探討N-hydroxycinnamoylphenalkylamides衍生物及Cinnamophilin抑制人類嗜中性白血球及單核球活化之作用機轉

Investigation of the Inhibitory Mechanisms of N-hydroxycinnamoylphenalkylamides Analogues and Cinnamophilin on the Activation in Human Neutrophils and THP-1 Cells

葉宇涵(Yu-Hen Yeh)

指導教授 ： 蕭哲志

臺北醫學大學/醫學院/醫學科學研究所/碩士(2008年)

摘要

在人類的免疫系統(Immune system)中，嗜中性白血球(Neutrophils)以及單核球(Monocytes)是扮演主要的防禦角色。有許多證據顯示當人體遭受外來的病原如細菌、真菌、病毒等感染時，嗜中性白血球會表現化學趨化性的現象，進而產生去顆粒化作用、釋放出大量的發炎前驅性細胞激素及蛋白質分解?△左娃閮繹i行吞噬作用。另外單核球也能夠分化成為巨噬細胞，進行更為有力且較長期的免疫作用。在這些釋放出來的物質中主要包括腫瘤壞死因子(Tumor necrosis factor-??, TNF-??)、介白素(Interleukin)等等，可以活化許多基因表現，像是合成和組織分解有關的基質金屬蛋白酵素(Matrix metalloproteinases, MMPs)。但是當身體正常的調控機制發生缺陷，使得上述的免疫反應在未受控制的情形下被誘導出來，則會造成許多發炎及自體免疫性疾病的發生，例如急性肺部損傷(Acute lung injury, ALI)、類風濕性關節炎(Rheumatoid arthritis, RA)及再灌流傷害(Reperfusion injury)等症狀。然而當以上的病理情況發生時，循環系統中的嗜中性白血球及單核球會被快速且過度的活化，造成人體內的組織受到傷害，因此在本篇論文中將評估生物活性成分抑制活化的作用機轉之探討。
由研究結果發現，由天然物萃取出的cinnamophilin (MA-1)及N-hydroxycinnamoylphenalkylamides的衍生物(EK8)具有抑制嗜中性白血球和單核球細胞(THP-1)誘導呼吸爆發作用(Respiratory burst)以及MMPs活性的能力。在實驗中我們利用化學冷光分析法(Chemiluminescence assay)得知，對於嗜中性白血球cinnamophilin和EK8可以抑制fMLP刺激超氧化自由基(Superoxide)的產生，但是EK8而非cinnamophilin也可以抑制PMA的刺激現象。由細胞內鈣離子分析法(Intracellular calcium analysis)、流式細胞技術(Flow cytometry analysis)及西方點墨法(Western blot)可觀察到cinnamophilin具有些微影響鈣離子濃度上升及黏附分子CD11b表現之效果，但對ERK1/2訊息傳遞的表現則無明顯影響。另外以電泳酵素分析法(Gelatin zymography)及西方點墨法發現EK8於THP-1細胞能夠抑制TNF-?恁BLPS、IL-1?狺垌GF-??1誘發之MMPs酵素活性表現，且細胞存活率的測定證明抑制作用並非藉由其細胞毒性產生。在轉錄(Transcription)層級方面，利用反轉錄-聚合酵素連鎖反應(RT-PCR)，EK8可以壓制TNF-???n刺激MMP-9 mRNA的表現，進一步探討發現其也抑制了I?羠?悛滬偶悝@用，且可能影響JNK的訊息路徑。但是對於其詳細的分子作用機轉還需要更深入的探討，而相關的活體動物試驗，以及在未來是否能夠應用到臨床的治療上仍是值得加以努力的方向。

關鍵字

嗜中性白血球

並列摘要

In the human innate immune system, the neutrophils and monocytes are the key cellular elements and serve as a critical line of defense. There are many evidences indicate that neutrophils can degranulate and release many kinds of pro-inflammatory cytokines, proteinases and express chemotaxis toward invading microorganisms like bacteria, fungus, and virus, whereupon they engulf them through the process of phagocytosis. On the other hand, the monocytes also can differentiate to macrophages and provide a more powerful, long-term immunoreaction. These mediators which are released from leukocytes by tumor necrosis factor-alpha (TNF-??) and interleukin (IL). They activate a variety of gene expression, such as matrix metalloproteinases (MMPs) involved in tissue degradation. Nevertheless, when the normal regulatory mechanisms was failed, the reaction will get out of control and cause many inflammatory, autoimmune diseases, such as acute lung injury, rheumatoid arthritis and reperfusion injury. However, the neutrophils and monocytes in the circulatory system will be activated rapidly and excessively when the pathological changes happened, then cause tissue injury.
The results here indicate that cinnamophilin extracted from herbal medicines and N-hydroxycinnamoylphenalkylamides analogues (EK8) showed obviously inhibitory effect on respiratory burst and MMPs activation in neutrophils and THP-1 cells. In this study, we found that cinnamophilin and EK8 was shown to inhibit the fMLP-induced superoxide production in neutrophils by chemiluminescence assay, but EK8 not cinnamophilin also inhibited the PMA-induced phenomenon. We also found that there are weak inhibitory effects in cinnamophilin on intracellular calcium rising, adhesion molecular-CD11b expression, but no effect on ERK1/2 signaling activation by intracellular calcium analysis, flow cytometry analysis and western blot. In addition, EK8 inhibited the activation of MMPs activation and expression induced by TNF-??, LPS, IL-1?? and TGF-??1 in THP-1 cells by gelatin zymography and western blot, and these inhibitions which not resulted from the effect of cytotoxicity could be proved by MTT assay. In the transcription level, EK8 could suppressed the TNF-??-induced MMP-9 mRNA expression by using RT-PCR. It also inhibit the TNF-??-induced I?羠?? degradation, and furthermore it might affect the JNK signaling pathway. However, the detailed inhibitory mechanisms of cinnamophilin and EK8 are still need to be investigated further. It will be worth studing the related experiments of animal models and clinical trials in the future.

並列關鍵字

Neutrophils

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