以人類臍靜脈內皮細胞評估香茹草萃取物抑制動脈粥狀硬化之作用及其機轉探討

Anti-atherogenic effects of Glossogyne tenuifolia extract in human umbilical endothelial cells and its mechanisms

宣錦峰(Chin-Feng Hsuan)

指導教授 ： 洪哲穎

義守大學/理工學院/化學工程學系暨生物技術與化學工程研究所/博士(2016年)

摘要

動脈粥狀硬化是造成心血管疾病的主因之一，其病理機制非常複雜。當血液中低密度脂蛋白濃度過高或血管壁受到危險因子的刺激，低密度脂蛋白會滲入血管內皮下並氧化。氧化態的低密度脂蛋白會刺激其上的內皮細胞分泌chemotactic factor，以及一些吸附因子促使單核球聚集並吸附到血管內皮上，進一步移動到內皮下間隙，並轉變成巨噬細胞。巨噬細胞所分泌的細胞激素及生長因子，會促使平滑肌細胞的增生並移行至內皮下間隙。在此，平滑肌細胞會分泌細胞外間質，同時，也會如同巨噬細胞表現吸附因子及細胞激素吸引其他發炎細胞至內皮下。巨噬細胞及平滑肌細胞會吸收低密度脂蛋白形成載脂細胞，最終促進動脈硬化斑塊的形成。
血管內皮的一氧化氮系統在動脈粥狀硬化的過程中扮演保護的角色，此一氧化氮是由一氧化氮合成酶 (eNOS) 將L-arginine轉換成L-citrulline所產生，具有抗血栓形成及抗動脈粥狀硬化的作用。其中，抗血栓形成主要是藉由抑制血小板的凝集和吸附。抗動脈粥狀硬化的機制主要包括：(1)防止白血球對血管內皮細胞的吸附及移行到血管壁內；(2)減少血管內皮細胞對脂蛋白的滲透及抑制低密度脂蛋白的氧化；(3)抑制血管平滑肌細胞的增生。一氧化氮合成酶失去活性或功能異常會導致一氧化氮生成量下降，促進動脈粥狀硬化的生成。
香茹(Glossogyne tenuifolia)廣泛分布於南亞、澳洲與新喀里多尼亞等地，台灣則以澎湖地區分布最廣。香茹具有退熱、解毒、保肝、抗發炎的作用。香茹萃取物含有許多多元酚化合物，如luteolin、luteolin-7-glucoside和oleanolic acid，具有抗發炎及抗氧化的作用。本研究首先針對香茹草得到的乙醇粗萃物，以及單離出之主要活性物luteolin及luteolin-7-glucoside，測試其是否能有效抑制在人類臍靜脈內皮細胞內由細胞激素刺激所產生的附著分子，並進一步探討其可能的作用機轉。第二部分為測試香茹草各分層萃取物作用到人類臍靜脈內皮細胞後，是否會增加其一氧化氮的產生及eNOS蛋白的表現；並探討其可能的作用機制。
實驗結果顯示香茹草乙醇粗萃物、luteolin和luteolin-7-glucoside能減少以TNF-α活化的人類臍靜脈內皮細胞吸附因子ICAM-1及VCAM-1的表現，並能抑制單核球吸附到人類臍靜脈內皮細胞上。ICAM-1及VCAM-1的mRNA表現也受到抑制，顯示這些物質的抑制作用是在基因轉譯的層次。另外，香茹草粗萃物、luteolin和luteolin-7-glucoside會阻止TNF-α引起的IkB的分解，表示這些物質是藉由抑制NF-kB的活化來抑制吸附分子的表現。第二部份的實驗結果顯示，香茹草根部EA層、香茹草地上部、根部和全株的methanol層和ethanol層萃取物作用在人類臍靜脈內皮細胞皆能增加其一氧化氮的生成。其中，香茹草根部methanol層萃取物作用效果最強。進一步由西方點墨法及RT-PCR分析結果發現，香茹草根部methanol層萃取物可以增加一氧化氮合成酶蛋白質及mRNA的表現，並且增加一氧化氮合成酶在serine1177這個位置的磷酸化，表示香茹草根部methanol層萃取物可以增加一氧化氮合成酶的表現活性。
總括而言，香茹草萃取物和其活性成分可以有效抑制單核球吸附到受細胞激素活化的內皮上，其機制是藉由抑制NF-kB的活化來抑制吸附分子的表現。此外，香茹草萃取物可以增加人類臍靜脈內皮細胞中一氧化氮的生成。香茹草根部methanol層萃取物提高一氧化氮生成的機制是增加一氧化氮合成酶的表現及增加一氧化氮合成酶在serine1177這個位置的磷酸化以增加其活性。

