探討evodiamine在okadaic acid誘導的阿茲海默症模型中神經保護的作用

Abstract

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。吳茱萸成分evodiamine被指出具有抗發炎、抗氧化等許多藥理活性，只有少數關於抗阿茲海默症之研究發表。 本研究建立阿茲海默症模式是利用外加okadaic acid (OA) 至分別為人類神經母細胞瘤–SH-SY5Y和老鼠神經母細胞瘤–Neuro-2a兩種細胞株，抑制蛋白質磷酸酶，進而引發tau蛋白的磷酸化。OA會抑制protein phosphatase 2a (PP2A) 活性和誘發細胞產生氧化壓力引起一系列蛋白激酶如glycogen synthase kinase 3 beta (GSK3β) 和cyclin-dependent kinase 5 (CDK5) 的異常活化，進而使得tau蛋白磷酸化，以此建立阿茲海默症模式。本研究將觀察evodiamine改善氧化壓力的情況以及利用西方墨點法觀察evodimaine在神經細胞株中對於蛋白激酶異常活化之抑制狀況，進而觀察evodiamine減少tau蛋白磷酸化與其蛋白異常聚集之情形，更進一步以流式細胞儀觀察細胞凋亡情形。結果顯示evodiamine能顯著降低細胞氧化壓力及顯著抑制GSK3β、CDK5和mitogen-activated protein kinases (MAPK) /extracellular signal-regulated kinases (ERK) 路徑異常活化的蛋白激酶之效果，進而觀察到evodiamine可以顯著地減少tau過度磷酸化的狀況，更進一步地減少tau在細胞中堆積並且降低細胞凋亡情形；而且呈現時間和劑量相關。在動物實驗中藉由使用莫氏水迷宮和被動式逃避學習來測試動物記憶相關的行為模式，我們可以觀察到evodimaine具有改善小鼠認知記憶學習能力，而西方墨點法之結果顯示在小鼠腦中evodiamine 減少tau蛋白的磷酸化，且如上述提到的像是GSK3β、CDK5和MAPKs這些異常活化的激酶也具有抑制效果。 總結來說，利用外加OA至細胞株的阿茲海默症模式，evodiamine會透過抑制GSK3β和CDK5活性及減少c-Jun N-terminal kinase (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。證明evodiamine具有神經保護效果，為具有潛力能改善阿茲海默症之天然活性物質。

Alzheimer’s disease (AD) is the most common neurodegenerative disease that displays as progressive memory loss and cognitive deterioration. The pathogenesis of AD includes β-amyloid (Aβ) accumulation, tau aggregations, cholinergic deficits and excessive N-methyl-D-aspartate (NMDA) stimulation. Currently, there is still an urgent need to find potential strategies to treat for AD. Evodiamine (Evo), one of the main bioactive ingredients of Evodia rutaecarpa, has been reported to demonstrate anti-inflammation and cardiovascular protective effects. However, few research studies are about the relationship with AD. Consequently, this study is to examine the neuroprotective effects of evodiamine for AD. In this study, we used the in vitro model by treating okadaic acid (OA) to induce phosphorylation of tau to mimic AD-like models in neuronal cells. OA inhibited protein phosphatase 2A (PP2A) and led to active glycogen synthase kinase 3 beta (GSK3β) and cyclin-dependent kinase (CDK5), two main kinases to cause phosphorylation of tau; as well as increase mitogen-activated protein kinases (MAPK) activity due to oxidative stress levels upregulation; and all of which enhanced the hyperphosphorylation of tau. Results by western blot analysis showed that evodiamine significantly reduced the expression of phosphor-tau in a time- and concentration -dependent manner and further decreased the tau aggregation in response to OA treatment; and this inhibition was through the inhibition of GSK3β, CDK5 and MAPK pathways since evodiamine treatment the increase of pGSK3βS9 (the inactive form), the decrease of pGSK3βY216 (the active form), the decline of CDK5 plus p25 and the reduction of phosphorylated MAPK via descending oxidative stress levels. Subsequently, the results from flow cytometry were observed that evodiamine treatment diminished OA-mediated neuronal cell death. Furthermore, the in vivo results indicated that evodiamine treatment ameliorated learning and memory impairments in mice via Morris water maze and passive avoidance test; and the results of western blot analysis by mice brain also demonstrated reduction of phosphorylated tau and inhibition of hyperactive kinases such as GSK3β and ERK1/2. In AD-like animal model, there were the behavioral test and biochemical parameter to confirm the neuroprotective effects of evodiamine. In conclusion, evodiamine can reduce tau phosphorylation, decrease the neurotoxicity of tau aggregation, and acquire the neuroprotective effect. Our results demonstrate evodiamine has therapeutic potential for AD treatment.