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研究生: 王雪娥
Wang, Sheue-Er
論文名稱: 中草藥對於亨丁頓舞蹈症模式小鼠的神經保護機制
Neuroprotection of Chinese herbal medicine in the R6/2 mouse model of Huntington's Disease
指導教授: 吳忠信
Wu, Chung-Hsin
學位類別: 博士
Doctor
系所名稱: 生命科學系
Department of Life Science
論文出版年: 2016
畢業學年度: 104
語文別: 英文
論文頁數: 70
中文關鍵詞: 亨丁頓舞蹈症中國傳統醫藥亨丁頓蛋白聚集神經保護血管新生興奮性毒殺作用氧化壓力發炎反應細胞凋亡基因轉殖小鼠模型
英文關鍵詞: Huntington’s disease, Traditional Chinese medicine, huntingtin aggregation, neuroprotection, angiogenesis, excitotoxicity, oxidative stress, inflammation, apoptosis, transgenic mouse model
DOI URL: https://doi.org/10.6345/NTNU202204498
論文種類: 學術論文
相關次數: 點閱:158下載:1
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  • 亨丁頓舞蹈症(HD)是一種無法治癒的神經退化性疾病,儘管多年的研究,關於亨丁頓舞蹈症神經退化病變的機制仍然不清楚。中國傳統醫藥可以提供新的見解或HD的新療法。中草藥配方的B401是一個眾所周知的台美專利配方,由六種中草藥成分組成。本論文研究主要探討中草藥配方B401對於亨丁頓舞蹈症的神經保護作用。本實驗比較口服中草藥配方B401實驗組以及控制組之R6 / 2小鼠的壽命和體重;透過行為實驗比較口服中草藥配方B401對R6 / 2小鼠的改善情形;利用moorFLPI雷射都普勒成像儀測量R6 / 2小鼠的皮下微循環;利用核磁共振成像技術測量R6 / 2小鼠的腦部體積變化;利用化學發光方法測量R6 / 2小鼠血液中的反應性氧物質(ROS);利用免疫組織化學染色和西方轉漬技術分析10週齡R6 / 2小鼠腦組織中的關於神經保護、血管生成、氧化壓力,發炎反應和細胞凋亡相關的蛋白質表現,我們的研究結果顯示R6 / 2小鼠口服B401後會延長生存時間、降低體重損失、並且提高運動能力。此外,B401處理也會增強R6 / 2小鼠皮下的微循環;緩解大腦、中腦和小腦的萎縮;並且降低血液中的ROS生成。從我們的研究顯示,口服B401治療顯著增加R6 / 2小鼠腦組織當中的BDNF、VEGF、以及SOD2的表現;但是降低亨丁頓蛋白聚集以及TNF-α的表現。此外,口服B401治療也顯著增加R6 / 2小鼠腦中Bcl-2的表現,但是顯著降低BAX、Calpain、以及Caspase 3的表現。我們的研究顯示口服B401治療亨丁頓舞蹈症R6 / 2小鼠的神經保護機制主要是通過增強神經與血管的新生,但是抑制興奮性毒殺,氧化壓力,發炎反應與細胞凋亡。因此我們認為中草藥配方B401應該可以開發成為改善亨丁頓舞蹈症神經退化性疾病的有效保健品。

    Huntington’s disease (HD) is a neurodegenerative disease characterized by motor dysfunction, and early death. Despite years of research, the mechanisms responsible for chronic neurodegeneration of HD remain elusive. Chinese traditional medicines might provide new insights or new therapy for HD. The Chinese herbal formula B401 is a well-known Taiwan-US patent formula and consists of six herbal ingredients. My study aimed to elucidate neuroprotective effects of the Chinese herbal formula B401 in the syndrome of HD. We compared the lifespan and body weight of R6/2 mice with and without oral B401 treatment. The ameliorative effects of B401 on symptom of HD mice were investigated through behavior tests. We measured the microcirculation of R6/2 mice by using moorFLPI Laser Doppler Imager, the brain volume of R6/2 mice by using magnetic resonance imaging, the reactive oxygen species (ROS) levels in the blood of R6/2 mice by using chemiluminescence methods, and expressions of proteins for neuroprotection, angiogenesis, oxidative stress, inflammation, and apoptosis in the brain tissue of 10-week-old R6/2 mice by using immunohistochemical staining and western blotting analysis. Our results showed oral B401 treatment increases duration of survival, reduces loss of body weight, and improves motor ability. In addition, B401 treatment enhances subcutaneous microcirculation, alleviates atrophy of cerebrum, midbrain and cerebellum, and reduces blood ROS of R6/2 mice. Evidences from our study show that oral B401 treatment significantly increases the brain-derived neurotrophic factor (BDNF), vascular endothelial growth factor (VEGF), and superoxide dismutase 2 (SOD2), but reduces the huntingtin and tumor necrosis factor-alpha (TNF-α) levels in the brain of R6/2 mice. Furthermore, oral B401 treatment significantly increases B-cell lymphoma 2 (Bcl-2), but reduces Bcl-2-associated X protein (Bax), calpain and caspases-3 in the brain of R6/2 mice. Our study demonstrated the neuroprotection of oral B401 treatment within the brain tissue of R6/2 mice is via enhancing neurogenesis and angiogenesis, but suppressing excitotoxicity, oxidative stress, inflammation and apoptosis. We suggest that the herbal formula B401 can be developed as a potential health supplement for ameliorating the neurodegenerative symptoms of HD.

