Ellagic acid保護人類皮膚角質細胞對抗光氧化壓力之機制探討

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Title:	Ellagic acid保護人類皮膚角質細胞對抗光氧化壓力之機制探討 Protective Effect of Ellagic Acid on UVA-Irradiated Human Keratinocytes HaCaT Cells
Authors:	傅可婷
Contributors:	營養學系碩士班
Keywords:	紫外線A;鞣花酸;人類皮膚細胞株;活性氧化物 Ultraviolet A;Ellagic acid;Nrf2;ROS
Date:	2011-07-27
Issue Date:	2011-10-17 16:52:01 (UTC+8)
Publisher:	中國醫藥大學
Abstract:	陽光紫外線照射會導致多種急性和慢性的不良影響，會導致皮膚炎症、造成免疫抑制、皮膚過早老化（光老化）甚至可能發展成為皮膚惡性腫瘤。許多對皮膚細胞造成的傷害是經由皮膚接觸到紫外線A（UVA）照射引起細胞內活性氧化物（ROS）生成增加。而過量的ROS生成已被證實與細胞凋亡和對細胞的蛋白質、脂質和核酸氧化損傷相關。先前的研究中已證實鞣花酸Ellagic acid具有顯著的抗氧化活性。本研究目的是探討鞣花酸在人類皮膚角質細胞（HaCaT）對UVA刺激造成之氧化損傷的影響以及Ellagic acid可能是透過何種機制達到保護細胞的作用。本篇研究以HaCaT細胞人類皮膚角質細胞照射UVA和鞣花酸觀察對細胞存活率、活性氧的產生、細胞膜破損情形、DNA片段化和脂質過氧化反應的影響。並且探討保護機制。　　在本篇研究中，UVA照射HaCaT細胞會導致細胞存活率下降和增加活性氧的產生。鞣花酸能有效地提高HaCaT細胞照射UVA後的細胞存活率，並降低UVA刺激造成的氧化壓力。Ellagic acid同時也降低氧化壓力造成的細胞膜損傷、DNA斷裂以及脂質過氧化情形。以TUNEL assay檢測細胞，其觀察結果也發現預先添加Ellagic acid再照射UVA具有降低細胞凋亡，保護細胞的作用。此外，鞣花酸增加Nrf2的蛋白量和促進Nrf2在細胞核內堆積，因此也觀察到Nrf2下游調節的抗氧化蛋白（HO - 1和SOD）也上升。以siNrf2抑制Nrf2表現會使Ellagic acid造成的抗氧化作用被抑制。而細胞經由添加Ellagic acid 24小時之後，細胞質中抑制Nrf2的KEAP1蛋白減少。顯示Ellagic acid能經由影響Nrf2-Keap1路徑，提升細胞防禦能力。　　總結上述結果，鞣花酸能降低KEAP1蛋白生成，並且提高細胞內轉錄因子Nrf2蛋白量和透過促使細胞核內Nrf2聚積增加下游抗氧化蛋白HO-1和SOD，從而保護 HaCaT細胞降低 UVA刺激產生的氧化壓力。所以，鞣花酸也許可作為天然的補充品，保護人類皮膚細胞降低UVA引起的損傷。　　UV radiation from sunlight causes a number of acute and chronic detrimental skin effects which can result in inflammation, immunosuppression, premature skin aging(photoaging) and the development of skin malignancies.Much of the skin cell injury caused by exposure of the skin to ultraviolet A (UVA) irradiation,which is associated with the cellular levels of reactive oxygen species (ROS).Overproduction of ROS has been linked to apoptosis and to the damage of proteins, lipids, and nucleic acids. Ellagic acid is demonstrated to possess remarkable antioxidant activity in the organism.In this study,the aim was to investigate the protective role of ellagic acid in human keratinocytes (HaCaT) against UVA-induced oxidative damage and the possible mechanism of the translocation of NF-E2-related factor-2 (Nrf2) into the nucleus. The HaCaT cells were UVA-irradiated and the effects of ellagic acid on cell viability, reactive oxygen species generation, DNA fragmentation and lipid peroxidation were measured. The proteins of Nrf2 and Kelch-like-ECH-associated protein 1 (Keap1) were determined by immunoﬂuorescence staining and Western blot. 　　In this study, UVA exposure led to a decrease in cell viability and an increase in reactive oxygen species generation in HaCaT cells. Ellagic acid could effectively increase the viability of HaCaT cells after UVA exposure and protect them from UVA-induced oxidative stress. Moreover, ellagic acid increased the level of Nrf2 protein and facilitated Nrf2 accumulation in the nucleus; as a result, the level of antioxidant proteins (HO-1 and SOD) was also upregulated. Importantly, these beneﬁcial effects were attenuated by downregulating Nrf2 expression. The mainﬁnding was that Keap1 protein, a repressor of Nrf2 in the cytoplasm, was decreased by ellagic acid treatment 24 h. 　　Our results suggest that ellagic acid can degrade Keap1 protein and elevate intracellular antioxidative protein via the enhanced accumulation in the nucleus of a transcription factor, Nrf2, thereby protecting HaCaT cells from UVA-induced oxidative stress. Ellagic acid could be a more useful natural medicine for the protection of epidermal cells from UVA-induced damage.
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