A Novel Function of Pseudane-VII
- Issued Date
- 2017
- Abstract
- 생물체의 이차대사산물은 성장, 발달 및 생식에는 직접 관여하지 않지만, 주위 환경에서 생존하기 위해서 외부 자극으로부터의 침입 또는 공격에 대항하기 위해 생성하여 방어 역할을 하는 것으로 알려져 있다. 최근 연구에 따르면 다양한 이차대사산물들이 항염증, 항산화, 항균 및 항암 활성을 가지고 있다는 것이 보고되었다. Pseudane-VII은 붉은 멍게의 장에서 분리한 해양 박테리아 pseudoalteromonas sp. M2의 이차대사산물로부터 추출된 화합물이며, 항멜라닌 활성을 가지고 있다고 보고되었다. 하지만, pseudane-VII의 대식세포와 미세아교세포에서의 항염증 및 항신경염증 활성에 관한 분자적 기전과 전임상 효능은 아직 알려지지 않았다. 따라서, 이 연구에서 pseudane-VII의 항염증 효과 및 항신경염증 활성을 연구하였다.
지질 다당체 (LPS)에 의해 자극된 대식세포와 미세아교세포에서 생성된 NO가 pseudane-VII을 처리함으로써 현저하게 감소되었으며, i-NOS, COX-2, IL-6 및 IL-1β 의 mRNA 발현을 억제하였다. 또한, Pseudane-VII은 i-NOS 단백질 발현과 IL-1β 분비를 감소시켰으며, 이는 LPS에 의해 증가된 MAPK와 NF-κB의 인산화 억제를 기인하였다. 나아가 LPS로 자극된 대식세포와 미세아교세포의 활성화에 대한 pseudane-VII의 억제효과를 C57BL/6 마우스의 생체 내에서 확인하였다. 그 결과, LPS로 유도된 염증 모델에서 pseudane-VII도 처리하였을 시 비장에서 LPS에 의해 증가된 IL-1β와 i-NOS의 발현이 저해됨을 관찰하였다. 더욱이, 마우스의 뇌에서도 LPS에 의해 증가된 i-NOS 발현과 미세아교세포 활성화 인자인 Iba-1 억제함을 관찰하였다. 따라서, 본 연구는 대식세포와 미세아교세포에 대한 pseudane-VII의 면역약리학적인 기능을 규명하고, 항염증 및 항신경염증과 그에 관련된 질병에 대한 치료제를 개발하는 데에 잠재적인 효과를 기여할 수 있음을 제시한다.| Recent studies reported that various compounds derived from natural products can regulate immune responses including inflammation and neuroinflammation. Pseudane-VII is a compound isolated from the Pseudoalteromonas sp. M2 metabolite. Also, it has been reported that pseudane-VII possesses anti-melanogenic activity. However, the anti-inflammatory effects and anti-neuroinflammatory activities of pseudane-VII in macrophages and microglia cells are still unknown. In the part I, I investigated the anti-inflammatory effects of pseudane-VII. I found that lipopolysaccaride (LPS)-induced nitric oxide (NO) production was significantly decreased by pseudane-VII treatment. Moreover, pseudane-VII treatment dose-dependently reduced mRNA levels of pro-inflammatory cytokines including interleukin-6 (IL-6), IL-1β,tumor necrosis factor (TNF-α), cyclooxygenase-2 (COX-2), and inducible nitric oxide synthase (i-NOS) in LPS-stimulated macrophages. Pseudane-VII also diminished i-NOS protein levels and IL-1β secretion. In addition, pseudane-VII elicited anti-inflammatory effects by inhibiting extracellular signal-regulated kinases (ERK), c-Jun N-terminal kinases (JNK), p38, and nuclear factor (NF)-κB-p65 phosphorylation. Consistently, pseudane-VII was also shown to inhibit the LPS-stimulated release of IL-1β and expression of i-NOS in mice.
Microglia are activated by immunological stimulation or brain injury. Activated microglia release pro-inflammatory mediators that can damage neurons. Neuroinflammation appears to be associated with certain neurological diseases, including Parkinson’s disease (PD) and Alzheimer’s disease (AD). Natural compounds that suppress microglial inflammatory responses could potentially be used to prevent neurodegenerative diseases or slow their progression. In the part II, this study show that pseudane-VII suppresses inflammation in LPS-stimulated BV-2 microglial cells and decreases the production of NO in a dose-dependent manner. Moreover, pseudane-VII was shown to inhibit the LPS-stimulated the expression of IL-6, COX-2, i-NOS, and IL-1β mRNA in a dose-dependent manner. A dose-dependent reduction in i-NOS and COX-2 protein levels and IL-1β secretion was also observed.
