帕金森氏症是現今常見的神經退化性疾病，其特徵主要表現在運動功能上的缺失包含靜止時振顫、步態不穩及肢體僵硬等特徵。而基因研究指出當PTEN-induced putative kinase 1 (PINK1)及Parkin基因發生突變可能會導致粒腺體功能失常，進而導致帕金森氏症之神經退化性疾病，另外PINK1基因突變的果蠅亦會產生嗅覺辦識功能障礙。在環境因素方面，一種廣效使用的除草劑百草枯(paraquat)被認為與誘發帕金森氏症有關。根據中醫生理學概念，消化系統功能紊亂，不但影響腸道功能亦會影響其他系統的運作,包含中樞神經系統。因此本實驗採用中草藥紫蘇初萃取物作為實驗藥物，紫蘇(Perilla frutescens)在中醫臨床的主要功能用於治療因飲食不節而導致的腸胃氣滯，進而減緩其他表症的不適感，另外紫蘇另一項功能在於能解食魚蟹之毒。因此我們評估紫蘇可能對於野生型及突變型果蠅因百草枯引發之毒性具有保護作用。實驗結果顯示，紫蘇可以有效的降低因曝露在百草枯(24-72小時)下的致死率，不論在野生型Oregon-R品系及PINK1B9或Parkin25的突變型(年齡：3-7天, 10-14天)。然而紫蘇對於果蠅的運動爬行功能只有在施予百草枯後的野生型Oregon-R 品系(年齡：3-7天)有些微改善，但是在嗅覺辨識功能被百草枯破壞的PINK1B9 突變果蠅，則能經由紫蘇而減少嗅覺辨識的損害。綜合以上實驗結果得知紫蘇對於百草枯所造成的毒性具有保護效果，期望未來可作為治療帕金森氏症的潛在藥物。 / Parkinson’s disease (PD) is the most common neurodegenerative disease characterized by motor deficits including resting tremor, akinesia, and rigidity. The olfactory disturbance appears to be an earlier symptom prior to the onset of motor dysfunction in PD. Genetic research has shown that mutations of the PTEN-induced putative kinase 1 (PINK1) and Parkin genes could lead to mitochondrial dysfunction and neuronal degeneration in PD. Moreover, the environmental neurotoxin paraquat (PQ), a widely used pesticide, is known to induce PD-like symptoms. According to Traditional Chinese medicine physiology theory, that the central nervous system and gut interact bidirectionally in functional gastrointestinal disorders. Perilla frutescens, a traditional herbal medicine, is mainly prescribed for the treatment of gastrointestinal discomfort after eating and symptom of qi stagnation. In addition, perilla has an important role in detoxification of seafood intake. Therefore, this study evaluated the protective effects of perilla on paraquat-induced toxicity in Drosophila PINK1 and parkin mutants and wild type flies. Result showed that perilla can effectively alleviate lethality of drosophila, including PINK1 and parkin mutant flies, exposed to paraquat for 24-72 hours. However, their motor dysfunctions induced by paraquat were little ameliorated by perilla. Importantly, the olfactory dysfunction, particularly olfactory discrimination, elicited by paraquat in PINK1 mutant fly was improved by perilla. Taken together, our findings provide the protective potential of perilla for treatment of PD syndromes.