    TABLE OF CONTENTS I LIST OF FIGURES IV 中文摘要 VI ABSTRACT VII CHAPTER 1 INTRODUCTION 2 1.1 HUNTINGTON'S DISEASE 3 1.2 TRANSGENIC MOUSE MODELS OF HD 3 1.3 THE HERBAL FORMULA B401 4 CHAPTER 2 RESEARCH AIMS 6 CHAPTER 3. MATERIALS AND METHODS 8 3.1 CHROMATOGRAPHIC FINGERPRINT ANALYSIS FOR THE B401 9 3.2 SH-SY5Y CELL VIABILITY ASSAY FOR THE B401 9 3.3 R6/2 TRANSGENIC HD MICE PREPARATION AND MONITOR 10 3.4 BEHAVIOR ANALYSIS IN R6/2 MICE 11 3.5 ANALYSIS OF SKIN BLOOD FLOW AND ROS IN R6/2 MICE 12 3.6 ANALYSIS OF BRAIN MORPHOLOGY IN R6/2 MICE 13 3.7 WESTERN BLOTTING IN THE BRAIN OF R6/2 MICE 13 3.8 HISTOLOGY IN THE BRAIN OF R6/2 MICE 14 3.9 STATISTICAL ANALYSIS 15 CHAPTER 4. RESULTS 16 4.1 THE B401 ALLEVIATES EARLY DEATH AND BODY WEIGHT LOSS IN R6/2 MICE 17 4.2 THE B401 ALLEVIATES MOTOR DISORDERS IN R6/2 HD MICE 17 4.3 THE B401 ENHANCES SUBCUTANEOUS MICROCIRCULATION AND REDUCES BLOOD ROS OF R6/2 MICE 18 4.4 THE B401 ALLEVIATES BRAIN ATROPHY IN R6/2 MICE 19 4.5 THE B401 REDUCES MHTT LEVELS IN THE BRAIN OF R6/2 MICE 19 4.6 THE B401 IMPROVES NEUROPROTECTION AND ANGIOGENESIS IN THE BRAIN OF R6/2 MICE 20 4.7 THE B401 ALLEVIATES HYPOXIA, INFLAMMATION AND OXIDATIVE STRESS IN THE BRAIN OF R6/2 MICE 21 4.8 THE B401 ALLEVIATES APOPTOSIS IN THE BRAIN OF R6/2 MICE 22 CHAPTER 5. DICUSSION 24 CHAPTER 6. CONCLUSION 32 REFERENCES 34 FIGURES 42

    Altar CA, Cai N, Bliven T, Juhasz M, Conner JM, Acheson AL, Lindsay RM, Wiegand SJ (1997) Anterograde transport of brain-derived neurotrophic factor and its role in the brain. Nature 389(6653): 856–860.
    Ayala GX, Tapia R (2005) Late N-methyl-D-aspartate receptor blockade rescues hippocampal neurons from excitotoxic stress and death after 4-aminopyridine-induced epilepsy. Eur J Neurosci 22(12): 3067–3076.
    Bates G, Harper P, Jones L (2002) Huntington’s Disease. Oxford, UK: Oxford Univ Press 2002.
