지질다당류에 의해 활성화된 대식세포에서의 4-tert-부틸 페닐 살리실산의 항염증 효과

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Inflammation may lead to onset of a variety of diseases if it runs uncontrolled. Over-expression of both inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) proteins is well known to be crucially related to stimulative effect on some severe chronic inflammatory diseases as well as various tumors. Nuclear factor-κB (NF-κB) is also known to play a critical role in transcriptional regulation of these proteins. This study aims to investigate the anti-inflammatory effect of 4-tert-butyl phenyl salicylic acid (TBPS), one of the salicylic acid derivatives, and to figure out its mechanism by using the model of lipopolysaccharides (LPS)-stimulated Raw 264.7 mouse macrophage cells.

MTT assay, Western blot analysis, ELISA analysis, and Reverse transcription and polymerase chain reaction (RT-PCR) analysis were performed in order to explore the anti-inflammatory effect

of TBPS. It was found that TBPS meaningfully regulated nitric oxide (NO) production without cytotoxic effects on Raw 264.7 cells in a stable state of 1∼15 µg/㎖. TBPS dose-dependently reduced iNOS expression, and COX-2 expression significantly in a range of 1∼15 µg/㎖. Moreover, expressions of iNOS mRNA and COX-2 mRNA meaningfully decreased in a dose dependent manner. In addition, TBPS significantly inhibited the production of inflammatory mediators or pro-inflammatory cytokines such as tumor necrosis factor-a (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6). Moreover, mRNA gene expressions of TNF-α, IL-1β and IL-6 attenuated in a dose dependent manner.

Furthermore, it was found that TBPS suppressed the LPS-stimulated DNA binding activity of NF-κB. TBPS potently inhibited the translocation of NF-κB into the nucleus by IκBα degradation following IκBα phosphorylation. It indicates that the TBPS inhibits NF-κB activation.

In conclusion, the results show the first that TBPS exerts the anti-inflammatory effect in vitro, both by inhibiting the expressions of iNOS and COX-2 in Raw 264.7 cells, and by suppressing the activation of the NF-κB signaling pathway. Therefore, TBPS may be a potential therapeutic candidate for the regulatory agents of inflammation.
Alternative Title
Anti-inflammatory effect of 4-tert-butyl phenyl salicylic acid in lipopolysaccharides-stimulated Raw 264.7 mouse macrophage cells
Alternative Author(s)
Na, Baek Hee
일반대학원 보완대체의학과
Awarded Date
Table Of Contents
목 차

도목차 iii

표목차 iv


List of Abbreviations vii

Ⅰ. 서론 1

Ⅱ. 재료 및 방법 12
1. 시약 12
2. 대식세포 배양 12
3. 세포 생존율 13
4. NO 생성 측정 13
5. Western blot analysis 14
6. ELISA를 이용한 IL-6, IL-1β, TNF-α 생성량 측정 14
7. RT-PCR을 이용한 iNOS, COX-2 및 사이토카인 mRNA 발현 측정 15
8. 통계분석 16

Ⅲ. 결과 18
1. TBPS의 Raw 264.7 세포의 증식에 미치는 영향 18
2. TBPS의 NO 생성 저해 효과 20
3. TBPS의 iNOS 발현 억제 효과 22
4. TBPS의 COX-2 발현 억제 효과 24
5. TBPS의 세포 내 염증 관련 단백질의 mRNA 발현에 미치는 영향 26
6. TBPS의 염증 매개성 사이토카인 저해 효과 28
7. TBPS의 염증 매개성 사이토카인의 mRNA 발현 억제 효과 30
8. TBPS의 NF-kB, IkBα, p-IkBα 발현에 미치는 영향 32

Ⅳ. 고찰 35

Ⅴ. 결론 40

Ⅵ. 참고문헌 42

Ⅶ. 감사의 글 49
조선대학교 대학원
나백희. (2014). 지질다당류에 의해 활성화된 대식세포에서의 4-tert-부틸 페닐 살리실산의 항염증 효과.
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