關鍵字

香茹 ； 木樨草素 ； 動脈粥狀硬化 ； 吸附分子 ； 一氧化氮

並列摘要

Atherosclerosis is a major cause of cardiovascular disease and its mechanism is complex. After damage to the vascular wall, low density lipoprotein (LDL) permeates the sub-endothelial space where it is oxidized, triggers an inflammatory response, recruits monocytes, and transform them to macrophages. Cytokines and growth factors secreted by macrophages induce the migration and proliferation of smooth muscle cells to the sub-endothelial space where they secrete extracellular matrix, and express adhesion molecules and cytokines that recruit other inflammatory cells. Both macrophages and smooth muscle cells accumulate oxidized LDL until they become lipid laden and an integral part of atherosclerotic plaque.
The vascular nitric oxide (NO) system plays a protective role in atherosclerosis. NO is generated from the endothelial NO synthase (eNOS)-mediated conversion of L-arginine to L-citrulline. Endothelial NO exerts its anti-thrombotic and anti-atherosclerotic effects by inhibiting platelet adhesion and aggregation, leukocyte adhesion to and infiltration of the vascular wall, and vascular smooth muscle cell proliferation. Influx and oxidation of LDL are also inhibited by NO. Endothelial dysfunction and reduction of eNOS-derived NO contribute to atherogenesis.
A plant native to Penghu, Taiwan, Glossogyne tenuifolia (GT) is used to make a traditional healthy drink and herbal remedy with antipyretic, hepatoprotective and anti-inflammatory properties. The ethanol extract of GT contains polyphenolic flavonoids, such as luteolin (lut), luteolin-7-glucoside (lut-7-g), and oleanolic acid (OA). These ingredients possess the anti-inflammatory and antioxidant activity. The present study explored the effects of GT ethanol extract and its major components, lut, lut-7-g, and OA on TNF-α-induced expression of adhesion molecules in human umbilical vein endothelial cells (HUVECs), and the possible mechanism. Moreover, this study examined the effects of ethyl acetate (EA), n-hexane, ethanol and methanol extracts of the aerial part, root, and whole GT plant on the regulation of NO production in HUVECs and evaluated their potential for preventing endothelial dysfunction.
Experimental results demonstrated that GT ethanol extract (GTE), lut, and lut-7-g attenuated the expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in TNF-α-activated HUVECs, and inhibited the adhesion of monocytes to TNF-α-activated HUVECs. The TNF-α-induced mRNA expression of ICAM-1 and VCAM-1 was also suppressed, revealing their inhibitory effects at the transcriptional level. Additionally, GTE, lut, and lut-7-g blocked the TNF-α-induced degradation of nuclear factor-kB inhibitor (IkB), an indicator of the activation of nuclear factor-kB (NF-kB). At the second part, the treatment of HUVECs with EA extract from the root of GT, methanol and ethanol extract from the aerial part, root, and whole plant increased NO production in a dose-dependent manner. Among these extracts, the methanol extract from the root had the most potent effect on NO production. Furthermore, the Western blot and RT-PCR analysis showed that the methanol extract from the root could increase eNOS protein and mRNA expression. The treatment with this extract increased eNOS phosphorylation at the serine residue 1177 (Ser1177), indicating that this extract could promote eNOS activity.
In summary, GTE and its bioactive components were effective in preventing the adhesion of monocytes to cytokine-activated endothelium by blocking the activation and nuclear translocation of NF-kB and thereby inhibiting the expression of adhesion molecules. GT extracts also increased NO production in HUVECs and the methanol extract from the root of GT increased NO production via increasing eNOS expression and activation by phosphorylation of eNOS at serine1177.

並列關鍵字

Glossogyne tenuifolia ； luteolin ； atherosclerosis ； adhesion molecule ； nitric oxide

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