桑葉萃取物及活性成分對抗親聚集Tau中介發炎和粒線體功能障礙的潛力

Abstract

阿茲海默氏症(Alzheimer’s disease, AD)是一種與年齡相關的神經退化性疾病，其特徵包括大腦中堆積的Amyoild β斑塊及Tau蛋白聚集的神經纖維纏結(Neurofibrillary tangles, NTF)，影響突觸和認知功能。神經纖維纏結主要由過度磷酸化的Tau蛋白組成，該蛋白會自我聚集並失去微管穩定功能。Tau聚集體可活化小膠質細胞使其釋放發炎因子，並進一步促進神經元中Tau蛋白過度磷酸化。此外，過度磷酸化的Tau蛋白會破壞線粒體功能，從而導致突觸功能障礙。因此，減少小膠質細胞介導的神經炎症，可能有利於改善病理性Tau介導的線粒體功能障礙。由於桑葉(Morus alba L.)已被報導具有抗氧化和抗炎活性，因此本研究聚焦在桑葉萃取物及其七種報導的抗氧化成分。通過HPLC分析桑葉萃取物的化學特徵，色譜圖顯示對應於槲皮素-3-O-芸香苷(QR)、槲皮素-3-O-葡萄糖苷(QG)、山奈酚-3-O-葡萄糖苷(KG)、綠原酸(CGA)和隱綠原酸(4-CQA)的峰值。MTT細胞活力測定顯示桑葉萃取物及其活性成分對BV-2和SH-SY5Y細胞的細胞毒性非常低(IC50大於10 mg/ml或100 μM)。所有七種測試化合物在生化測定中均表現出自由基清除活性和/或氧自由基吸收能力。本研究利用大腸桿菌製備His標記的ΔK280 Tau重複結構域(TauRD)原纖維(Fibril)，並且在冷凍電子顯微鏡(Cryo-TEM)下觀察到寡聚的Tau聚集體。生化硫黃素T螢光測定顯示，QR、QG、槲皮素-3-O-(6-乙酰基)-葡萄糖苷(QAG)、CGA和4-CQA可顯著減少ΔK280 Tau蛋白的聚集。製備的ΔK280 Tau原纖維誘導BV-2微膠細胞活化，這一點可通過細胞型態的改變、一氧化氮(NO)產生的增加和細胞中IBA1和MHCII的表達增加來揭示。桑葉萃取物可以抑制NO、IL-1β、IL-6、TNF-α等促炎介質的產生，並降低BV-2細胞中NLRP3發炎小體及凋亡蛋白酶-1的活化。收集激活的BV-2細胞的條件培養液(Condition media)，並應用於ΔK280 TauRD-DsRed SH-SY5Y細胞引發細胞炎症，桑葉萃取物能降低細胞釋放乳酸脫氫酶、減少細胞活性氧的產生來減輕粒線體損傷、並增加神經突的生長，達到神經保護作用。該研究結果可能支持桑葉萃取物作為改善AD疾病症狀的新療法的概念。

Alzheimer’s disease (AD) is an age-related neurodegenerative disease characterized by aggregated Aβ plaques and neurofibrillary tangles (NFTs) which affects synaptic and cognitive function. NFTs are mainly composed of hyperphosphorylated Tau protein which self-aggregates and loses its function of microtubule stabilization. Tau aggregates activate microglia to release inflammatory factors and promote Tau hyperphosphorylation in neurons. In addition, hyperphosphorylated Tau disrupts mitochondrial function, thereby causing synaptic dysfunction. Therefore, the reduction of microglia-mediated neuroinflammation may be beneficial in ameliorating pathological Tau-mediated mitochondrial dysfunction. Since mulberry (Morus alba L.) leaves have been reported with antioxidant and anti-inflammatory activities, this study focused on the mulberry leaf extract and its seven reported active constituents. The chemical profile of mulberry leaf extract was analyzed by HPLC and chromatographic patterns showed peaks corresponding to the quercetin-3-O-rutinoside (QR), quercetin-3-O-glucoside (QG), kaempferol-3-O-glucoside (KG), chlorogenic acid (CGA) and cryptochlorogenic acid(4-CQA). MTT cell viability assay revealed very low cytotoxicity of mulberry leaf extract and its active constituents in BV-2 and SH-SY5Y cells (IC50>10 mg/ml or 100 μM). All the seven test compounds displayed free radical scavenging activity and/or oxygen radical absorbance capacity in biochemical assays. Then His-tagged ΔK280 Tau repeat domain (TauRD) fibril was prepared from E. coli and oligomeric Tau aggregates were readily seen under cryogenic transmission electron microscopy (cryo-TEM). Tau aggregation was significantly reduced by QR, QG, quercetin-3-O-(6-acetyl)-glucoside (QAG), CGA, and 4-CQA as revealed by biochemical thioflavin T fluorescence assay. The prepared Tau fibril induced BV-2 microglia activation, as revealed by the altered morphology, increased the production of nitric oxide (NO) and IBA1 and MHCII. Mulberry leaf extract suppressed the production of pro-inflammatory mediators, including NO, IL-1β, IL-6, and TNF-α, and reduced the activation of NLRP3 inflammasome and caspase-1 in BV-2 cells. Conditioned media of activated BV-2 cells were collected and applied to ΔK280 TauRD-DsRed SH-SY5Y cells to induce cellular inflammation. Mulberry leaf extract reduced the release of lactate dehydrogenase, decreased cellular reactive oxygen species production to alleviate mitochondrial damage, and enhanced neurite outgrowth, achieving neuroprotective effects. The study results may support the notion of mulberry leaf extract and/or active constituents as a new treatment to improve the symptoms of AD disease.