探討新型組織蛋白去乙醯酶抑制劑PBHA降低內毒素脂多醣體誘發人類單核球細胞基質蛋白酵素活化之作用機轉

Investigation of Inhibitory Mechanisms of a New Histone Deacetylase Inhibitor, PBHA, on Lipopolysaccharide-activated Matrix metalloproteinase Expression in Human Monocytic Cells

王文君(Wen-Chun Wang)

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(2011年)

摘要

葛蘭氏陰性菌之致病成分脂多醣體(Lipopolysaccharide, LPS)會活化單核球中的MAPK及NF-?羠訊息傳遞路徑進而產生基質金屬蛋白酵素(Matrix Metalloproteinase, MMPs)以進行組織重組與發炎反應，其中以MMP-9與全身性發炎疾病之病程最有相關性。組織蛋白去乙醯酶抑制劑(Histone deacetylase inhibitor, HDACi)具有抗發炎特性，它們會經由抑制不同訊息活化路徑如影響NF-?羠及MAPK活性或直接抑制轉錄階層以降低發炎物質表現。
在本研究中探討新型去乙醯酶抑制劑phenylbutyrylhydroxamic acid (PBHA)降低內毒素所誘發之MMP-9的能力及其抑制機轉。由實驗結果顯示，PBHA不具細胞毒殺性，其相關抑制作用均具有濃度梯度效應。另外在NF-?羠活化之相關實驗中，PBHA不具明顯抑制作用，而在MMP-9 mRNA的表現及ERK磷酸化上則有明顯的抑制。PBHA藉由抑制ERK磷酸化及其mRNA之轉錄達到抑制THP-1細胞之MMP-9釋放及生成活性與蛋白表現，其中ERK磷酸化的減少可能與降低AP-1之活性有關。PBHA於MMP-9之抑制作用具有細胞選擇性，須在較高濃度下才能抑制人類初級單核球細胞之MMP-9釋放及生成活性。而PBHA是否會經由其它之分子機轉達到抑制MMP-9及其活體相關作用，仍須相關實驗進一步探討。

關鍵字

組織蛋白去乙醯酶抑制劑 ； 脂多醣體 ； 基質蛋白酵素

並列摘要

Lipopolysaccharide (LPS) is a major component in the outer membrane of Gram-negative bacteria. LPS activates mitogen-activated protein kinase (MAPKs) and NF-?羠 signaling pathway, leading to the production of metalloproteinases (MMPs). Among of all MMPs, MMP-9 is relevant to the development of sepsis. Histone deacetylase (HDAC) inhibitors were recently shown to suppress inflammatory responses on LPS-stimulated macrophages and the mechanism of anti-inflammatory effect may through the inhibition of different signaling pathways, for instance, NF-?羠 and MAPKs pathways. Thus, we investigated the inhibitory effect of new HDAC inhibitor, phenylbutyrylhydroxamic acid (PBHA), on the LPS-activated macrophages.
In this study, we found PBHA showed the concentration-dependent inhibitory effect on MMP-9 production with no cytotoxicity in THP-1 cells. Because there is no inhibition of NF-?羠 activation, the inhibitory effect may be caused by directly decreasing mRNA expression and p42/p44 extracellular signal receptor kinases (ERKs) phosphorylation. However, the inhibitory effect in human primary monocyte is only observed in the higher concentration manner. We predicted that PBHA may inhibit the activation of activator proteins-1 (AP-1) to achieve the inhibition of MMP-9. These results showed that PBHA has the potent anti-inflammatory activity in monocytic cells and it is necessary to investigate the effects of PBHA in the animal model in the future.

並列關鍵字

histone deacetylase inhibitor ； lipopolysaccharide ； matrix metalloproteinase

參考文獻

Abraham, E., Matthay, M. A., Dinarello, C. A., Vincent, J. L., Cohen, J., Opal, S. M., Glauser, M., Parsons, P., Fisher, C. J., Jr. and Repine, J. E. 2000. Consensus conference definitions for sepsis, septic shock, acute lung injury, and acute respiratory distress syndrome: time for a reevaluation. Crit Care Med 28: 232-5.

Abraham, E. and Singer, M. 2007. Mechanisms of sepsis-induced organ dysfunction. Crit Care Med 35: 2408-16.

Adib-Conquy, M., Adrie, C., Fitting, C., Gattolliat, O., Beyaert, R. and Cavaillon, J. M. 2006. Up-regulation of MyD88s and SIGIRR, molecules inhibiting Toll-like receptor signaling, in monocytes from septic patients. Crit Care Med 34: 2377-85.

Akashi, S., Shimazu, R., Ogata, H., Nagai, Y., Takeda, K., Kimoto, M. and Miyake, K. 2000. Cutting edge: cell surface expression and lipopolysaccharide signaling via the toll-like receptor 4-MD-2 complex on mouse peritoneal macrophages. J Immunol 164: 3471-5.

Alessi, D. R., Deak, M., Casamayor, A., Caudwell, F. B., Morrice, N., Norman, D. G., Gaffney, P., Reese, C. B., MacDougall, C. N., Harbison, D., Ashworth, A. and Bownes, M. 1997a. 3-Phosphoinositide-dependent protein kinase-1 (PDK1): structural and functional homology with the Drosophila DSTPK61 kinase. Curr Biol 7: 776-89.