探討新型組織蛋白去乙醯酶抑制劑及海藻萃取物LTH1-122-1 抑
制人類白血球活化與小鼠腦內出血損傷之作用探討

在人類的免疫系統中，嗜中性白血球(neutrophils)與單核球 (monocytes)扮演著最主要的初級防禦角色。有相當多的證據顯示，當人體遭受外來病原或內生性壓迫(stress)時，嗜中性白血球將會產生化學趨化的現象，而進一步產生了去顆粒化作用、釋放大量的發炎前驅性細胞激素及蛋白質分解酶等物質並進行吞噬作用。而在單核球方面，單核球會分化成巨噬細胞，進行較為長期的發炎與免疫反應。在這些釋放出來的物質中包括了腫瘤壞死因子(tumor necrosis factor-α,TNF-α)、介白素(interleukin)等，可以活化許多基因的表現，例如和組織分解有關的基質金屬蛋白酵素(matrix metalloproteinases,MMPs)。但許多發炎疾病的發生，如腦內出血之發炎併發症、急性肺部損傷(acute lung injury)等會使得嗜中性白血球及單核球被過度的活化，反而造成人體組織受到傷害。因此，適當抑制白血球的作用，將可以降低疾病傷害。由藥物篩選結果實驗發現，海藻成分LTH1-122-1 具有抑制嗜中性白血球釋放超氧化自由基(superoxide)的能力。在實驗中得知，對於嗜中性白血球，LTH1-122-2 可以抑制N-formyl-Met-Leu-Phe(fMLP)刺激superoxide 的產生，但是LTH1-122-11 僅部分抑制Phorbol12-myristate 13-acetate(PMA)的刺激現象。在對於MMPs 活性的能力方面，在嗜中性白血球及單核球細胞(THP-1)中，均無明顯抑制的效果。另一方面，在組織蛋白去乙醯酵素藥物研究上，PBHA 雖然不能抑制superoxide 的產生，但在活體實驗中，PBHA 對於腦內出血後的 MMP-9 的活性及腦水腫方面，則有抑制的功用。在N96 方面，以電泳酵素分析法(Gelatin zymography)及西方墨點法發現N96 於THP-1 細胞能夠抑制lipopolysaccharide (LPS)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。針對轉錄(Transcription)層級方面，利用反轉錄-聚合酵素連鎖反應 (RT-PCR)，N96 可以抑制LPS 刺激MMP-9 mRNA 的表現，進一步探討發現也可能抑制IκBα 的降解作用，且可能影響JNK，ERK 的訊息路徑。但是對於其詳細的分子作用機轉仍需深入的探討，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是值得加以努力的方向。

關鍵字

組織蛋白去乙醯酶抑制劑；白血球；腦內出血

並列摘要

In human innate immune system, neutrophils and monocytes play key roles and serve as a critical line of defense. There are many evidences indicate that neutrophils can degranulate and release various pro-inflammatory cytokines, proteinases and express chemotaxis toward invading microorganisms by the process of phagocytosis. On the other hand, the monocytes can differentiate to macrophages and provide a long-term immunoreaction. These mediators are released from leukocytes by tumor necrosis factor-alpha (TNF-α) and interleukin (IL). They activate a variety of gene expression, such as matrix metalloproteinases (MMPs) involved in tissue degradation. But in many inflammatory diseases, including intracerebral haemorrhage and acute lung injury, are associated with increasing activation of neutrophils and monocytes which could lead tissue injury. Therefore, the inhibitions of leukocytes activation are warrant study for the treatment of inflammatory diseases. In the present study, we found that algal natural product LTH1-122-1 inhibits neutrophils activations by inhibiting superoxide production. LTH1-122-1 markedly inhibits N-formyl-Met-Leu-Phe (fMLP)-induced superoxide, but it partially inhibited Phorbol 12-myristate 13-acetate (PMA)-induced superoxide. On MMP activity, we found LTH1-122-1 had no effect in activated neutrophils and THP-1 cells. On the other hand, the bioactive function of two histone deacetylase inhibitors, phenylbutyryl-hydroxamic acid (PBHA) and its amino acid derivative N96, were investigated. PBHA could not inhibit either fMLP-induced or PMA-induced superoxide in neutrophils. But in vivo studies, we found that PBHA could partially reduce brain edema and MMP activity. According to the cell studies, N96 inhibited the activation of MMPs activation and expression induced by lipopolysaccharide (LPS) in THP-1 cells by gelatin zymography and Western blot. And such inhibitory activity was not resulted from the cytotoxicity as revealed by the MTT assay. In the transcription level, N96 could suppress the LPS-induced MMP-9 mRNA expression by using reverse transcription - polymerase chain reaction. We also foud N96 is able to attenuate the LPS-induced IκBα degradation, and it might affect the JNK and ERK signaling pathways. It was proposed the inhibitory mechanisms of N96 may through NF-κB inhibition and need to be investigated further. It will be worth studing the related experiments of animal models and clinical trials in the future.