| 摘要: | 動脈粥狀硬化是一種發炎的疾病，在動脈粥狀硬化的早期，氧化型低密度脂蛋會造成血管壁的傷害，已有研究指出氧化型低密度脂蛋白會調控細胞內訊息傳遞而誘發壞死及凋亡。當細胞內自由基過高或是細胞內抗氧化酵素功能不張時，就容易引起氧化壓力，氧化壓力過高已被認為是造成動脈粥狀硬化很重要的因素之一。本實驗主要探討白英，來自於中藥的萃取物，兒茶素，來自於綠茶的多酚類化合物，是否能保護氧化型低密度脂蛋白誘發內皮細胞傷害，及其可能的機轉為何，本實驗以用人類臍帶靜脈細胞，利用氧化型低密度脂蛋白誘發內皮細胞傷害時，來討論天然物是否有保護的作用。首先利用種不同的抗氧化實驗來証明天然物之抗氧化能力。分別是利用DPPH 自由基清除試驗、抑制低密度脂蛋白的氧化變性、抑制銅離子及內皮細胞氧化人類低密度脂蛋白的能力。利用螢光染劑DCF-AM來檢測天然物對氧化型低密度脂蛋白所引起自由基產生之抑制效果。相關蛋白表現，如lectin-like oxidized LDL receptor-1(LOX-1) ， cyclooxygenase-2 (COX-2)， p38磷酸化， Akt磷酸化， endothelial nitric oxide synthase (eNOS)， intercellular adhesion molecule-1 (ICAM-1)， vascular cell adhesion molecule-1 (VCAM-1)， monocyte chemoattractant protein-1 (MCP-1) and Chemokine (C-X-C motif) receptor 6 (CXCR6) 及人類血球細胞株THP1黏著實驗。此外，一些與凋亡相關的訊息傳遞，如細胞質內鈣離子濃度，粒腺體膜電位改變，細胞色素C的釋出及相關蛋白表現。最後，發炎相關蛋白表現，如NF-kappaB引起之細胞激素釋放均有探討。實驗結果顯示， 白英及兒茶素可有效的清除自由基及抑制脂質氧化反應，並可以降低氧化型低密度脂蛋白對一氧化氮合成酶的抑制作用，保護細胞表面黏著蛋白表現及THP1粘黏。白英及兒茶素均可調節細胞質內鈣離子濃度進而穩定細胞之粒腺體膜電位、細胞色素C的釋出及Caspase-3的活化，避免氧化型低密度脂蛋白誘發之細胞凋亡。白英及兒茶素亦可增進抗氧化酵素表現藉此降低氧化壓力。白英及兒茶素降低氧化型低密度脂蛋白活化之NF-kappaB及下游發炎反應。我們的研究發現，白英及兒茶素皆可降低氧化型低密度脂蛋白引發內皮細胞傷害。此實驗結果証明白英及兒茶素有很好的抗氧化及抗發炎能力，而具有預防動脈粥狀硬化及心血管疾病的效果。
Atherosclerosis is an inflammatory disease. Atherogenic lipoproteins, such like oxidized low-density lipoprotein (oxLDL), cause injury to the vascular wall in the early stage of atherogenesis. Studies have shown that ox-LDL elicits both necrotic and apoptotic cell death and several mechanisms in endothelial cells have been proposed. The oxidative stress plays a central role in endothelial dysfunction, which is caused by an overabundance of reactive oxygen species (ROS) or a decline in antioxidant ability against them. In this study, we investigated the protective effects of Solanum Lyratum Thumb , one of the traditional medicines in Asia, and (-)-Epigallocatechin-3-gallate (EGCG) , the main catechin in green tea on oxLDL-indcued endothelial dysfunction. Possible mechanisms were explored. Primary human umbilical vein endothelial cell cultures (HUVECs) treated with oxLDL were used to explore the protective effects of SLE and EGCG. SLE were assayed with three antioxidant capacity methods, namely, the DPPH free radical scavenging effect and inhibition oxidative modification of LDL, the inhibition of copper- and endothelial cells-mediated oxidation of human LDL. OxLDL-induced reactive oxygen species (ROS) production were determined by using fluorescent probe 2’,7’-dichlorofluorescein acetoxymethyl ester (DCF-AM).The protein expression of lectin-like oxidized LDL receptor-1(LOX-1), cyclooxygenase-2 (COX-2), phosphorylation of p38, phosphrylation of Akt, endothelial nitric oxide synthase (eNOS) and intercellular adhesion molecule-1 (ICAM-1) vascular cell adhesion molecule-1 (VCAM-1), monocyte chemoattractant protein-1 (MCP-1) and Chemokine (C-X-C motif) receptor 6 (CXCR6) and adherence of monocytic THP1 cells were investigated. Furthermore, several apoptotic signaling pathways including cytosolic calcium, alteration of mitochondrial membrane potential, cytochrome c release and protein expression were investigated. As last, pro-inflammatory signaling pathways were investigated also, such the NF-kappaB-mediated cytokine release. The results showed that SLE and EGCG could inhibit the ROS generation and LDL oxidation, enhance eNOS expression and suppress adhesion molecules well as adherence of monocytic THP1 cells to HUVECs. SLE and EGCG attenuated oxLDL-induced cytotoxicity via regulation of calcium homeostasis and subsequent mitochondrial membrane potential collapse, cytochome c release and activation of caspase-3, thereby suppress endothelial cells apoptosis.SLE and EGCG could enhance antioxidant enzyme expression which contribute suppress of oxidative stress. Furthermore, SLE and EGCG prevented the activation of NF-kappaB and related downstream inflammatory response. Our study demonstrated that SLE and EGCG protect against oxLDL-induced endothelial dysfunction, implying that SLE and EGCG have potential roles in the prevention of atherosclerotic vascular disease via anti-oxidant and anti-inflammatory action. |
