探討石斛成分Moscatilin在阿茲海默症的神經保護作用之研究

Abstract

實驗目的 阿茲海默症 (Alzheimer’s disease, AD) 是一種神經退化性疾病，是最常造成失智 (dementia) 的疾病因素，會表現漸進式記憶喪失及認知障礙。臨床上，阿茲海默症的病理特徵為β-amyloid (Aβ) 老年斑塊之沉積、神經細胞內表現過度磷酸化Tau蛋白所聚集成的神經纖維糾結 (NFTs) 及乙醯膽鹼 (acetylcholine) 含量減少。美國食品藥物管理局 (Food and Drug Administration, FDA) 認可已上市的藥物有五種分別為donepezil、rivastigmine、galantamine、memantine、namzaric，然而這些藥物僅能減緩症狀無法完全根治阿茲海默症；因此科學家們仍致力於找尋有效治療阿茲海默症的藥物。石斛成分moscatilin被指出具有抗發炎、解熱等許多藥理活性，然而還沒有關於抗阿茲海默症之研究發表。 實驗方法 本研究利用兩種阿茲海默症模式細胞株，其一為細胞轉染pRK5-EGFP-Tau P301L 和pCAX-FLAG-APP兩質體，將神經細胞株過度表現Aβ前身蛋白APP與易磷酸化之突變tau蛋白，建立阿茲海默症模式神經細胞株；另一種為外加okadaic acid (OA) ，抑制蛋白磷酸酶，進而磷酸化tau蛋白，兩種模式皆會引起tau蛋白磷酸化以及誘發一系列激酶如GSK3β的異常活化，以此建立阿茲海默症模式。以西方墨點法觀察moscatilin在神經細胞株中減少Tau蛋白磷酸化與其蛋白異常聚集之情形；並觀察moscatilin對於抑制蛋白激酶磷酸化tau蛋白之作用，更進一步以流式細胞儀觀察細胞凋亡情形。在活體實驗中，利用腹腔注射scopolamine誘發阿茲海默症，建立認知障礙之小鼠模型，藉由莫式水迷宮與舉臂式十字迷宮確認moscatilin改善認知能力，並以西方墨點法及免疫組織染色法確認神經保護之效果。 實驗結果 結果顯示moscatilin能顯著減少tau磷酸化表現量，更進一步減少tau在細胞中堆積的程度，並呈現time-和dose-dependent的情形。而後又觀察到moscatilin具有抑制GSK3β和MAPK/ERK路徑異常活化的激酶之效果，以這樣的抑制效果改善tau過度磷酸化的情形，接著實驗結果顯示moscatilin有減少細胞凋亡之能力。動物試驗結果透過莫式水迷宮及舉臂式十字迷宮呈現，moscatilin具有改善小鼠認知記憶學習之能力，而後西方墨點法與免疫組織染色法之結果皆顯示小鼠腦中moscatilin減少tau蛋白的磷酸化，且異常活化的激酶也與細胞實驗有相同的被抑制效果。 結論 利用細胞轉染pRK5-EGFP-Tau P301L及pCAX-FLAG- APP兩質體與外加OA兩種阿茲海默症模式，moscatilin會透過抑制GSK3β活性及減少JNK、ERK、p38磷酸化，顯著地減少tau蛋白磷酸化，並減少細胞內tau蛋白聚集，更進一步減少神經細胞凋亡情形。而後動物實驗證明moscatilin能改善小鼠空間學習記憶，並改善小鼠腦中病理變化，證明moscatilin具有神經保護效果，因此證實moscatilin是具有潛力能改善阿茲海默症之天然活性物質。

Objective：Alzheimer’s disease (AD) is a neurodegenerative disease which is the most common cause of dementia that manifests as progressive memory loss and cognitive decline. Pathologically, AD is characterized by β-amyloid (Aβ) accumulation, tau aggregations and dysfunction of Ach and NMDA receptor. Currently, there is still an urgent need to find potential strategies to treat for AD. This study is to examine the neuroprotective effects of moscatilin, a bibenzyl extraction from orchid Dendrobium loddigesii or Dendrobium nobile. Moscatilin was reported used to reduce fever and has exerted anti-inflammatory actions, yet none research studies about the relationship with AD. Methods：In this study, we used two methods to mimic AD-like models in vitro. One is co-transfection of pRK5-EGFP-Tau P301L and pCAX-FLAG-APP plasmids making the neuron cells overexpress amyloid precursor protein (APP) and mutant tau protein. The other is adding okadaic acid (OA) to induce phosphorylation of tau protein. Both of the methods result in the hyperphosphorylation of tau protein and trigger the activity of GSK3β, the main kinase indicated to cause phosphorylation of tau protein. We used western blot analysis to detect the protein expression and flow cytometry assay to investigate the apoptotic cells. Furthermore, we used a scopolamine-induced model as cognitive impairment in mice to investigate the neuroprotective effects of moscatilin. Result：We found that moscatilin significantly reduces the expression of phospho-tau in a time- and dose-dependent manner and further decreased the tau aggregation. Results showed that moscatilin reverses the tauopathy through the inactivation of GSK3β and MAPK/ERK pathway. Furthermore, we observed that moscatilin decreased the percentage of apoptotic cells induced by OA. The in vivo results indicated that moscatilin ameliorates learning and memory impairments in mice via Morris water maze and elevated plus maze. The IHC staining of mice hippocampus and western blot results of mice brain also confirmed the neuroprotective effects of moscatilin. Conclusion：Moscatilin significantly decreased tau phosphorylation after co-transfection of pRK5-EGFP-Tau P301L and pCAX-FLAG-APP and OA treatment. The inhibition of tau phosphorylation is through GSK3β inactivation and downregulation of JNK, ERK, p38 phosphorylation. Moscatilin decreased cellular tau aggregation and also reduced neuron cell apoptosis in vitro. Furthermore, moscatilin significantly ameliorated the spatial memory impairment in vivo. Moscatilin therefore demonstrates therapeutic potential for AD.