    Beal MF (1995) Aging, energy, and oxidative stress in neurodegenerative diseases. Ann Neurol 38(3): 357–366.
    Beal MF (1996) Mitochondria, free radicals and neurodegeneration. Curr Opin Neurobiol 6(5): 661–666.
    Carter RJ, Lione LA, Humby T, Mangiarini L, Mahal A, Bates GP, Dunnett SB, Morton AJ (1999) Characterization of progressive motor deficits in mice transgenic for the human Huntington's disease mutation. J Neurosci 19(8): 3248–3257.
    Cepeda C, Wu N, Andre VM, Cummings DM, Levine MS (2007) The corticostriatal pathway in Huntington’s disease. Prog Neurobiol 81(5-6): 253–271.
    Cepeda-Prado E, Popp S, Khan U, Stefanov D, Rodríguez J, Menalled LB, Dow-Edwards D, Small SA, Moreno H (2012) R6/2 Huntington's disease mice develop early and progressive abnormal brain metabolism and seizures. J Neurosci 32(19): 6456–6467.
    Cha JH (2007) Transcriptional signatures in Huntington’s disease. Prog Neurobiol 83(4): 228–248.
    Chien CT, Lee PH, Chen CF, Ma MC, Lai MK, Hsu SM (2001) De novo demonstration and co-localization of free-radical production and apoptosis formation in rat kidney subjected to ischemia/reperfusion. J Am Soc Nephrol 12(5): 973–982.
    Chiu E, Alexander L (1982) Causes of death in Huntington’s disease. Med J Aust 1(4): 153.
    Chong ZZ, Li F, Maiese K (2005) Oxidative stress in the brain: novel cellular targets that govern survival during neurodegenerative disease. Prog Neurobiol 75(3): 207–246.
    Davies SW, Turmaine M, Cozens BA, DiFiglia M, Sharp AH, Ross CA, Scherzinger E, Wanker EE, Mangiarini L, Bates GP (1997) Formation of neuronal intranuclear inclusions underlies the neurological dysfunction in mice transgenic for the HD mutation. Cell 90(3): 537–548.
    Desai Bradaric B, Patel A, Schneider JA, Carvey PM, Hendey B (2012) Evidence for angiogenesis in Parkinson’s disease, incidental Lewy body disease, and progressive supranuclear palsy. J Neural Transm 119(1): 59–71.
    Desai BS, Schneider JA, Li JL, Carvey PM, Hendey B (2009) Evidence of angiogenic vessels in Alzheimer’s disease. J Neural Transm 116(5): 587–597.
    Estrada Sánchez AM, Mejía-Toiber J, Massieu L (2008) Excitotoxic neuronal death and the pathogenesis of Huntington’s disease. Arch Med Res 39(3): 265–276.
    Fan MM, Raymond LA (2007) N-methyl-D-aspartate (NMDA) receptor function and excitotoxicity in Huntington’s disease. Prog Neurobiol 81(5-6): 272–293.
    Ferrante RJ, Gutekunst CA, Persichetti F, McNeil SM, Kowall NW, Gusella JF, MacDonald ME, Beal MF, Hersch SM (1997) Heterogeneous topographic and cellular distribution of huntingtin expression in the normal human neostriatum. J Neurosci 17(9): 3052–3063.
    Gervais FG, Singaraja R, Xanthoudakis S, Gutekunst CA, Leavitt BR, Metzler M, Hackam AS, Tam J, Vaillancourt JP, Houtzager V, Rasper DM, Roy S, Hayden MR, Nicholson DW (2002) Recruitment and activation of caspase-8 by the Huntingtin-interacting protein Hip-1 and a novel partner Hippi. Nat. Cell Biol 4(2): 95–105.
    Gowdak LH, Poliakova L, Wang X, Kovesdi I, Fishbein KW, Zacheo A, Palumbo R, Straino S, Emanueli C, Marrocco-Trischitta M, Lakatta EG, Anversa P, Spencer RG, Talan M, Capogrossi MC (2000) Adenovirus-mediated VEGF(121) gene transfer stimulates angiogenesis in normoperfused skeletal muscle and preserves tissue perfusion after induction of isch¬emia. Circulation 102(5): 565–571.
    Gutekunst CA, Li SH, Yi H, Mulroy JS, Kuemmerle S, Jones R, Rye D, Ferrante RJ, Hersch SM, Li XJ (1999) Nuclear and neuropil aggregates in Huntington’s disease: relationship to neuropathology. J Neurosci 19(7): 2522–2534.
