探討N-hydroxycinnamoylphenalkylamides衍生物及Aristolochic Acid抑制MCP-1誘發人類單核球細胞活化之作用機轉

Investigation of the Inhibitory Mechanisms of N-hydroxycinnamoylphenalkylamides Analogues and Aristolochic Acid on MCP-1-Activated Monocytic cells

王欣玉(Hsin-Yu 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(2009年)

https://doi.org/10.6831/TMU.2009.00117

摘要

在人類的初級免疫系統中，單核球 (monocyte) 扮演相當重要的角色。有許多證據顯示當人體遭受外來的病原如細菌、黴菌、病毒等感染時，單核球能夠分泌細胞生長激素(cytokine)、抗微生物因子 (antimicrobial factors)、表現CCR2 chemokine受體，單核球細胞之CCR2 chemokine受體可與發炎部位大量表現的第一型單核球細胞趨化蛋白(monocyte chemoattractant protein-1, MCP-1) 行交互作用移動到感染部位分化成為巨噬細胞，進行更為有力且較長期的免疫作用。當MCP-1不恰當地過量表現，則會造成許多人類疾病相關的病理及生理病變，例如動脈粥狀硬化 (atherosclerosis)、類風濕性關節炎 (rheumatoid arthritis)、癌症 (cancer) 等疾病。
　　本研究利用體外細胞遷徙實驗 (In vitro migration assay) 及體外細胞侵襲實驗 (In vitro matrix invasion assay) 觀察單核球細胞化學趨化(chemotaxis) 的能力，發現N-hydroxycinnamoylphenalkylamides衍生物 (如EK5、EK8) 及中草藥 (如 Aristolochia spp.) 所萃取出的馬兜鈴酸 (Aristolochic acid) 具有抑制單核球細胞化學趨化(chemotaxis) 的能力，且MEK 抑制劑PD98059及PI3K抑制劑LY294002對於MCP-1誘導之細胞侵襲亦具有抑制作用，可推測ERK1/2及PI3K訊息傳遞路徑的活化與THP-1細胞進行細胞侵襲有關。另外藉由酵素電泳分析法發現馬兜鈴酸及EK5具有抑制MMP-9活性的能力。
本研究更進一步利用西方墨點法發現馬兜鈴酸及EK5可以抑制Akt訊息路徑的活化，推測馬兜鈴酸及EK5可能抑制單核球細胞利用integrin與血管內皮細胞進行緊密的黏附及在血管壁上滾動(rolling) 的過程及MMP-9的活化。且馬兜鈴酸也可抑制ERK1/2的活化，推測馬兜鈴酸可能可以抑制單核球細胞利用integrin與血管內皮細胞進行緊密黏附以及細胞遷徙時肌動蛋白 (actin) 重新排列的過程，但EK8並不會抑制Akt及ERK1/2的活化。

關鍵字

單核球 ； 趨化蛋白 ； 馬兜鈴酸

並列摘要

In the human innate immune system, the monocytes are the key cellular elements and serve as a critical line of defense. Inflammatory monocytes respond rapidly to microbial stimuli by secreting cytokines and antimicrobial factors, express the CCR2 chemokine receptor, and traffic to sites of microbial infection in response to monocyte chemoattractnat protein (MCP-1) secretion. On the other hand, the monocytes also can differentiate to macrophages and proceed a more powerful, long-term immunoreaction. When normal regulatory mechanisms fail, the monocyte is also responsible for immunologically induced many physiological and pathological changes, like atherosclerosis, rheumatoid arthritis (RA) and cancer.
In in vitro invasion and migration assay, we found that N-hydroxycinnamoylphenalkylamides analogues (EK5 and EK8) and Aristolochic acid (AsA) showed obviously inhibitory effect on MCP-1-induced monocyte invasion and migration. The MEK inhibitor PD98059 and PI3K inhibitor LY294002 also inhibited MCP-1-induced monocyte invasion, so monocyte invasion might be regulated by ERK1/2 and PI3K signaling pathway.
We also found AsA and EK5 might inhibit MCP-1-induced monocyte tethering (rolling) to firm adhesion via integrin activation and MMP-9 activity by affecting Akt signaling pathway. And AsA might inhibit MCP-1-induced monocyte ??1 integrin activation and actin rearrangement by affecting ERK1/2 signaling pathway. But EK8 had no effect on Akt and ERK1/2 signaling.

並列關鍵字

Monocyte Chemoattractant Protein- ； aristolochic acid ； N-hydroxycinnamoylphenalkylamides ； monocytes

參考文獻

Ajuebor, M. N., Flower, R. J., Hannon, R., Christie, M., Bowers, K., Verity, A. & Perretti, M. 1998. Endogenous monocyte chemoattractant protein-1 recruits monocytes in the zymosan peritonitis model. J Leukoc Biol 63: 108-16.

Albert, M. L., Sauter, B. & Bhardwaj, N. 1998. Dendritic cells acquire antigen from apoptotic cells and induce class I-restricted CTLs. Nature 392: 86-9.

Ancuta, P., Rao, R., Moses, A., Mehle, A., Shaw, S. K., Luscinskas, F. W. & Gabuzda, D. 2003. Fractalkine preferentially mediates arrest and migration of CD16+ monocytes. J Exp Med 197: 1701-7.

Ashida, N., Arai, H., Yamasaki, M. & Kita, T. 2001. Distinct signaling pathways for MCP-1-dependent integrin activation and chemotaxis. J Biol Chem 276: 16555-60.

Auffray, C., Fogg, D., Garfa, M., Elain, G., Join-Lambert, O., Kayal, S., Sarnacki, S., Cumano, A., Lauvau, G. & Geissmann, F. 2007. Monitoring of blood vessels and tissues by a population of monocytes with patrolling behavior. Science 317: 666-70.