中文摘要 厚朴，採用厚朴植物之樹皮，是ㄧ種已知可應用於治療精神疾病的傳統天然藥物，例如：憂鬱症等。厚朴主要的有效多酚環成分已被證實具有抗氧化、抗發炎及抗興奮性毒殺等神經保護作用，因此，推測厚朴可作為一種潛在治療像是帕金森氏症這累神經退化性疾病之藥物。本研究之目的為探討厚朴是否可以預防與治療因百草枯及MPTP所誘導的毒害及學習、記憶和運動功能缺失等行為異常現象。本研究監測Oregon-R品系之果蠅(年齡：1-2, 20天或30天)之壽命在長期暴露於百草枯(5-20 mM)並先給予厚朴(100, 300或600 mg/L)治療之變化。其結果顯示，厚朴無法延長暴露在百草枯環境下之果蠅壽命。另外，我們給予雄性ICR小鼠(30-35 g)，連續五天，每日一劑MPTP(25 mg/kg, i.p.)，誘導神經毒性及行為異常現象。在共同投藥組別，在給予MPTP注射前一小時，先以灌餵方式給予小鼠厚朴(100或300 mg/kg)預防，連續五天後，只單獨給予厚朴治療連續十四天。後投藥組別，在給予最後一劑MPTP後，連續十四天給予厚朴(100或300 mg/kg).治療。在控制組別中，給予生理食鹽水(0.9%, i.p.)及灌餵玉米油。結果顯示，MPTP與厚朴並不影響小鼠之運動協調功能，然而，可利用新位置辨識能力測試及新物體辨識認知行為測試，檢測因MPTP所引起之認知功能障礙現象，由我們結果中顯示，不論是與MPTP共同給予厚朴治療抑或是後處理厚朴皆可恢復因MPTP所造成的認知功能障礙現象，此外，厚朴也可恢復因MPTP所造成多巴胺神經元及多巴胺轉運子受損之情形，另外，我們也初步發現，厚朴可在海馬迴中使Nrf2表現量提升。因此，初步結果表明，厚朴將可成為未來治療帕金森氏症之天然藥物。 / Cortex Magnoliae, the bark of Magnolia officinalis, has been prescribed in the traditional herbal medicine to treat a variety of mental disorders including depression. The main constituents of cortex Magnoliae contain the biphenyl compounds such as honokiol and magnolol. Both biphenyl compounds were shown to have the neuronal protective effect which is related to the anti-oxidation, anti-inflammation, and anti-excitatory toxicity. Thus, it was proposed that cortex Magnoliae may act as the potential therapeutic agent for the treatment of neurodegenerative disorders such as Parkinson’s disease (PD). The aim of the present study was to examine whether cortex Magnoliae exhibits the neuroprotective and therapeutic action against the neuronal toxicity and behavioral deficits in learning, memory, and motor function induced by neurotoxin paraquat and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in PD-like models. The lifespan of flies from Oregon-R strain of Drosophila melanogaster (age: 1-2, 20 or 30 days) chronically exposed to paraquat (5-20 mM) with pre-treatment of Cortex Magnoliae (100, 300 or 600 mg/L) were measured. Results showed that pre-treatment of Cortex Magnoliae could not extend the lifespan of the flies reduced by paraquat. On the other hand, male ICR mice (30-35g) were administered with MPTP (25 mg/kg, i.p.) once daily for 5 consecutive days to induce neurotoxicity and behavioral impairment. In co-treatment group, male mice were orally administrated with cortex Magnoliae (100 or 300 mg/kg) 1 hour before MPTP injection for 5 days and then followed by oral administration of cortex Magnoliae alone for consecutive 14 days. Mice in post-treatment group were orally administered with cortex Magnoliae (100 or 300 mg/kg) for consecutive 14 days after the final injection of MPTP. Mice in control group were injected with saline (0.9%, i.p.) and orally administrated with vehicle (corn oil). Our results showed that MPTP and cortex Magnoliae did not affect mouse coordination and balance in beam walking test. However, cortex Magnoliae improved the cognitive impairments determined by novel-location recognition task (NLRT) and novel-object recognition task (NORT) in MPTP-induced PD mouse. Additionally, cortex Magnoliae restored MPTP-induced loss of dopaminergic neurons and recovered MPTP-induced loss of dopamine transporters in striatum. Cortex Magnoliae also activated Nrf2 in hippocampus. Therefore, the preliminary results suggest that cortex Magnoliae may be a novel candidate for the treatment of Parkinson's disease in the future. The pharmacological mechanism of cortex Magnoliae in PD treatment needs further study.