    Halliwell B (2001) Role of free radicals in the neurodegenerative diseases: therapeutic implications for antioxidant treatment. Drugs Aging 18(9): 685–716.
    Hickey MA, Chesselet MF (2003) Apoptosis in Huntington's disease. Prog Neuropsychopharmacol Biol Psychiatry 27(2): 255–265.
    Ho LW, Brown R, Maxwell M, Wyttenbach A, Rubinsztein DC (2001) Wild type Huntingtin reduces the cellular toxicity of mutant Huntingtin in mammalian cell models of Huntington’s disease. J Med Genet 38(7): 450–452.
    Huettner JE, Bean BP (1987) Block of N-methyl-D-aspartate-activated current by the anticonvulsant MK-801: selective binding to open channels. Proc Natl Acad Sci USA 85(4): 1307–1311.
    Jenkins BG, Koroshetz WJ, Beal MF, Rosen BR (1993) Evidence for impairment of energy metabolism in vivo in Huntington’s disease using localized 1H NMR spectroscopy. Neurology 43(12): 2689–2695.

    Kubli S, Waeber B, Dalle-Ave A, Feihl F (2000) Reproducibility of laser Doppler imaging of skin blood fl ow as a tool to assess endothelial function. J Cardiovasc Pharmacol 36(5): 640–648.
    Lanska DJ, Lavine L, Lanska MJ, Schoenberg BS (1988) Huntington’s disease mortality in the United States. Neurology 38(5): 769–772.
    Leavitt BR, Guttman JA, Hodgson JG, Kimel GH, Singaraja R, Vogl AW, Hayden MR (2001) Wild-type huntingtin reduces the cellular toxicity of mutant huntingtin in vivo. Am J Hum Gene 68(2): 313–324.
    Mangiarini L, Sathasivam K, Mahal A, Mott R, Seller M, Bates GP (1997) Instability of highly expanded CAG repeats in mice transgenic for the Huntington’s disease mutation. Nat Genet 15(2): 197–200.
    Mangiarini L, Sathasivam K, Seller M, Cozens B, Harper A, Hetherington C, Lawton M, Trottier Y, Lehrach H, Davies SW, Bates GP (1996) Exon 1 of the HD gene with an expanded CAG repeat is sufficient to cause a progressive neurological phenotype in transgenic mice. Cell 87(3): 493–506.
    Martin JB, Gusella JF (1986) Huntington’s disease. Pathogenesis and manage¬ment. N Engl J Med 315(20): 1267–1276.
    McCoy MK, Tansey MG (2008) TNF signaling inhibition in the CNS: implications for normal brain function and neurodegenerative disease. J Neuroinflammation 5: 45
    Miller BR, Walker AG, Shah AS, Barton SJ, Rebec GV (2008) Dysregulated information processing by medium spiny neurons in striatum of freely behaving mouse models of Huntington’s disease. J Neurophysiol 100(4): 2205–2216.
    Nakao N, Brundin P (1998) Neurodegeneration and glutamate induced oxidative stress. Prog Brain Res 116: 245–263.
    Portera-Cailliau C, Hedreen JC, Price DL, Koliatsos VE (1995) Evidence for apoptotic cell death in Huntington disease and excitotoxic animal models. J Neurosci 5(Pt2): 3775–3787.
    Rao RV, Bredesen DE (2004) Misfolded proteins, endoplasmic reticulum stress and neurodegeneration. Curr Opin Cell Biol 16(6): 653–662.
    Rattray I, Smith E, Gale R, Matsumoto K, Bates GP, Modo M (2013) Correlations of behavioral deficits with brain pathology assessed through longitudinal MRI and histopathology in the R6/2 mouse model of HD. PLoS One 8(4): e60012
    Raymond LA (2003) Excitotoxicity in Huntington disease. Clin Neurosci Res 3(3): 121–128.
    Reiner A, Albin RL, Anderson KD, D'Amato CJ, Penney JB, Young AB (1988) Differential loss of striatal projection neurons in Huntington disease. Proc Natl Acad Sci USA 85(15): 5733–5737.
