木犀草素抑制過氧化氫所誘導內皮細胞的氧化傷害及凋亡

氧化壓力最近被指出與冠狀動脈硬化的發病機制有關，補充抗氧化劑有利於預防冠狀動脈硬化。造成氧化壓力的自由基主要包含O2-、H2O2、．OH，會使內皮細胞功能受損進而導致冠狀動脈硬化及其它心血管方面之疾病。木犀草素（luteolin）屬於類黃酮之一，是一種天然的多酚類（polyphenolic compounds），廣泛的存於植物中，已知的生物效應包括抗發炎和免疫調控等。然而有關木犀草素是否具心血管保護作用，目前為止仍不清楚。因此本實驗利用過氧化氫（600 M）誘發內皮細胞氧化傷害為模式，探討木犀草素（2.5-20 M）是否具有血管內皮的保護作用及其可能之作用機轉。實驗方法為利用MTT及LDH release測定細胞存活率、螢光染劑DCF-AM、DHE來測定細胞內自由基產生，以西方轉漬法分析木犀草素保護過氧化氫傷害之訊息傳遞路徑進一步利用siRNA技術將AMPK蛋白降解，以釐清木犀草素保護內皮細胞的作用機制是否經由AMPK而抑制PKC所活化之NADPH oxidase。利用螢光染劑JC1測定粒腺體膜電位，以caspase 3活性測試及TUNEL染色法來測定木犀草素是否保護過氧化氫所誘發內皮細胞凋亡。實驗結果顯示，木犀草素可保護過氧化氫所造成的細胞死亡，並有效的清除自由基及回復過氧化氫所對抗氧化酵素SOD-I之抑制。木犀草素抑制過氧化氫誘發之NADPH Oxidase complex的活性是透過AMPK對PKC的抑制。此外過氧化氫經由MAPK 之活化造成細胞質IkB降解導致轉錄因子NF-kB轉移到細胞核並啟動下游發炎相關基因之如誘發行一氧化氮合成酶（iNOS）表現。過氧化氫經由轉錄因子P53磷酸化而增加促凋亡蛋白Bax及抑制抗凋亡蛋白Bcl-2導致粒腺體膜電位下降，電子傳遞蛋白細胞色素Ｃ（cytochrome C）釋放到細胞質而活化凋亡執行蛋白caspase 3而導致細胞凋亡。上述之傷害木犀草素呈現劑量關係的保護作用。本實驗結果有助瞭解木犀草素保護氧化壓力誘發血管內皮凋亡之分子機制，已提升未來應用於預防心血管疾病的價值。

關鍵字

木犀草素；內皮細胞

並列摘要

Oxidative stress has recently been implicated in the pathogenesis of atherosclerotic cardiovascular diseases. Dietary supplement of antioxidants has been reported to have beneficial effects on prevention of atherogenic diseases. Reactive oxygen species (ROS) such as superoxide anion (O2−), hydrogen peroxide (H2O2) and hydroxyl radical (•OH) are continuously produced in cells as products of cellular oxidation–reduction processes and as the mechanisms of biophylaxis. Luteolin, 3,4,5,7-tetrahydroxyflavone, a polyphenolic compound, usually occurs as glycosylated forms in celery, green pepper, perilla leaf and camomile tea. It has been found to possess antimutagenic, antitumorigenic antioxidant and anti-inflammatory properties. However, the effects of luteolin on cardiovascular systems are still largely unknown. Therefore, the aim of the present study is to test whether luteolin could protect against oxidative stress-induced endothelial cell injury and explore the possible mechanisms. Primary human umbilical vein endothelial cells (HUVECs) were pretreated with luteolin (2.5-20 uM) for 2 hours followed by hydrogen peroxide (600 uM) for indicated time period. Cell viability was determined by MTT and LDH release. The generation of reactive oxygen species (ROS) and superoxide were determined by using the fluorescent probe 2’, 7’-dichloro-fluorescein acetoxymethyl ester (DCF-AM), dihydroethidium (DHE), respectively. The protein levels of ROS-mediated signaling pathways were measured by Western blot. In addition, AMPK knockdown were employed to explore whether the AMPK/PKC/NADPH oxidase involved in the process. Furthermore, several apoptotic features which showed NF-kB activation, alteration of mitochondria membrane potential, cytochrom c release and subsequent activation of caspase 3 were also inverstigated. Our results showed that luteolin protected hydrogen peroxide-induced cell death in a dose-dependent manner. Luteolin ameliorated ROS production and impaired antioxidant enzyme SOD-I caused by hydrogen peroxide. Luteolin suppressed hydrogen peroxide-induced superoxide anion radical generation and membrane assembly of NADPH oxidase subunits via modulating AMPK-suppressed PKC activation. Application of AMPK1-specific siRNA resulted in increased activation of PKC and p47phox. In addition, hydrogen peroxide also decreased AMPK-mediated Akt/eNOS/NO signaling and induced the phosphorylation of MAPK which in turn activated NF-B-mediated inflammatory responses such as the iNOS expression leading to a overproduction of NO and protein nitrosylation. We also found that hydrogen peroxide increased phosphorylation levels of transcription factor P53 which in turn disturbed the balance of Bcl-2 family proteins, destabilized mitochondrial membrane potential, and triggered subsequent cytochrome c release into the cytosol and activation of caspase 3. Pretreatment with luteolin, however, exerted significant cytoprotective effects in a dose-dependent manner. Results from this study may provide insight into a possible molecular mechanism underlying luteolin suppression of the oxidative stress-induced endothelial dysfunction.

並列關鍵字

Endothelial cells ； Luteolin

參考文獻

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木犀草素抑制過氧化氫所誘導內皮細胞的氧化傷害及凋亡

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