MAP Kinases, Akt以及eNOS等蛋白在氧化性膽固醇造成人類臍靜脈內皮細胞死亡之訊息傳遞中所扮演的角色

The Role of MAP Kinasesand Akt, eNOS Phosphorylation Pathways in Oxysterols Induced Cytotoxicity in Human Umbilical Vein Endothelial Cells

王智琦

指導教授 ： 鄭幼文

臺北醫學大學/藥學院/藥學研究所/碩士(2004年)

摘要

氧化性膽固醇已被證實存在於氧化性低密度脂蛋白與粥狀動脈硬化的區域。在本實驗中，我們呈現三種膽固醇氧化物：Cholesterol-5α,6α-epoxide（Epoxide）、7-Keto-cholesterol（7-Keto）及Cholesterol-3β,5α,6β-triol（Triol）會在HUVECs造成細胞毒性，而此作用是透過不同的傳導路徑調節MAP kinases、Akt、eNOS的磷酸化而來。 這三種氧化膽固醇在HUVECs調節的機制皆不盡相同，而且都會同時調節了兩個相反的路徑：apoptosis或necrosis以及cell protection。Epoxide在36個小時並不會對HUVECs造成細胞毒性，並且同時活化了與細胞保護機制相關的蛋白：ERK及Akt，此外，Epoxide能調節ERK與eNOS之間的回饋機制，此一回饋機制可能因此使得Epoxide不具細胞毒性，而這一個獨特的調節機制因此使得Epoxide與Triol及7-Keto區隔開來。Triol有明顯的雙向調節作用，其一為細胞毒性作用，包括了apoptosis和necrosis，而且Triol會誘發JNK、c-Jun、p38及caspase-3的活化因此貢獻了細胞毒性。此外，Triol所誘發的ROS的生成也與其細胞毒性有關，像是N-acetylcystein (NAC)、維他命C及維他命E等抗氧化劑都會減弱Triol的細胞毒性作用。另外一個作用即是會誘發走向細胞保護機制的蛋白像是p-ERK和p-Akt的表現。7-Keto則是很明顯的會誘發HUVECs的apoptosis作用且也會誘發JNK、c-Jun、p38及caspase-3的活化，7-Keto也同時會誘發ERK的磷酸化，而且誘發的p-JNK蛋白表現似乎是走向細胞保護機制的。 綜合以上的結果，我們發現了膽固醇氧化物在HUVECs的作用是多樣化的。Triol及7-Keto能造成細胞的毒性，而在同時間也會誘發另一條具細胞保護作用的路徑，與Triol及7-Keto不同的是，Epoxide在本實驗我們並沒有看到嚴重的毒性作用，誘發的作用似乎都是細胞保護作用。我們發現氧化性膽固醇在HUVECs的作用機轉似乎是很複雜的，還需要更多的研究來進一步釐清。

關鍵字

氧化性膽固醇 ； 人類臍靜脈內皮細胞 ； 訊息傳遞 ； 細胞凋亡

並列摘要

Oxidation products of cholesterol have been shown to be present in oxidized low density lipoprotein and in atherosclerotic lesions. Here we showed three oxysterols, Cholesterol-5a-6a-epoxide (Epoxide), 7-keto-cholesterol (7-Keto) and Cholesterol-3b-5a-6b-triol (Triol) induce cytotoxicity in human umbilical endothelial cells (HUVECs). And the cytotoxicity effect were regulated by MAP kinases, Akt, and eNOS phosphorylation through diversal pathways. Three oxysterols showed different mechanisms and dual aspects of regulation pathways, apoptosis or necrosis, and cell protection, in HUVECs. Epoxide did not cause cytotoxicity within 36 hours and showed the induction of cell protection proteins, ERK, Akt in HUVECs. Besides, Epoxide showed a feedback effect between ERK and eNOS, and thus it might show no cytotoxicity in HUVECs. This unique effect caused by Epoxide made it different from other two oxysterols. Triol showed obvious dual effects in HUVECs. One is cytotoxicity effect including apoptosis and necrosis. JNK, c-Jun, p-38 and caspase-3 were activated by Triol and those were believed to contribute the cytotoxicity. In addition, the generation of ROS also involved in Triol-induced cytotoxicity. Antioxidants such as N-acetylcystein (NAC), vitamin C and vitamin E could diminish the cytotoxicity of Triol. The other is the induction of cell protection proteins, like ERK and Akt. 7-Keto significantly induced apoptosis and JNK, c-Jun, p-38, caspase-3 activation in HUVECs. 7-Keto also activated ERK phosphorylation, and the p-JNK activation seems to contribute the beneficial effect in HUVECs. In summary, the role of oxysterols in HUVECs is controversial. We found that Triol and 7-Keto could induce cytotoxicity, but there is still a cell protection pathway going in the same time. Different with Triol and 7-keto, Epoxide only showed the beneficial effect in this study. The effect of oxysterols on HUVECs seems to be complicated and need further investigation.

並列關鍵字

MAP Kinases ； eNOS ； HUVEC ； Oxysterol ； apoptosis

參考文獻

Addis PB.(1986)Occurrence of lipid oxidation products in foods. Food Chem Toxicol. 24(10-11): 1021-30.

Addis PB, Emanuel HA, Bergmann SD, Zavoral JH.(1989)Capillary GC quantification of cholesterol oxidation products in plasma lipoproteins of fasted humans. Free Radic Biol Med. 7(2): 179-82.

Ares MP, Porn-Ares MI, Moses S, Thyberg J, Juntti-Berggren L, Berggren P, Hultgardh-Nilsson A, Kallin B, Nilsson J.(2000)7beta-hydroxycholesterol induces Ca2+ oscillations, MAP kinase activation and apoptosis in human aortic smooth muscle cells. Atherosclerosis. 153(1): 23-35.

Aupeix K, Weltin D, Mejia JE, Christ M, Marchal J, Freyssinet JM, Bischoff P.(1995)Oxysterol-induced apoptosis in human monocytic cell lines. Immunobiology. 194(4-5): 415-28.

Ayajiki K, Kindermann M, Hecker M, Fleming I, Busse R.(1996)Intracellular pH and tyrosine phosphorylation but not calcium determine shear stress-induced nitric oxide production in native endothelial cells. Circ Res. 78(5): 750-8.