- 學位論文
新型組蛋白去乙醯酶抑制劑在類風濕性關節炎之功能與機轉探討
Functional and Mechanistic Studies of A Novel Histone Deacetylase Inhibitor in Rheumatoid Arthritis
摘要
實驗目的：類風溼性關節炎是一種全身性的自體免疫疾病，其病理方面包括滑液 膜細胞的增生、發炎物質的表現及免疫細胞的持續性浸潤，而這些現象可能源自 於HDAC (組蛋白去乙醯酶)對於表觀遺傳調控的異常；因此，HDAC 抑制劑未來 可能可以用來治療發炎性疾病。此篇論文的研究目的為探討一個新型的HDAC 抑 制劑MPT0G009 在類風溼性關節炎滑液膜細胞以及實驗動物模型中的抗風溼效果 及其機轉。 實驗方法：利用HDAC assay kit 測定HDAC 抑制劑對於各種HDAC 亞型的抑制活性能力，並利用西方點墨法觀察HDAC 抑制劑是否會影響各種HDAC 亞型的蛋白 質表現；利用SRB 試驗測定藥物對於細胞增生的抑制效果，並利用流式細胞儀探 討藥物對於細胞週期的影響及機轉；發炎反應中的細胞激素則是用ELISA (酵素免 疫分析法)測定；利用酒石酸具耐受性酸性磷酸酵素染色法觀察噬骨細胞的生成分 化以及型態，並利用流式細胞儀檢測噬骨細胞生物標記的表現量。另外，我們也 於實驗中將細胞過量表現HDAC 來探討藥物的抗發炎及抑制噬骨細胞生成的效果 是否直接透過抑制HDAC。我們更利用動物實驗模型來驗證藥物的抗風溼效果與 機轉。 實驗結果：MPT0G009 相較於已核准上市的其中一種HDAC 抑制劑，SAHA，具 有更強的HDAC 活性抑制能力，並且在類風溼性關節炎滑液膜細胞中能夠抑制 HDAC3 的蛋白質表現。在類風溼性關節炎滑液膜細胞與巨噬細胞中，MPT0G009 均顯示出很好的抗發炎效果，並且可以抑制滑液膜細胞增生。另外，在利用巨噬 細胞分化成噬骨細胞的實驗中，我們發現MPT0G009 在3 nM 的濃度下能有效抑制 噬骨細胞生成，而SAHA 在30 nM 下卻無法有明顯的抑制效果。在動物實驗中， 我們發現MPT0G009 在25 mg/kg 的口服給予下，能達到與SAHA 在200 mg/kg 給予下相似的抗風溼效果。 結論：我們的體內與體外實驗結果顯示MPT0G009 具有很強的抗發炎、抑制滑液 膜細胞增生、抑制噬骨細胞生成及減少硬骨與軟骨損壞的效果，迄今尚未有HDAC 抑制劑被核准上市或是進入臨床試驗來治療類風濕性關節炎，因此MPT0G009 可 望在未來能發展成為治療類風濕性關節炎的藥物。
並列摘要
Objective. The pathology of rheumatoid arthritis (RA) includes synoviocytes proliferation, expression of inflammatory mediators, and persistent recruitment of immune cell, and these might result from dysregulation of epigenetic control by histone deacetylase (HDAC). Therefore, HDAC inhibitor may act as a new treatment for inflammatory disease. The aim of this study is to examine the anti-arthritic effect of MPT0G009, a histone deacetylse (HDAC) inhibitor, by in vitro and in vivo models. Methods. HDACs activity and protein expression were determined by HDAC assay kit and western blot. Anti-proliferative effect was determined by SRB assay, and the cell cycle arrest was determined by flow cytometry. Cytokines production was studied by ELISA. Osteoclastogenesis was evaluated by TRAP stain and flow cytometry. In vivo study was determined by Adjuvant Induced Arthritis rat model and IHC stain. Result. The IC50 values of MPT0G009 against enzymatic activity of HDACs were significantly lower than SAHA, and MPT0G009 could inhibit HDAC3 protein expression in RA synovial fibroblasts, indicating that MPT0G009 is more potent than SAHA. Treatment of RAW264.7 macrophages and RA synovial fibroblasts with MPT0G009 inhibited cytokines secretion in a concentration-dependent manner, and MPT0G009 also had anti-proliferative effect on RA synovial fibroblasts. In addition, MPT0G009 dramatically suppressed RANKL/M-CSF induced osteoclastogenesis from macrophages at low concentration (3 nM). The in vivo anti-arthritic effects of MPT0G009 were evaluated in a Mycobacterium butyricum-induced arthritis model in rats. Oral administration of MPT0G009 (25 mg/kg) significantly inhibited paw swelling,bone destruction and reduced serum cytokine levels. Conclusion. Our results demonstrate that HDAC inhibitor MPT0G009 inhibits the development of arthritis, suggesting that it might provide a new therapeutic approach to inflammatory arthritis diseases.
參考文獻
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