茶多酚對甲基乙二醛誘導人類神經細胞毒性之保護功效

The Protective Effect of Tea Polyphenol against Methylglyoxal-induced Neurotoxicity

黃孜立(Tzu-Li Huang)

指導教授 ： 潘敏雄

共同指導教授 ： 李銘仁(Ming-Jen Lee)

國立臺灣大學/生物資源暨農學院/食品科技研究所/碩士(2016年)

https://doi.org/10.6342/NTU201603033

摘要

甲基乙二醛 (methylglyoxal, MG) 為一種高反應活性的雙羰基醛類化合物，已有許多研究證實MG造成神經損傷進一步導致神經退化性疾病的發生。茶葉是一種傳統的飲品，流行病學研究指出，長期飲茶能有效降低罹患神經退化性疾病的風險。文獻提出綠茶中的主要活性物質:表沒食子兒茶素沒食子酸酯「(-)-epigallocatechin-3-gallate, EGCG」以及烏龍茶經發酵後產生的EGCG二聚物: 聚酯型兒茶素A (theasinensin A, TSA) 具有抗氧化等功效，然而其在神經保護之功效與分子機制仍不清楚。因此，本篇研究擬以人類神經瘤細胞SH-SY5Y探討茶多酚EGCG以及TSA對於MG誘導的神經毒性之保護作用及其分子機轉。本研究顯示，EGCG與TSA對於MG誘導的神經毒性具有相當的保護效果。EGCG與TSA皆可透過阻斷MG誘導之活性氧物質 (reactive oxygen species, ROS) 產生以及維持胞內GSH含量，藉此減少MG誘導的氧化壓力。透過caspase 3活化及其基質PARP降解可知EGCG能抑制MG誘導之細胞凋亡現象，然而TSA抑制MG誘導細胞凋亡現象並不顯著。透過acridine orange (AO) 染色於共軛焦顯微鏡下觀察、AO染色後透過流式細胞儀測量螢光強度以及LC3蛋白質的表現判斷MG在TSA存在下會促使細胞走向自噬作用進而保護神經細胞。在MG誘導神經毒性之情形下，EGCG抑制p53、MAPK訊息傳遞路徑進而抑制細胞凋亡，另一方面TSA透過抑制Akt訊息傳遞路徑達到促進細胞自噬作用之功效。綜合上述發現在MG誘導之神經細胞死亡中 EGCG和TSA扮演不同角色與功能，EGCG透過抑制MG誘導的細胞凋亡而TSA則是透過誘發細胞自噬進而保護細胞免於MG誘導的細胞毒性。我們期望此研究成果未來能提供茶多酚開發為預防MG誘導神經相關疾病發展之重要依據。

關鍵字

甲基乙二醛 ； 表沒食子兒茶素沒食子酸酯 ； 聚酯型兒茶素A ； 細胞凋亡 ； 細胞自噬 ； 氧化壓力

並列摘要

Methylglyoxal (MG) is a highly reactive dicarbonyl aldehyde. MG has been proved to be toxic to neuron and may be the reason of many neurodegenerative diseases. Tea is a traditional drinking and previous studies have observed that chronic tea consumption may reduce the risk of neurodegeneration. One of the most well-known tea catechin, (-)-epigallocatechin-3-gallate (EGCG), and theasinensin A (TSA), the dimer of EGCG had been found to have many bioactivities, such as anti-oxidation. However, the effect of the neuroprotective effect of tea polyphenol remains unclear. The aim of this study is to investigate the neuroprotective effects and the molecular mechanism of tea polyphenol against MG-induced toxicity in SH-SY5Y cell model. The cell viability assay demonstrated that EGCG and TSA treatment protected cells from MG-induced neurotoxicity. Besides, evidences from flow cytometry showed that EGCG and TSA inhibited MG-induced oxidative stress via reducing MG-induced reactive oxygen species (ROS) generation and induce intracellular GSH level. Through activation of caspase 3 and cleavage-PARP, EGCG would inhibit MG-induced apoptosis but TSA did not inhibit MG-induced apoptosis. The result of acridine orange (AO) stain, detecting AO fluoresce intensity and the level of LC3, showed that TSA would induce autophagy in MG-treated neuron cell. EGCG inhibited p53 and MAPK pathway to increase cell viability via western blotting analysis; on the other hand, TSA inhibited Akt pathway to induce autophagy. In conclusion, both EGCG and TSA has the ability to protect cell from MG-induced cytotoxicity via different mechanisms. We expect that EGCG and TSA could be reagents to protect neurodegenerative diseases.

並列關鍵字

Methylglyoxal (MG) ； (-)-Epigallocatechin-3-gallate (EGCG) ； Theasinensin A (TSA) ； Apoptosis ； Autophagy ； Oxidative stress

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