可延緩Aβ誘發神經不正常去極化之中草藥純物質

Abstract

阿茲海默氏症是一種隨著時間漸漸惡化的重度失智症。目前對於造成阿茲海默氏症發病原因的假說是：因為神經細胞類澱粉前驅蛋白APP的不正常剪切修飾以及神經細胞內骨架蛋白tau的過度磷酸化，最終造成神經細胞壞死，進而使病患失智。有研究指出，β類澱粉蛋白所具有的神經毒性會影響大腦皮質神經元及海馬迴區域麩胺酸的訊息傳遞路徑，並破壞麩胺酸訊息傳遞所調控的學習以及記憶。神經訊息傳導物質麩胺酸自突觸前神經元大量釋出後，會與突觸後神經元細胞膜上的麩胺酸受器AMPA、NMDA、Kainate、mGluR1/3/5結合。為了抑制β類澱粉蛋白誘發的神經毒性，有一種NMDA受器的拮抗劑名為Memantine的藥物，已在臨床中使用於治療重症阿茲海默氏症患者。由於中草藥已被廣泛使用數千年，此研究想從中草藥篩選出能減緩β類澱粉蛋白所誘發的神經損害。實驗使用出生一到三天的小鼠，分離出其大腦神經元進行初級培養，使用免疫螢光染色法和西方墨點法檢驗如vGLUT1/2、AMPAR、PSD95、Type III tubulin這些特殊的標記來確認麩胺酸神經元的純度以及神經軸的形成。再者，使用感應膜電位變化的螢光染劑DiBAC4(3)來偵測神經細胞膜電位的變化。我們使用這個平台來篩選抑制β類澱粉蛋白誘導去極化的中草藥。從四十多種中草藥目前找到六種具有此特性的中草藥單方，其中兩種命名為P1031及1057，在分析主成分其中之純物質P1031-PN及P1057-CC做進一步作用機轉的探討，發現P1031-PN可抑制β類澱粉蛋白所誘發神經不正常的去極化，其作用機轉之一為透過抑制NMDA受器所誘發的去極化，更進一步探討此純物質作用的訊息傳遞機制後，將可能發展成為治療阿茲海默氏症的藥物。Alzheimer disease (AD), a deteriorate neurodegenerative disease, is the most common form of dementia. Abnormal β-amyloid (Aβ) and tau protein plaque accumulation can be observed in patients’ brains by which cause neuronal death and finally become dementia. Currently, the prevailing hypothesis of AD is thought to due to β-amyloid precursor protein (APP) abnormally splicing and tau protein hyperphosphorylation. Glutamate, the neurotransmitter at many excitatory synapses in the central nervous system, binds to glutamate receptor such as AMPA, NMDA, Kainate, and mGluR1/3/5. Researches indicate that the neurotoxicity of Aβ results from the over-stimulation of glutamate through NMDA receptor at postsynapses. Therefore memantine, a NMDA receptor inhibitor, is effective on treating the moderate-to-severe AD patients. In this study, we screen Chinese herbal medicines for ameliorating the Aβ-induced depolarization in neurons. We established the primary culture of cortical neurons by sacrificing postnatal mice at day 1-3. These neurons had been characterized by markers of glutamatergic neurons and neuronal networks such as vGLUT1/2, AMPAR, PSD95, type III tubulin. After stimulated by glutamates or Aβ, these neurons become depolarization indicating by voltage sensitive dye DiBAC4(3). Using this plateform, we found 6 out of 40 Chinese herbal medicines which are capable to block the Aβ-induced depolarization. We focus on two of them named P1031 and P1057 and their pure compounds. We found P1031-PN can inhibit Aβ-induced abnormal depolarization. The mechanism is through the inhibition of AMPAR/ NMDAR pathway. This pure compound is hopefully to become a lead compound on therapeutic purpose of Alzheimer disease.