells. DJ alone induced the expression of TNF-a and COX-2 genes, but neither induced the NO production nor iNOS gene expression. IL-1b gene expression was induced only in a higher concentration (25 mg/ml) of DJ. Western blot, RT-PCR and ELISA revealed that DJ in combination with INF-g showed a strongly synergistic induction on NOS and IL-1b gene expression. Electrophoretic mobility shift assay showed that DJ alone and in combination with IFN-g could activate NF-kB/Rel in a slightly synergistic manner. In addition, DJ alone strongly induced p44/42mapk, and SAPK/JNK phosphorylation but not p38mapk phosphorylation. In contrast, DJ in combination with IFN-g synergistically activated p38mapk, but only slightly activated p44/42mapk and SAPK/JNK. The specific p38 inhibitor SB203580 partly inhibited NO production and iNOS gene expression. These data indicate that DJ stimulates RAW 264.7 cells through increasing gene expression of TNF-a, IL-1b and COX-2, which can be regulated by p44/42mapk and SAPK/JNK pathways, and that DJ in combination with IFN-g induce strongly NO production and iNOS gene expression in a synergistic manner, which can be mediated by p38 mapk. It could be implicated that DJ could be used as an immune modulator.
Key words: Dioscorea japonica; Macrophages; TNF-a; IL-1b; COX-2; iNOS; IFN-g; MAPKs
- Alternative Author(s)
- Pham Thi Thu Huong
- Affiliation
- Graduate School of Chosun University
- Department
- 일반대학원 바이오신약개발학과
- Awarded Date
- 2008-02
- Table Of Contents
- TABLE OF CONTENT
TABLE OF CONTENT I
LIST OF FIGURES III
ABBREVIATIONS IV
ABSTRACT VI
CHAPTER 1: INTRODUCTION 1
CHAPTER 2: MATERIALS AND METHODS 4
2.1. MATERIALS 4
2.1.1. DJ 4
2.1.2. RT-PCR assay 4
2.1.3. Antibodies 4
2.1.4. SDS-PAGE and Western blot 5
2.1.5. EMSA . 6
2.1.6. Cell culture 6
2.2. METHODS 6
2.2.1. NO measurement 6
2.2.2. Determination of IL-1b and TNF-a levels 7
2.2.3. Determination of protein concentration 7
2.2.4. SDS-PAGE and Western blot 7
2.2.5. RT-PCR assay 8
2.2.6. EMSA 9
2.2.7. Statistic analysis 9
CHAPTER 3: RESULTS 10
Part I: Macrophage activation by DJ
3.1.1. Effect of DJ on nitrite production and iNOS expression 10
3.1.2. Effect of DJ on gene expression of some cytokines 13
3.1.3. The activation of NF-kB/Rel by DJ 16
3.1.4. The activation of p38, p44/42 and JNK by DJ 18 Part II: Synergistic macrophage activation by DJ and IFN-g
3.2.1. The synergistic effect of DJ and IFN-g on NO production and iNOS gene expression 20
3.2.2. The synergistic effect of DJ and IFN-g on gene expression of some cytokines 24
3.2.3. The synergistic activation of DJ and IFN-g on NF-kB/Rel 27
3.2.4. The synergistic effects of DJ and IFN-g on the phosphorylation of p38mapk, p44/42 and SAPK/JNK 29
3.2.5. The effect of p38 inhibitor SB203580 on iNOS gene expression in RAW 264.7 cells 31
CHAPTER 4: DISCUSSION 33
CHAPTER 5: CONCLUSION 37
CHAPTER 6: REFERENCES 39
LIST OF FIGURES
Fig. 1: Effect of DJ on NO production and iNOS gene expression in RAW 264.7 cells 12
Fig. 2: Effect of DJ on gene expression of some cytokines in RAW 264.7 cells 15
Fig. 3: Fig. 3. The activation of NF-kB/Rel by DJ in RAW 264.7 cells 17
Fig. 4: Effect of DJ on the phophorylation of p38, p44/42, and JNK/SAPK in RAW 264.7 cells 19
Fig. 5: The synergistic effect of DJ and IFN-g on the NO production and iNOS gene expression in RAW 264.7 cells 21
Fig. 6: The synergistic effect of DJ and IFN-g on the morphology of RAW 264.7 cells 22
Fig. 7: Immunohistochemical staining of iNOS in RAW 264.7 cells 23
Fig. 8: The synergistic effect of DJ and IFN-g on gene expression of some cytokines in RAW 264.7 cells. 26
Fig. 9: The synergistic activation of DJ and IFN-g on NF-kB/Rel in RAW 264.7 cells 28
Fig. 10: The synergistic effects of DJ and IFN-g on the phosphorylation of p38mapk, p44/42 and SAPK/JNK in RAW 264.7 cells 30
Fig. 11: The effect of p38 inhibitor SB203580 on iNOS gene expression in RAW 264.7 cells. 32
ABBREVIATIONS
BSA Bovine serum albumin
DJ Glycoprotein from Dioscorea japonica
DMEM Dulbecco’s modified eagle’s medium
DTT Dithiothreitol
EDTA Ethylenediamine tetraacetic acid
EGTA Ethyleneglycol tetraacetic acid
ELISA Enzyme-linked immunosorbent assay
EMSA Electrophoretic mobility shift assay
FBS Fetal bovine serum
HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
iNOS Inducible nitric oxide synthase
LPS Lipopolysaccharide
MAPKs Mitogen-activated protein kinases
NO Nitrite oxide
PKA Protein kinase A (or cAMP-dependent protein kinase (cAPK)
PKC Protein kinase C
PMSF Phenylmethanesulphonylfluoride
RT-PCR Reverse transcription-polymerase chain reaction
SD Standard deviation
SDS-PAGE Sodium dodecyl sulfate-polyacrylamide gel electrophoresis
TBE Tris/boric acid EDTA buffer
TEMED N, N, N’, N- Tetramethylethylenediamine
- Degree
- Master
- Publisher
- 조선대학교
- Citation
- 팜띠뚜흐엉. (2007). Glycoprotein isolated from Dioscorea japonica activates mouse macrophages.
- Type
- Dissertation
- URI
- https://oak.chosun.ac.kr/handle/2020.oak/6988
http://chosun.dcollection.net/common/orgView/200000235875
- Appears in Collections:
- General Graduate School > 3. Theses(Master)
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