Journal of Life Science (생명과학회지)

Korean Society of Life Science (한국생명과학회)
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Proteome Analysis of Responses to Ascochlorin in LPS-induced Mouse Macrophage RAW264.7 Cells by 2-D Gel Electrophoresis and MALDI-TOF MS.

LPS로 자극된 macrophage RAW264.7 세포에서 ascochlorin에 대한 단백질체 분석

  • Chang, Young-Chae (Research Institute of Biomedical Engineering and Department of Pathology, Catholic University of Daegu School of Medicine)
  • 장영채 (대구가톨릭대학교 의용생체공학연구소, 대구가톨릭대학교 의과대학 병리학교실)
  • Published : 2008.06.30
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Abstract

Ascochlorin (ASC) is prenyl-phenol compound that was isolated from the fungus Ascochyta viciae. ASC reduces serum cholesterol and triglyceride levels, and suppresses hypertension, tumor development, ameliorates type I and II diabetes. Here, to better understand the mechanisms by which ASC regulates physiological or pathological events and induces responses in the pharmacological treatment of inflammation, we performed differential analysis of the proteome of the mouse macrophage RAW264.7 cells in response to ASC. In this study, we used a proteomic analysis of LPS-induced RAW264.7 cells treated by ASC, to identify proteins potentially involved in inflammatory processes. The RAW264.7 cell proteomes with and without treatment with ASC were compared using two-dimensional electrophoresis (2-D SDS-PAGE), matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF-MS) and bioinformatics. The largest differences in expression were observed for the calreticulin (4-fold decrease), ${\beta}-actin$ (4-fold decrease) and vimentin (1.5-fold decrease). In addition, rabaptin was increased 3-fold in RAW264.7 cells treated with ASC. The expression of some selected proteins was confirmed by RT-PCR analysis.

아스코크로린(Ascochlorin, ASC)은 Ascochyta viciae로부터 추출된 프레닐페놀 물질로, 혈청 콜레스테롤과 트리글리세라이드 수치를 감소시키고 종양 성장을 억제한다는 연구 결과가 보고되어 있다. 본 논문에서는 아스코크로린이 생리학적 혹은 병리학적인 작용과 염증반응에서 약리학적으로 유도되는 반응을 어떻게 조절하며, 이러한 메커니즘에 대해 이해하기 위해 mouse macrophage Raw264.7 세포에 아스코크로린을 처리하여 이에 대한 프로테옴의 특이적인 발현에 대해 분석하였다. 따라서 본 연구는 LPS를 처리한 mouse macrophage Raw264.7 세포에 아스코크로린을 처리하여 염증과정에 관련된 단백질의 발현 양상을 확인하기 위해 프로테오믹스를 시행하였다. Mouse macrophage RAW264.7 세포에 아스코크로린을 처리한 조건과 무처리한 조건으로 나누어 two-dimensional electrophoresis (2-D SDS-PAGE), matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF-MS)와 bioinformatics 방법으로 아스코크로린을 처리한 mouse macrophage Raw264.6 세포의 프로테옴을 분석하였다. 그 결과 mouse macrophage Raw264.7 세포에 아스코크로린 처리 시 Calreticulin이 4배 감소, ${\beta}-actin$도 4배 감소 그리고 vimentin이 1.5배 감소함을 확인 할 수 있었다. 그러나 rabaptin 아스코크로린 처리에 의해 3배 증가함을 확인 할 수 있었다. 이러한 단백질 발현은 RT-PCR을 수행하여 결과에 대해 재확인 하였으며, 프로테오믹스와 동일한 결과를 얻을 수 있었다. 따라서 본 연구를 통해 LPS 처리에 의해 활성화된 mouse macrophage RAW264.7 세포에 ASC를 처리한 후 이차원 전기영동법을 이용하여, 단백질의 발현 변화 및 양상을 규명하고 단백질 지도를 확립 하였으며, RAW264.7 세포를 이용한 면역세포 모델에서 ASC의 항염증 작용을 중심으로 생리활성 조절기능을 확인 할 수 있었다. 향후 분자 기능 조절 연구와 전 임상 연구를 통해 ASC의 생리활성 조절 기능을 규명한다면 ASC는 항염증 및 항암활성을 갖는 약물로 개발될 것으로 기대된다.

Keywords

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