探討新型組織蛋白去乙醯酶抑制劑AN抑制內毒素誘發人類單核球細胞IL-6表現之作用機制

Studies of Novel Histone Deacetylase Inhibitor AN Inhibit Endotoxin-induced Interleukin-6 Expression in Human Monocytes

柯雅婷(Ya-Ting Ko)

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(2013年)

摘要

在風濕性關節炎或黏膜炎等發炎病症中，單核球於製造並分泌多種趨發炎激素 (pro-inflammatory cytokine) 如TNF-?恁BIL-6及基質金屬酵素酶 (matrix metalloproteinase, MMP) 皆會影響病程的進展。組織蛋白去乙醯酶 (Histone deacetylases, HDAC) 會藉由乙醯化和去乙醯化組織蛋白進而調控基因表現，近期的文獻中指出，HDAC inhibitor在脂多醣體 (Lipopolysaccharide, LPS) 所誘發人類單核球細胞的發炎反應中，具有抑制發炎物質產生的效果。

初步實驗結果顯示 HDACi (AN) 可以有效抑制由LPS誘發人類單核球細胞THP-1製造及釋放MMP-9，且不具有細胞毒性，其相關抑制作用具濃度梯度效應。而酵素連結免疫吸附分析法 (Enzyme-Linked Immunosorbent Assay, ELISA) 發現AN可抑制受LPS活化之THP-1細胞釋放多種趨發炎細胞激素如TNF-α與IL-6。利用西方墨點法可以發現AN有濃度梯度效應的降低THP-1細胞內的IL-6及MMP-9蛋白表現，而Real-time qRT-PCR結果顯示AN可降低IL-6及MMP-9之mRNA的表現量。根據實驗結果推測AN藉由抑制ERK磷酸化達到抑制THP-1細胞之MMP-9釋放及生成活性與蛋白表現。AN是否會經由其他分子機轉達到抑制IL-6和MMP-9作用，仍需要更多的實驗加以證實。

關鍵字

組織蛋白去乙醯酶內毒素

並列摘要

In many inflammatory diseases such as mucositis and rheumatoid arthritis (RA) , monocytes influence disease progress via secreting a number of pro-inflammatory cytokine such as TNF-???n, IL-6 and matrix metalloproteinase (MMP) . Histone deacetylases (HDAC) regulate gene expression by acetylation and deacetylation of histone, and recently report show HDAC inhibitor (HDACi) suppress inflammatory mediators production in lipopolysaccharide (LPS) induces human monocyte inflammatory response.
The preliminary experimental results show the HDACi (AN) has no cytotoxicity can significant concentration-dependently inhibits LPS induced MMP-9 release and production of human monocytes. In enzyme-linked immunosorbent assay (ELISA) , we found that the AN reduce the production of various pro-inflammatory cytokine such as TNF-α and IL-6 in LPS-activate THP-1 cells. Western blot analysis show that AN concentration-dependently suppressed of IL-6 and MMP-9 expression in THP-1 cells, moreover AN suppressed TNF-α、IL-6 mRNA expression in LPS-activate THP-1 cells showed in result of real-time qRT-PCR. Western blot analysis also show that AN suppress phosphorylation of MAPK kinase ERK. Those data suggest that AN inhibit MMP-9 release, production enzyme activity via suppression on phosphorylation of ERK. The detailed molecular mechanism of AN still is needed more experiments to be confirmed.

並列關鍵字

HDACi ； LPS

參考文獻

Aleksander, M., Grabiec, Olexandr., Korchynskyi, Paul., P. Tak., Kris, A. Reedquist. Histone deacetylase inhibitors suppress rheumatoid arthritis fibroblast-like synoviocyte and macrophage IL-6 production by accelerating mRNA decay. 2012. Ann Rheum Dis 71: 424–431

Akira, S. and Takeda, K. Toll-like receptor signalling. 2004. Nat Rev Immunol 4: 499-511

Auwerx, J. 1991. The human leukemia cell line, THP-1: a multifacetted model for the study of monocyte-macrophage differentiation. Experientia 47: 22-31.

Annemieke, J., M. DE, RUIJTER., Albert, H. VAN, GENNIP., Huib, N. CARON., Stephan KEMP and Andre B. P. VAN KUILENBUR. Histone deacetylases (HDACs) : characterization of the classical HDAC family. Biochem. J. 2003. 370: 737-749

Barton GM, Medzhitov R. Toll-like receptor signaling pathways.Science. 2003 ;300 (5625) : 1524-5.