探討氧化態低密度脂蛋白與phenytoin對小膠質細胞產生誘發型一氧化氮合成酵素之影響

Investigation of the effect of Oxidized Low-Density Lipoprotein and Phenytoin on the Expression of the Inducible Nitric Oxide Synthase in microglia cells

鄭景文(Ching-Wen Cheng)

指導教授 ： 蕭哲志

臺北醫學大學/醫學院/藥理學研究所/碩士(2007年)

https://doi.org/10.6831/TMU.2007.00035

摘要

有許多證據顯示誘導出誘發型一氧化氮合成酵素(iNOS)的表現以及一氧化氮(NO)的產生對於抗病毒、免疫調節以及抗腫瘤情況是具有幫助的。但假如在不對的地方及時間誘導出來，則會造成許多人類疾病相關的病理生理病變。在中樞神經系統當中，一氧化氮的大量增加會造成一些神經發炎性及神經退化性疾病的發生，例如：多發性硬化症(multiple sclerosis)、愛滋痴呆症候群(HIV dementia)、腦部局部缺血、帕金森氏症(Parkinson's disease)以及阿滋海默症(Alzheimer's disease)等。然而在以上的病理情況發生下，中樞神經系統中小膠質細胞會快速地被過度活化，而在本篇研究中，我們則會加以探討氧化態低密度脂蛋白(ox-LDL)與phenytoin對於小膠質細胞產生誘發型一氧化氮合成酵素之影響機轉。
氧化態低密度脂蛋白對於動脈粥狀硬化的發生扮演著很重要的角色，而ox-LDL透過與清道夫受體A型的高度親合力以及內化作用之後，進而影響一些慢性發炎疾病的發生。經由我們的結果證實，ox-LDL可以誘發小膠質細胞產生大量的一氧化氮，並發現ox-LDL也能誘發大量iNOS蛋白及其mRNA的表現，代表著這ox-LDL對於iNOS的影響可能在基因表現層面。因此我們對於ox-LDL在iNOS的表現機轉上做進一步的探討，利用不同抑制劑的觀察，發現誘導出iNOS蛋白表現主要是經由p38 MAPK的活化，並且其活化的p38能進入細胞核內，促使轉錄因子ATF-2的磷酸化。綜合以上結論，ox-LDL活化小膠質細胞，主要是經由活化p38來活化轉錄因子ATF-2，進而啟動iNOS的基因表現，最終使得iNOS蛋白及NO大量產生。另一方面也發現ox-LDL所引發的訊息傳導與dynamin有著相當的關係，即透過endocytosis抑制劑可以降低ox-LDL所誘發的iNOS蛋白表現。在相關的受體方面，發現ox-LDL可能與fucoidan所結合的受體並不相同，由於fucoidan並無法誘發小膠質細胞產生iNOS蛋白表現。
phenytoin為一種抗痙攣藥物，臨床上用來治療癲癇相當有效。近來有文獻指出，在腦脊髓炎與多發性硬化症中，phenytoin能夠改善其中小膠質細胞活化的情況，而目前對於此相關機轉還不甚清楚。我們從實驗結果中顯示，phenytoin能夠降低經由LPS刺激小膠質細胞所產生的NO，並且不影響其細胞存活率。此外，我們還發現phenytoin能夠抑制LPS刺激小膠質細胞所產生的iNOS蛋白表現，而且是有濃度依賴性的抑制效果。在機轉方面，我們證實phenytoin並不能有效抑制經LPS活化所造成ΙκB-α的降解，因此，我們推論phenytoin的抑制作用可能不是經由NF-kappaB這條路徑。

關鍵字

氧化態低密度脂蛋白 ； 小膠質細胞 ； 誘發型一氧化氮合成酵素 ； phenytoin

並列摘要

Accumulating evidence indicates that induction of inducible nitric oxide synthase (iNOS) expression and hence nitric oxide (NO) production has been described to have beneficial antiviral, immunomodulatory, and antitumoral affect. However, induced at the wrong place or at the wrong time, iNOS has detrimental consequences and seems to be involved in the pathophysiology of different human diseases. As such, elevated levels of NO produced within the central nervous system (CNS) are associated with the pathogenesis of neuroinflammatory and neurodegenerative human diseases such as multiple sclerosis, HIV dementia, brain ischemia, trauma, Parkinson's disease, and Alzheimer's disease. However, the overactivation of microglia can occur rapidly after the above pathologies of the CNS. In this study, we have observed that oxidized low-density lipoprotein (ox-LDL) and phenytoin affect microglial functions in association with the mechanisms of iNOS expression in microglial cells.
It is well documented that ox-LDL have been shown to play a crucial role in atherosclerosis. The class A scavenger receptor (SR-A) mediates the high affinity binding and internalization of ox-LDL implicated in the development of chronic inflammation. Our results demonstrate that ox-LDL stimulates NO production in microglial cells, and it also stimulates both protein levels and mRNA of iNOS in concentration-dependent manner, indicating that this oxidative components may affect iNOS gene expression. Therefore, we investigated the molecular mechanism of ox-LDL on iNOS expression. Using different pharmacological inhibitors, we found the induction of iNOS by p38 mitogen-activated protein kinase activation. Besides, we even more found that activated p38 in the nucleus induced phosphorylation of activating transcription factor-2 (ATF-2). Taken together, our data clearly demonstrated that ox-LDL-induced microglial cells production of NO and iNOS was mediated mainly by phosphorylation of p38 and ATF-2. On the other hand, we also found that dynamin inhibitor decreased iNOS production in ox-LDL stimulated microglial cells, and fucoidan, SR-A ligand, couldn’t stimulate iNOS production in microglial cells.
Phenytoin is an anticonvulsant drug which can be useful in the treatment of epilepsy clinically. The recent report indicated that sodium channels are important for activation of microglia in encephalomyelitis (EAE) and multiple sclerosis (MS), but the anti-inflammatory mechanisms remain poorly understood. We showed here that phenytoin decreased NO production in LPS-stimulated microglial cells, without affecting cell viability. Besides, we even more found that phenytoin suppressed the expression of iNOS protein induced by LPS in a dose dependent way. Further, we found that phenytoin couldn’t inhibit the LPS-induced IkappaB-alpha degradation suggesting that this drug might not affect LPS-induced activation of NF-kappaB by abrogating the LPS-induced IkappaB-alpha?ndegradation.

並列關鍵字

Oxidized Low-Density Lipoprotein ； microglia cells ； Inducible Nitric Oxide Synthase ； phenytoin

參考文獻

1. Lee P., Lee J., Kim S., Lee M.S., Yagita H., Kim S.Y., Kim H., and Suk K., NO as an autocrine mediator in the apoptosis of activated microglial cells: correlation between activation and apoptosis of microglial cells. Brain Res. 892: 380-385, 2001.

2. Davalos D., Grutzendler J., Yang G., Kim J.V., Zuo Y., Jung S., Littman D.R., Dustin ML, and Gan WB, ATP mediates rapid microglial response to local brain injury in vivo. Nat. Neurosci. 8: 752-758, 2005.

3. Fetler L. and Amigorena S., Neuroscience. Brain under surveillance: the microglia patrol. Science 309: 392-393, 2005.

4. Nimmerjahn A., Kirchhoff F., and Helmchen F., Resting microglial cells are highly dynamic surveillants of brain parenchyma in vivo. Science 308: 1314-1318, 2005.

5. Vilhardt F., Microglia: phagocyte and glia cell. Int. J. Biochem. Cell Biol. 37: 17-21, 2005.