巴西洋菇萃取物與數種純化合物在PC-12細胞模式中抗阿茲海默症之潛力

Abstract

阿茲海默症是常見於老年人的神經退化疾病，導致患者記憶喪失與腦部萎縮。該病症的主因是由於患者腦部呈現類澱粉蛋白β胜肽amyloid-beta peptide (Aβ) 堆積，造成氧化壓力上升、發炎反應、神經元的損傷，進而造成腦部功能退化。PC-12細胞是一種類似副交感神經細胞的細胞株，能夠表現神經細胞的部分生理特性，廣泛應用於阿茲海默症與神經細胞相關研究。本研究將PC-12 細胞培養於含有合成的Aβ之培養基中，以模擬阿茲海默症中Aβ造成神經細胞損傷的情形。利用數種具有抗氧化能力或免疫調節功效的天然物，包含gallic acid、tetrahydrocurcumin、sesamol、proanthocyanidin A2、arachidin-1、以及巴西洋菇(Agaricus blazei) 液態發酵凍乾物的水萃物及乙醇萃物，與Aβ同時處理PC-12細胞，探討樣品保護PC-12細胞免受Aβ損傷的能力。多次進行長片段Aβ (Aβ1-40) 之聚集都未能展現細胞毒性，因此改以可展現毒性的Aβ25-35片段以及過氧化氫進行對PC-12之氧化傷害。結果顯示，分化後的PC-12細胞對於樣品的毒性更為敏感，然而對於過氧化氫的毒性卻較不敏感。Proanthocyanidin A2與arachidin-1都具有良好的保護效果，能減少Aβ25-35對於未分化PC-12 (nPC-12) 造成之傷害；同時在過氧化氫誘導的氧化壓力下，也都展現良好的保護能力。巴西洋菇的水萃物及乙醇萃物對於Aβ25-35毒性並無明顯保護效果，然而能夠保護過氧化氫對未分化PC-12所造成之傷害。在分化的PC-12 (dPC-12) 細胞中，所有樣品都未能有效減少過氧化氫所造成的毒性。在所使用的純化合物中，gallic acid、tetrahydrocurcumin、proanthocyanidin A2、arachidin-1都展現優於正控制組trolox的DPPH清除能力，sesamol及α-tocopherol為其次，而巴西洋菇萃取物DPPH清除能力則最差。結果顯示，proanthocyanidin A2與arachidin-1能夠有效的保護Aβ25-35對未分化PC-12細胞造成的毒性，並且推測樣品能夠有效的降低氧化壓力，進而保護細胞。巴西洋菇的水萃物及乙醇萃物能夠有效保護未分化PC-12細胞免於H2O2的傷害，並推測保護效果並非由於該樣品之抗氧化能力。

Alzheimer’s disease (AD) is the most common form of dementia caused by accumulation of amyloid-beta (Aβ) peptide in the brain, leading to elevated oxidative stress, inflammation, and neuronal loss in the brain. PC-12 cells, a neuronal-like cell line, were treated with Aβ to simulate the toxicity of Aβ towards neuron cells in AD. Several samples with high antioxidative activity including gallic acid, proanthocyanidin A2, arachidin-1, and sesamol, which is able to pass through the blood-brain-barrier (BBB), and samples with immunomodulating activities including tetrahydrocurcumin, Agaricus blazei, were used in Aβ treated PC-12 cells to evaluate the potential of these samples in the prevention of AD. Several methods for the preparation of toxic aggregates of Aβ1-40 fragment failed to exhibit significant toxicity towards PC-12 cells. The shorter active fragment Aβ25-35 was used instead, and H2O2 was also applied to mimic the elevated oxidative stress observed in neurons treated by Aβ. Result showed that differentiated PC-12 cells formed neurites and were more sensitive to toxicity of samples, but more resistance towards oxidative stress induced by H2O2. Both proanthocyanidin A2 and arachidin-1 exhibited protective effect in naïve PC-12 cells towards both Aβ25-35 and H2O2 damage. Ethanol and water extracts of A. blazei did not protect naïve PC-12 cells from Aβ25-35 toxicity, but was effective in protection of H2O2 damage. However, all the samples failed to protect differentiated PC-12 cells from H2O2 insult. Of the pure compound used, gallic acid, tetrahydrocurcumin, proanthocyanidin A2, arachidin-1 all exhibited stronger DPPH scavenging ability than trolox, followed by sesamol and α-tocopherol, with the extracts of A. blazei exhibiting poor scavenging abilities. The results suggest that proanthocyanidin A2, arachidin-1 exhibited protection against Aβ25-35 in naïve PC-12 cells, possibly by decreasing the elevated oxidative stress caused by Aβ25-35. Ethanol and water extracts of A. blazei were poor in DPPH scavenging ability yet protected naïve PC-12 cells from H2O2, implicating mechanism of actions other than direct antioxidative activity.