    Rigamonti D, Bauer JH, De-Fraja C, Conti L, Sipione S, Sciorati C, Clementi E, Hackam A, Hayden MR, Li Y, Cooper JK, Ross CA, Govoni S, Vincenz C, Cattaneo E (2000) Wildtype huntingtin protects from apoptosis upstream of caspase-3. J. Neurosci 20(10): 3705–3713.
    Rigamonti D, Sipione S, Goffredo D, Zuccato C, Fossale E, Cattaneo E (2001) Huntingtin’s neuroprotective activity occurs via inhibition of procaspase-9 processing. J Biol Chem 276(18): 14545–14548.
    Rissanen TT, Korpisalo P, Markkanen JE, Liimatainen T, Ordén MR, Kholová I, de Goede A, Heikura T, Gröhn OH, Ylä-Herttuala S (2005) Blood flow remodels growing vasculature during vascular endothelial growth factor gene therapy and determines between capillary arterialization and sprouting angiogenesis. Circulation 112(25): 3937–3946.
    Rosas HD, Koroshetz WJ, Chen YI, Skeuse C, Vangel M, Cudkowicz ME, Caplan K, Marek K, Seidman LJ, Makris N, Jenkins BG, Goldstein JM (2003) Evidence for more widespread cerebral pathology in early HD: an MRI-based morphometric analysis. Neurology 60(10): 1615–1620.
    Ruiz de Almodovar C, Lambrechts D, Mazzone M, Carmeliet P (2009) Role and therapeutic potential of VEGF in the nervous system. Physiol Rev 89(2): 607-648.
    Satoh T, Takahashi T, Iwasaki K, Tago H, Seki T, Yaegashi N, Tobita M, Arai H (2009) Traditional Chinese medicine on four patients with Huntington’s disease. Mov Disord 24(3): 453–455.
    Schilling G, Becher MW, Sharp AH, Jinnah HA, Duan K, Kotzuk JA, Slunt HH, Ratovitski T, Cooper JK, Jenkins NA, Copeland NG, Price DL, Ross CA, Borchelt DR (1999) Intranuclear inclusions and neuritic aggregates in transgenic mice expressing a mutant N-terminal fragment of huntingtin. Hum Mol Genet 8(3): 397–407.
    Silvestroni A, Faull RL, Strand AD, Möller T (2009) Distinct neuroinflamma¬tory profile in post-mortem human Huntington’s disease. Neuroreport 20(12): 1098–1103.
    Stack EC, Kubilus JK, Smith K, Cormier K, Del Signore SJ, Guelin E, Ryu H, Hersch SM, Ferrante RJ (2005) Chronology of behavioral symptoms and neuropathological sequela in R6/2 Huntington's disease transgenic mice. J Comp Neurol 490(4): 354–370.
    Tatton WG, Olanow CW (1999) Apoptosis in neurodegenerative diseases: the role of mitochondria. Biochim Biophys Acta 1410(2): 195–213.
    The Huntington’s Disease Collaboration Group (1993) A novel gene containing a trinucleotide repeat that is expanded and unstable on Huntington’s disease chromosomes. Cell 72(6): 971–983.
    Vonsattel JP, Myers RH, Stevens TJ, Ferrante RJ, Bird ED, Richardson EP Jr (1985) Neuropathological classification of Huntington’s disease. J Neuropathol Exp Neurol 44(6): 559–577.
    Wang SE, Lin CL, Hsu CH, Sheu SJ, Chien CT, Wu CH (2015) Treatment of a herbal formula B401 enhances neuroprotection and angiogenesis in the R6/2 mouse model of Huntington's disease. Drug Des Devel Ther 9: 887–900.
    Wyttenbach A, Sauvageot O, Carmichael J, Diaz-Latoud C, Arrigo AP, Rubinsztein DC (2002) Heat shock protein 27 prevents cellular polyglutamine toxicity and suppresses the increase of reactive oxygen species caused by huntingtin. Hum Mol Genet 11(9): 1137–1151.
    Zhang Z, Yan J, Chang Y, ShiDu Yan S, Shi H (2011) Hypoxia inducible factor-1 as a target for neurodegenerative diseases. Curr Med Chem 18(28): 4335-4343.
    Zoghbi HY, Orr HT (2000) Glutamine repeats and neurodegeneration. Annu Rev Neurosci 23: 217–247.
    Zuccato C, Valenza M, Cattaneo E (2010) Molecular mechanisms and potential therapeutical targets in Huntington’s disease. Physiol Rev 90(3): 905–981.

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