探討atorvastatin在人類主動脈內皮細胞及餵食膽固醇 飼料兔子中血栓調節蛋白的表現及其相關機轉

The effects of atorvastatin on thrombomodulin expression in human aortic endothelial cells and cholesterol-fed rabbits.

林家琪(Chia-Chi Lin)

指導教授 ： 陳玉怜

國立臺灣大學/醫學院/解剖學研究所/碩士(2006年)

https://doi.org/10.6342/NTU.2006.02006

摘要

血栓調節蛋白 (thrombomodulin, TM) 為血管內皮細胞膜上的一種醣蛋白，可與血液中的血栓蛋白結合，使血栓蛋白無法開啟下游的凝血機轉。Atorvastatin (Lipitor®) 是一種三氫氧基三甲基穀胱胺基輔酶還原酶的抑制劑 (HMG-CoA reductase inhibitor)，在臨床上常被用來治療心血管疾病，這種藥物可顯著降低病患的罹患率及死亡率，過去報告指出statins藥物能夠拮抗由腫瘤壞死因子(tumor necrosis factor-alpha, TNF-α) 所抑制的TM表現，因此我們想要探討atorvastatin對於餵食膽固醇兔子或對於正常及TNF-α處理人類主動脈內皮細胞所誘發TM的表現及其影響的相關機制。
在動物實驗中，我們取正常、餵食三週及九週膽固醇飼料以及同時餵食atorvastatin及膽固醇飼料三週、九週的紐西蘭白兔的主動脈，可發現在正常或無動脈硬化斑塊病灶處的血管中，TM會表現在內皮細胞上。而在病灶區的TM除了表現在內皮細胞，也會在斑塊中的平滑肌細胞表現。另外我們將主動脈的蛋白質萃取出來進行西方轉漬法，發現除了餵食三週膽固醇的TM表現下降之外，餵食九週膽固醇、同時餵食atorvastatin及膽固醇飼料三週、九週的TM表現與控制組相比皆有上升的趨勢，且有餵食atorvastatin的組別的TM表現比單以餵食膽固醇兔子來的高。在細胞實驗我們發現以TNF-α處理人類主動脈內皮細胞，內皮細胞TM的表現會隨著TNF-α濃度及處理時間的增加，而有顯著減少的趨勢；以atorvastatin藥物處理人類主動脈內皮細胞，其TM的表現隨著藥物濃度及時間的增加而有增加的趨勢；若先以atorvastatin處理，再以TNF-α刺激，可發現atorvastatin能夠拮抗因TNF-α所造成TM表現降低的情形。進一步，我們發現以TNF-α處理人類主動脈內皮細胞，nuclear factor-kappa B (NF-κB)會被活化，而以atorvastatin前處理，再以TNF-α處理人類主動脈內皮細胞，NF-κB活化的情形顯著減少。若以NF-κB抑制劑 (parthenolide) 處理細胞時，可發現parthenolide也可拮抗因TNF-α所導致的TM表現下降情形，因此atorvastatin拮抗因TNF-α所導致的TM表現下降，是因為抑制NF-κB的活化。由動物實驗及細胞實驗結果可發現atorvastatin增加TM的表現，而這也許是atorvastatin保護血管內皮細胞的一種方法。

關鍵字

膽固醇飼料兔子 ； 人類主動脈內皮細胞 ； 血栓調節蛋白

並列摘要

TM is an integral membrane glycoprotein, which functions in anticoagulation by virtue of eliminating thrombin from the blood. Formation of the thrombin-TM complex can effectively activate protein C, which in turn catalyzes the proteolytic inactivation of blood coagulation factor Va and VIIIa, leading to downregulation of blood coagulation cascade. The main feature of the antithrombotic properties of endothelial cells is an increase in the expression of thrombomodulin (TM). The 3-hydroxyl-3-methyl-glutaryl coenzyme A reductase inhibitor, atorvastatin, can protect the vasculature independent of their lipid-lowering activity. We investigated the effect of atorvastatin on the expression of TM and their underlying mechanisms in cholesterol-fed rabbits and in human aortic endothelial cells (HAECs) with or without tumor necrosis-alpha (TNF-α) treatment. In vivo experiments, we found that the expression of TM appeared not only in endothelial cells but also in smooth muscle cells at atherosclerotic lesions.
In vitro experiments, TNF-α treatment decreased the expression of TM in a time- and dose-dependent manner in HAECs. Atorvastatin pretreatment upregulated the expression of TM in a time- and dose-dependent manner in HAECs with or without TNF-α treatment. TNF-α treatment increased the activation of nuclear factor-kappa B (NF-κB), and atorvastatin pretreatment could inverse the effect. In addition, blocking the activation of NF-TM is an integral membrane glycoprotein, which functions in anticoagulation by virtue of eliminating thrombin from the blood. Formation of the thrombin-TM complex can effectively activate protein C, which in turn catalyzes the proteolytic inactivation of blood coagulation factor Va and VIIIa, leading to downregulation of blood coagulation cascade. The main feature of the antithrombotic properties of endothelial cells is an increase in the expression of thrombomodulin (TM). The 3-hydroxyl-3-methyl-glutaryl coenzyme A reductase inhibitor, atorvastatin, can protect the vasculature independent of their lipid-lowering activity. We investigated the effect of atorvastatin on the expression of TM and their underlying mechanisms in cholesterol-fed rabbits and in human aortic endothelial cells (HAECs) with or without tumor necrosis-alpha (TNF-α) treatment. In vivo experiments, we found that the expression of TM appeared not only in endothelial cells but also in smooth muscle cells at atherosclerotic lesions.
In vitro experiments, TNF-α treatment decreased the expression of TM in a time- and dose-dependent manner in HAECs. Atorvastatin pretreatment upregulated the expression of TM in a time- and dose-dependent manner in HAECs with or without TNF-α treatment. TNF-α treatment increased the activation of nuclear factor-kappa B (NF-κB), and atorvastatin pretreatment could inverse the effect. In addition, blocking the activation of NF-κB could reverse the downregulation of TM by TNF-α treatment in HAECs. Atorvastatin-mediated increase in TM expression by endothelial cells may contribute to the beneficial effects on endothelial function.

並列關鍵字

atrovastatin ； human aortic endothelial cells ； choleseterol-fed rabbits

參考文獻

Aggarwal B.B. Tumor necrosis factors receptor associated signaling molecules and their role in activation of apoptosis, JNK and NF-kappa B. Ann Rheum Dis. 2000;59:i6- i16.

Aikawa M, Rabkin E, Okada Y, Voglic SJ, et al. Lipid lowering by diet reduces matrix metalloproteinase activity and increases collagen content of rabbit atheroma. Circulation. 1998;97:2433–2444.

Bajaj MS, Kuppuswamy MN, Manepalli AN, Bajaj SP. Transcriptional expression of tissue factor pathway inhibitor, thrombomodulin and von Willebrand factor in normal human tissues. Thromb Haemost. 1999;82:1047-1052.

Baldwin AS. The NF-kappa B and I kappa B proteins: new discoveries and insights. Annu Rev Immunol. 1996;14:649-683.

Bevilacqua MP, Stengelin S, Gimbrone MJ, Seed B. Endothelial leukocyte adhesion molecule 1: an inducible receptor for neutrophils related to complement regulatory proteins and lectins. Science. 1989;243:1160-1165.