To identify the signaling pathway targeted by pseudane-VII, I used western blog analysis to assess the LPS-induced phosphorylation state of p38, ERK1/2, JNK1/2, and NF-κB. I found that pseudane-VII attenuated LPS-induced phosphorylation of MAPK and NF-κB. Other results indicated that ERK1/2 phosphorylation is essential for the suppression of inflammation by pseudane-VII. Consistently, pseudane-VII was shown to suppress the LPS-induced i-NOS expression and microglial activation in mice brain. Taken together, these results show that pseudane-VII may be useful for the prevention or treatment of inflammatory and neuroinflammatory diseases.
- Alternative Title
- Pseudane-VII : 염증성 질환에 대한 잠재적 치료제
- Alternative Author(s)
- Inae Jung
- Department
- 일반대학원 생명과학
- Advisor
- 이준식
- Awarded Date
- 2018-02
- Table Of Contents
- CONTENTS
LIST OF TABLES...........................................................i
LIST OF FIGURES.........................................................ii
ABBREVIATIONS.........................................................ⅲ
ABSTRACT.....................................................................ⅴ
국문초록...........................................................................ⅶ
I. INTRODUCTION.........................................................1
1. Pseudanes.................................................................................1
(1) Pseudoalteromonas sp. M2..........................................................1
(2) Pseudane-VII...............................................................................2
2. Innate immunity.......................................................................5
(1) Peripheral systems......................................................................5
1) Inflammation......................................................................................5
2) Macrophage........................................................................................6
(2) Central nervous systems.............................................................8
1) Neuroinflammation............................................................................8
2) Microglia.............................................................................................8
3. Signaling pathway..................................................................10
(1) MAPK pathway.........................................................................10
(2) NF-κB pathway..........................................................................10
II. MATERIALS AND METHODS...............................12
1. Extraction and purification of secondary metabolites.......12
2. LC-MS analysis of pseudane-VII.........................................12
3. Mice.........................................................................................13
4. Chemical and reagents..........................................................13
5. Cell culture and pseudane-VII treatment............................14
6. Cytotoxicity assay..................................................................14
7. NO assay.................................................................................14
8. Isolation of total RNA and RT-PCR.........………………...15
9. Enzyme-linked immunosorbent assay (ELISA).................15
10. Western blot analysis...........................................................17
11. Treatment with inhibitors of ERK 1/2 and p38 signaling
pathway.................................................................................19
12. In vivo experiment...............................................................19
13. Immunohistochemistry........................................................19
14. Statistical analysis................................................................20
III. RESULTS..................................................................21
PART Ⅰ. Pseudane-VII isolated from Pseudoalteromonas sp.
M2 ameliorates LPS-induced inflammatory response in
vitro and vivo......................................................................21
1. LC-MS analysis of the secondary metabolite.........................21
2. Purification and analysis of pseudane-VII...............................21
3. Pseudane-VII inhibits NO production in LPS-stimulated
macrophages..............................................................................22
4. Pseudane-VII suppresses pro-inflammatory enzyme
expression..................................................................................27
5. Pseudane-VII diminishes pro-inflammatory cytokine
production..................................................................................27
6. Pseudane-VII inhibits LPS-induced MAPK activation and
NF-κB signaling........................................................................31
7. Pseudane-VII decreases i-NOS expression and IL-1β
production in LPS-treated mice..............................................34
PART Ⅱ. Pseudane-VII suppresses neuroinflammation in
LPS-stimulated BV-2 microglial cells via the ERK1/2
pathway..................................................................................37
1. Pseudane-VII inhibits LPS-induced NO production in BV-2
microglial cells...........................................................................37
2. Pseudane-VII decreases gene expression, protein levels of i-
NOS and COX-2......................................................................40
3. Pseudane-VII reduces LPS-stimulated expression of proinflammatory
genes and cytokines..........................................40
4 . Pseudane-VII suppresses MAPK and NF-κB
phosphorylation........................................................................44
5. Pseudane-VII ameliorates LPS-induced i-NOS expression in
brain...........................................................................................44
6. Pseudane-VII suppresses LPS-induced microglia activation in
brain hippocampus...................................................................47
IV. DISCUSSION............................................................50
V. REFERENCES...........................................................53
ACKNOWLEDGEMENT..............................................59
- Degree
- Master
- Publisher
- 조선대학교 대학원
- Citation
- 정인애. (2017). A Novel Function of Pseudane-VII.
- Type
- Dissertation
- URI
- https://oak.chosun.ac.kr/handle/2020.oak/13356
http://chosun.dcollection.net/common/orgView/200000266477
- Appears in Collections:
- General Graduate School > 3. Theses(Master)
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