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Immune Regulation Effects of Lipid Extracted from Aptocyclus ventricosus Eggs on RAW264.7 Macrophages

뚝지 알에서 추출한 지질의 RAW264.7 대식세포에서의 면역조절 효과

초록/요약 도움말

The lipids were extracted from Aptocyclus ventricosus eggs, which consisted of saturated fatty acids (SFAs, 24.37%), 20.90% monounsaturated fatty acids (MUFAs, 20.90%), and polyunsaturated fatty acids (PUFAs, 54.73%). They showed the high contents of DHA (C22:6n-3) and EPA (C20:5n-3) at approximately 25.91% and 22.05%, respectively, by gas chromatography analysis. The present study investigated the immune-regulatory activities, as well as the molecular mechanisms of A. ventricosus lipids on macrophage cells. Various concentrations (100-300 μg/mL) of A. ventricosus lipids were treated in RAW264.7 cells to evaluate the inflammatory responses. Firstly, the immune-enhancement activities of A. ventricosus lipids effectively enhanced nitric oxide (NO) production, and phagocytosis in a dose-dependent manner. A. ventricosus lipids increased the expression of inflammatory mediators (COX-2 and iNOS) and pro-inflammatory cytokines (IL-6, IL-1β and TNF-α). Moreover, A. ventricosus lipids upregulated the phosphorylation of MAPKs (JNK, ERK, and p38) as well as the expression of phosphorylated nuclear factor kappa B (NF-κB) subunit p65 in a concentration-dependent manner, indicating that these lipids activated MAPK and NF-κB signaling pathways. Secondly, the anti-inflammatory effects of A. ventricosus lipids significantly inhibited LPS-induced NO production. In LPS-stimulated RAW264.7 macrophages, mRNA expression of COX-2, TNF-α, IL-6, IL-1β, and iNOS mRNA was inhibited. It also significantly down-regulates LPS-induced expression of the cell surface CD86 molecule. In addition, A. ventricosus lipids remarkably downregulated LPS-induced phosphorylation of JNK, ERK, p38, and NF-κB p65 by suppressing MAPK and NF-κB activation. Collectively, these results suggest that the lipids from A. ventricosus eggs may have strong immune-regulatory biological activities on immune cells and could be potentially used as a source of immunomodulatory agents.

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초록/요약 도움말

A. ventricosus의 알 에서 추출한 지질은 가스 크로마토그래피 분석 결과 포화지방산포화지방산(SFAs) 24.37%, 단일불포화지방산단일불포화지방산(MUFAs) 20.90%, 다가불포화지방산다가불포화지방산(PUFAs) 54.73%로 구성되어있었으며 , DHA(C22:6n-3) 와 EPA(C20:5n-3) 는 각각 약약25.91%와 22.05%로 높은 함량을 보 였 다 . 본 연구에서는 대식세포에서 A. ventricosus알 에서 추출한 지질지질(100-300 μg/mL)이 가지는 분자 기전 뿐만아니라 면역 활성을 조사하였다. 첫번째로, A. ventricosus지질의 면역강화활성은 용량 의존적으로 NO 생성량과 식균작용을 효과적으로 향상시켰으며 , 염증매개체염증매개체(COX-2, iNOS) 및 전염증성 사이토카인사이토카인(IL-1β, IL-6, TNF-α)의 발현을 증가시켰다. 또한, 농도의존적 인 방식으로 MAPKs (JNK, ERK, p38)의 인산화와 NF-κB 신호전달 경로를 활성화 하는 것을 확인하였다. 두번째로, A. ventricosus알에서 추출한 지질의 항염효과는 LPS로 자극된 대식세포에서 NO생성을 유의미하게 억제 시켰으며 , COX-2, iNOS, IL-1β, IL-6, TNF-α의 mRNA 발현수준을 감소시켰다. 또한 LPS로 자극된 세포 표면 분자 (CD86)와 MAPKs, NF-κB 신호전달경로 의 인산화를 현저히 하향조절하는 것을 확인 할 수 있었다 . 종합적으로 이와 같은 결 과 A. ventricosus 알 에서 추출한 지질이 면역세포에 대한 강력한 면역조절 생물학적 활성을 가질수있으며, 면역조절제의 공급원으로 사용될 수 있는 잠재력을 내포하고있음을 시사하고있다

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목차 도움말

Table of contents I
List of Tables V
List of Figures VI
Acknowledgements VII
Abstract VIII
Chapter 1. Literature Review 1
1. Inflammation 1
2. Lipids 4
3. Aptocyclus ventricosus 6
4. References 7
Chapter 2. Immune-enhancing effects of lipid extracted from Aptocyclus ventricosus eggs on RAW264.7 cells 21
Abstract 21
1. Introduction 22
2. Materials and methods 23
2.1 Preparation of Aptocyclus ventricosus lipids 23
2.2 Determination of fatty acid compositions 23
2.3 Cell culture 24
2.4 Treatments of sample in RAW264.7 cells 24
2.5 Assay of cell viability 24
2.6 Measurement of NO production 24
2.7 RNA isolation and real-time PCR 25
2.8 Western blotting assay 26
2.9 Inhibition of NF-κB and MAPK using specific inhibitors 27
2.10 Determination of phagocytosis 27
2.11 Statistical analysis 27
3. Results and discussion 28
3.1 Fatty acid analysis of A. ventricosus egg lipids 28
3.2 Effects of A. ventricosus lipids on cell proliferation 30
3.3 A. ventricosus lipids enhance NO production in RAW264.7 cells 30
3.4 A. ventricosus lipids enhance immune-associated gene expression in RAW 264.7 cells 33
3.5 A. ventricosus lipids enhance the activation of NF-κB and MAPK signaling pathway 35
3.6 A. ventricosus lipids activate NF-κB and MAPK inhibited-RAW264.7 cells 37
3.7 A. ventricosus lipids enhance phagocytic activities of RAW264.7 cells 39
4. Conclusions 41
5. References 42
Chapter 3. Anti-inflammatory effects of lipid extracted from Aptocyclus ventricosus eggs on RAW264.7 cells 47
Abstract 47
1. Introduction 48
2. Materials and methods 49
2.1 Extraction of A. ventricosus lipids 49
2.2 Treatments of sample in LPS-inducted RAW264.7 cells 49
2.3 Determination of cell proliferation and NO production 49
2.4 Quantitative RT-PCR analysis (qRT-PCR) 50
2.5 Western blot analysis 50
2.6 Analysis of expression of surface molecules 50
2.7 Monitor TNF-α expressions of A. ventricosus with specific inhibitors 51
2.8 Statistical analysis 51
3. Results and discussion 52
3.1 Effect of A. ventricosus lipids on cell proliferation in LPS-stimulated RAW264.7 cells 52
3.2 A. ventricosus lipids inhibit LPS-induced NO production 53
3.3 A. ventricosus lipids inhibit LPS-induced gene expression 55
3.4 A. ventricosus lipids inhibit LPS-induced MAPK and NF-κB activation 57
3.5 A. ventricosus lipids inhibit LPS-induced cell surface expression 60
4. Conclusions 63
5. References 64
Chapter 4. Summary 70

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