|摘要: ||已知氧化壓力是造成神經退化性疾病的主要原因。越來越多的文獻顯示小分子類黃酮能有效通過血腦障壁，並能藉由清除神經細胞的自由基、提升神經細胞的抗氧化防禦力 、抑制神經細胞的凋亡、促進神經細胞的新生、神經突觸的生長，達到預防或治療神經退化疾病的功效。過去主持人已探討山奈酚(kaempferol) 和鼠李檸檬素 (rhamnocitrin)對PC12 細胞在缺乏血清及添加H2O2 所引起的氧化傷害的保護作用及機轉，並已於2009 年初發表在Toxicology and Applied Pharmacology。本研究之目的旨在利用PC12 細胞，深入探討其他類黃酮對神經細胞的保護功能及機轉，包括目前正在進行中的柑橘的多甲氧基類黃酮nobiletin 及其代謝物(3', 4'-didemethyl-NOB)及其他親脂性較高的類黃酮。計畫第一年主要以未分化的PC12 細胞當作in vitro model of immature neurons； 並以已受NGF 誘導分化的貼附型PC12 細胞當作in vitro model of differentiated neurons，利用無血清培養、H2O2、 6-OH-DA (模擬PD 的傷害)、 Aβ (25–35) (模擬AD 的傷害) 當做氧化傷害源，探討類黃酮保護氧化傷害的功效及抗氧化機轉。先以細胞存活率做篩選，找出有效之類黃酮化合物，再進一步探討抗氧化機轉，包括胞內ROS 的清除、HO-1 基因表達，ARE activation、Nrf-2 activation 及細胞訊息路徑 (MAPKs)。計畫第二年，繼續探討PI3-kinase/Akt pathway 與抗氧化路徑之關連，同時再探討類黃酮 anti-apoptotic 機轉，包括胞內Ca+2 的改變、對cell cycle、對粒腺體功能及genomic DNA fragmentation 形成的影響、及內生性路徑相關的pro-apoptotic、anti-apoptotic 蛋白及caspases 的活化的影響。第三年則著重於類黃酮的神經滋養功能，利用PC12 細胞在低血清條件下貼附培養，加入類黃酮，篩選出能促進PC12 分化的類黃酮。接著分析這些類黃酮對神經突觸的cell markers 及 neuropepetides 生合成的相關基因表現的影響。最後探討這些類黃酮可能參與PC12 細胞分化的路徑及受體，包括PLCγ/PKC, MAPK, PI3k/AKT, CREB (cAMP response element binding) activation, Trk phosphorylation 等。藉由此三年研究計畫我們希望對類黃酮的神經滋養及保護功能、機轉有深一層的了解，以做為未來動物或人體實驗的依據。|
Oxidative stress has been considered as a major cause of cellular injuries in a variety of clinical abnormalities, especially neural diseases. Emerging evidence suggests that dietary flavonoids, may exert beneficial effects in the central nervous system by protecting neurons against stress-induced injury, by suppressing neuroinflammation and by promoting neurocognitive performance, through changes in synaptic plasticity. It is likely that flavonoids which are able to traverse the BBB, exert such effects in neurons, through selective actions on different components within a number of protein kinase and lipid kinase signaling cascades, such as phosphatidylinositol-3 kinase (PI3K)/Akt, protein kinase C and mitogen-activated protein kinase. Previously, we have reported that kaempferol and rhamnocitrin can augment PC12 antioxidant defense capacity through regulation of heme oxygenase (HO)-1, the phase II antioxidant enzyme, expression and MAPK signal transduction. This proposal aims to extend our previous study by investing the neurotrophic activity, namely the ability to induce neurite outgrowth and to attenuate oxidative damage in rat pheochromocytoma PC12 cells, for other dietary flavonoids, eg. citrus polymethoxylated flavones and lipophilic flavonoids. In the first two years, oxidative damage and apoptosis will be imposed by serum deprivation, hydrogen peroxide (H2O2), 6-OHDA or Aβ (25–35) in undifferentiated and differentiated PC12 cells, which serve as models for immature and mature neurons, respectively. Candidate flavonoid which shows significant protective effect against any of the insults will be used for the subsequent intracellular ROS scavenging, GSH elevation and apoptotic analyses. Furthermore, the possible involvement of HO-1 and the activation of its transcription factor, Nrf2, will also be investigated. The activation of anti-apoptotic proteins and/or inhibition of pro-apoptotic proteins and of caspases activation will also be studied. As of the possible underlying signaling pathways, PI3K/Akt pathway is believed to elicit a survival signal against multiple apoptotic insults; ERK1/2 is usually associated with pro-survival signaling; while, JNK has been strongly linked to transcription-dependent apoptotic signaling. To elucidate the detailed signal transduction network involved, Western blotting and specific inhibitor for each kinase will be employed. In the third year, flavonoids which can promote PC12 differentiation and neurite outgrowth will be selected for further molecular and cellular analyses. The effect of the flavonoid on the expression of neural cell markers 及neuropepetides biosynthesis will be studied. The signaling pathway and tyrosine kinase receptor involved, such as PLCγ/PKC, MAPK, PI3k/AKT, CREB (cAMP response element binding) activation, Trk phosphorylation will also be investigated. In conclusion, it is evident that flavonoids are potent bioactive molecules and a clear understanding of their mechanism of action as modulators of cell signaling will be crucial in the evaluation of their potential to act as inhibitors of neurodegeneration or as neurotrophic agents.