| 摘要: | 神經退化性疾病逐年增多，其中以阿茲海默症為所佔比例最多。包含乙醯膽鹼假說、類澱粉胜肽假說、Tau蛋白假說、自由基與發炎等多重因子皆促進阿茲海默症的病程。文獻指出桑黃中的多酚活性化合物Hispolon具有抗腫瘤、免疫調節、改善糖尿病、保護肝臟、抗氧化及與神經保護多重活性。本實驗室先前以體外乙醯膽鹼酯酶抑制活性評估，同時進行東莨菪鹼誘導失憶 (amnesia)小鼠與配合行為模式評估，結果顯示不同的Hispolon及其結構類似物具有不同程度乙醯膽鹼酯酶抑制活性，而動物介入Hispolon衍生物No.6與No.7明顯改善東莨菪鹼誘導類似阿茲海默症的記憶與學習能力。本論文繼續探討以SH-SY5Y神經纖維母細胞瘤為細胞模式，探討Hispolon 衍生物No.6與No.7對於神經細胞突出生長影響，並與維生素A酸 (retinoic acid, RA) 進行比較。七種Hispolon 衍生物清除DPPH自由基與ORAC抗氧化活性、抑制非酵素性蛋白質醣化反應、並以 (carboxymethyl) lysine (CML) 誘導神經細胞 (以SH-SY5Y為模式細胞) 死亡，評估化合物神經保護活性。Hispolon衍生物No.6, No.7 (5 μM，10 μM)，在5%FBS或10%FBS培養液下處理SH-SY5Y細胞3天到7天後以光學顯微鏡觀察突出生長之形態，並利用image J軟體計算30條突出長度，結果顯示，Hispolon衍生物No.6, No.7在5%FBS或10%FBS培養液下，均有顯著性促進SH-SY5Y突出生長。另利用螢光套組進行非專一性的細胞膜螢光染色定量模式細胞突出生長，隨後利用類流式固態組織細胞儀 (TissueFAXS)掃描96孔盤取得螢光影像。實驗結果顯示，10 μM Hispolon衍生物No.6, No.7在5%FBS培養液下促進細胞突出生長; 5 μM Hispolon衍生物No.6, No.7在10%FBS培養液下促進細胞突出生長且有顯著差異(p<0.001)，神經細胞突出生長隨著天數增加而變多。且在5μM ~ 25μM濃度下可促進神經細胞突出生長。在清除DPPH自由基抗氧化活性方面，Hispolon衍生物均呈現濃度相關活性，衍生物No.3活性最強(IC50為0.3 µM)；在總抗氧化能力ORAC實驗中，Hispolon 衍生物No.1，No.3, No.4, No.7具有較高ORAC清除活性(在2.5 μM 濃度下相當於50 μM Trolox )。以牛血清蛋白和Galactose 為體外非酵素醣化反應模式，生成 (carboxymethyl) lysine (CML)醣化最終產物，而Hispolon衍生物 (100 μM 和500 μM)具有濃度相關抑制生成CML活性。另以CML誘導SH-SY5Y 細胞死亡，Hispolon衍生物可以經由降低細胞內ROS而提高細胞存活性，具有神經細胞保護活性。以上的結果，我們推測出Hispolon衍生物具有神經細胞保護活性及促進神經細胞突出生長的潛力，可能有助於延緩阿茲海默病程的發展，而相關機制須再進一步研究。
Neurodegenerative diseases have been increasing by year, and Alzheimer's disease has taken up the largest proportion. Multiple factors such as acetylcholine hypothesis, starch-like peptide hypothesis, Tau protein hypothesis, free radicals and inflammation had accelerated the course of Alzheimer's disease. The literature pointed out that Hispolon, a polyphenol active compound in Phellinus igniarius, has multiple activities for anti-tumor, immune regulation, improvement of diabetes, liver protection, antioxidant and neuroprotection. Our laboratory evaluated the inhibitory activity of acetylcholinesterase in vitro first, and also evaluated the behavioral patterns of scopolamine-induced amnesia in mice and cooperation. The results showed that different Hispolon and its structural analogs have different degrees of acetylcholine ester. Enzyme inhibitory activity, and animal intervention of Hispolon derivatives No.6 and No.7 significantly improved the memory and learning ability similar to Alzheimer's induced by scopolamine. This paper continues to explore the SH-SY5Y neurofibroblastoma as the cell model, discusses the effects of Hispolon derivatives No.6 and No.7 on the prominent growth of nerve cells, and compares it with retinoic acid (RA). Seven kinds of Hispolon derivatives scavenge DPPH free radicals and ORAC antioxidant activity, inhibit non-enzymatic protein glycation reaction, and use (carboxymethyl) lysine (CML) to induce neuronal cell death (using SH-SY5Y as a model cell), evaluate the neuroprotection of the compound active. Hispolon derivatives No.6, No.7 (5 μM, 10 μM), SH-SY5Y cells were treated in 5% FBS or 10% FBS medium for 3 to 7 days, and then the morphology of synaptic growth was observed under an optical microscope. The image J software was used to calculate the length of 30 synapses. The results showed that Hispolon derivatives No.6 and No.7 significantly promote the prominent growth of SH-SY5Y under 5% FBS or 10% FBS medium. furthermore, a fluorescence kit was used to perform non-specific cell membrane fluorescence staining to quantitatively pattern cell protruding growth, and then use a flow-like solid-state tissue cytometer (TissueFAXS) to scan a 96-well plate to obtain fluorescence images. The experimental results showed that 10 μM Hispolon derivative No. 6, No. 7 promoted cell growth in 5% FBS medium; 5 μM Hispolon derivative No. 6, No. 7 promoted cell protruding in 10% FBS medium The growth was significantly different (p<0.001), and the prominent growth of nerve cells increased with the increase of days. And at a concentration of 5μM ~ 25μM, it can promote the prominent growth of nerve cells. In terms of antioxidant activity in scavenging DPPH free radicals, Hispolon derivatives all show concentration-related activities, with derivative No.3 having the strongest activity (IC50 of 0.3 µM); in the ORAC experiment of total antioxidant capacity, Hispolon derivatives No.1, No. .3, No.4, No.7 have high ORAC scavenging activity (equivalent to 50 μM Trolox at a concentration of 2.5 μM). Using bovine serum albumin and 1M Galactose as the in vitro non-enzymatic saccharification reaction mode, the final product of (carboxymethyl) lysine (CML) saccharification is produced, and Hispolon derivatives (100 μM and 500 μM) have a concentration-related inhibitory activity to produce CML. In addition, CML induces SH-SY5Y cell death. Hispolon derivatives can improve cell viability by reducing intracellular ROS and have neuroprotective activity. From the results above, we speculate that Hispolon derivatives have the potential to protect nerve cells and promote the prominent growth of nerve cells, which may help to delay the progression of Alzheimer's disease, and the related mechanisms could be studied further. |