帕金森氏症 (Parkinson’s disease，PD) 為目前最普遍的神經退化性疾病之一，該病因主要是由於中腦黑質區的多巴胺細胞的死亡造成運動系統的失能。環境常用農藥百草枯 (Paraquat，PQ) 目前已知是導致帕金森氏症的環境因子之一。它主要作用在粒線體上，阻斷粒線體的功能、造成大量氧化自由基生成、並誘導細胞凋亡的發生。沉默調節因子蛋白Sirtuin家族 (Sirtuin Family，Sirt1-Sirt7) 是一群 Nicotinamide adenine dinucleotide (NAD+) 依賴性去乙醯化酶，具有抗老化、以及預防神經退化性疾病等能力。SRT1720是根據天然植物酚類白藜蘆醇製造出來的化學衍生物，具有活化Sirtuin的能力。先前研究也顯示SRT1720具有增加糖尿病小鼠的存活率、抗腫瘤、抗發炎等功能，但SRT1720對於神經退化性疾病的保護性並不清楚。為了解SRT1720是否具有對抗PQ的細胞毒性，用以評估SRT1720是否具有治療帕金森氏症的潛力，本研究使用人類神經瘤母細胞株 (SH-SY5Y) 作為帕金森氏症的離體外實驗模式，來探討SRT1720及PQ對於細胞的作用及影響。實驗結果顯示，PQ造成細胞存活率呈劑量反應地下降，而SRT1720可以回復因PQ所造成細胞存活率的下降、細胞凋亡的產生、粒線體的型態變化，以及降低氧化自由基的生成等。這證明SRT1720對細胞具有神經保護的效果。本研究也利用西方點墨法證實了當細胞暴露在PQ下，SRT1720會回復因PQ所導致Sirt1-Sirt7蛋白含量的下降。其中，大量表現Sirt1可以對抗PQ造成細胞的死亡。本研究也發現SRT1720可回復PQ自噬小體在細胞中的堆積情形，利用西方點墨法觀察SRT1720可以回復LC3-I/II的蛋白質再細胞間的堆積。此外，在對於20週大的C57BL/6小鼠注射PQ (10 mg/kg) 及SRT1720 (0.1mg/kg或1 mg/kg)，並利用滾輪及獨木橋試驗觀察其運動行為。結果顯示，SRT1720可以回復PQ所造成運動能力上的下降，並且減緩PQ所造成中腦區多巴胺神經元的傷害。綜觀以上結果，在細胞暴露在PQ時，SRT1720或許可以經由保護粒線體功能，使ROS生成量達到回復及降低細胞凋亡的發生。同時SRT1720也能保持自噬作用的平衡，降低自噬小體在細胞中的堆積。這些機轉也許與SRT1720可以保護多巴胺神經元有關。另一方面，由於Peroxisomal proliferator-activated receptor-coactivator 1α (PGC-1α) 與粒線體的生合成與神經保護有關，本實驗也發現SRT1720可改變PGC-1α去乙醯化的程度，但SRT1720對於Sirtuin蛋白以及Sirt1下游PGC-1α的活化與否還需做進一步的調查及研究。此研究顯示SRT1720對於保護細胞免於受到PQ所引發氧化壓力以及粒線體損傷之神經退化模型提供了一個具有潛力的治療方法。 / Parkinson’s disease (PD) is one of the most common neurodegenerative disorder and mainly affecting the motor system because of the dopamine neuronal death in the substantia nigra. The exposure to environmental neurotoxin paraquat (PQ) is a widely used herbicide. It induces the increase of ROS stress, leads to mitochondrial dysfunction, and results in apoptotic cell death. Epidemiologically, it could be the risk for PD incidence. Mammalian silent information regulator 2 Sirtuin Family (Sirt1-Sirt7) is a NAD+ dependent deacetylase enzyme and it protects against such as anti-aging and neurodegenerative disease. SRT1720 which derives from resveratrol is able to activate Sirt1. SRT1720 has been reported to improve survival in obese mice, anti-tumor, and anti-inflammatory, but the effect in the neurodegenerative disease it still unknown. We thus proposed if SRT1720 could have neuron- protective effect in PQ-induced toxicity. We used SH-SY5Y cell to evaluate the effect of SRT1720 and PQ. First, we confirmed that PQ could dose- and time-dependently decrease SH-SY5Y cell viability, increase ROS formation, and induce mitochondrial dysfunction. However, SRT1720 pretreatment improved cell viability, decreased apoptosis and ROS formation, and prevented mitochondrial dysfunction in PQ-treated SH-SY5Y cells. By Western blot analysis, SRT1720 pretreatment could preserve Sirt1-Sirt7 protein contents during PQ intoxication. In autophagy studies, we also found that SRT1720 could reduce PQ-induce autophagic vacuoles accumulation. Furthermore, we also found that intraperitoneally injection of 10 mg/kg PQ once a week in mice can decrease the level of motor activity after 6 weeks treatment. However, SRT1720 (0.1 mg/kg or 1mg/kg) treatment, reversed PQ- induced motor defect. Taken together, SRT1720 could protect mitochondrial function and improve cell survival during PQ intoxication. This work provided a promising therapeutic way for treating aging-related neurodegenerations, such as PD.