臺灣博碩士論文加值系統

先前研究證實，代謝性症候群患者血液中的極低密度脂蛋白 (very low density lipoprotein , 簡稱 VLDL) 具較高之陰電性，並造成血管內皮細胞較大之
損傷，然而其對中樞神經的影響尚未知。我們假設長期血液中過多的陰電性 VLDL 可能會傷害中樞神經細胞，進而造成認知功能下降。 本研究以動物認知行為模式，來看不同陰電性 VLDL 對於神經功能之影 響，並分析認知功能相關腦區來探討其相關機轉。從正常受試者 (normal) 及代 謝性症候群 (metabolic syndrome, met) 患者的血液中，我們分別分離出具有較 低 (nVLDL) 與較高 (metVLDL) 陰電性的 VLDL ，以 15 μg/g BW 的劑量連 續 6 週注射於 10 個月大小鼠之尾靜脈，而其中 1 組 metVLDL 動物在最後兩 週 (6-8 週) 餵食何首烏萃取物 (EH201)。 在活動度測試上，連續注射 6 週 nVLDL 或 metVLDL 的小鼠分別在中央 及周邊區域活動度表現增加。Y 迷宮行為測試結果顯示，小鼠注射 3 週的 metVLDL 即明顯的減少對於新奇區域探索之比例，顯示有認知功能退化之情 形。連續注射 6 週的 nVLDL 或 metVLDL 後，兩組小鼠均表現明顯認知功能 退化之情形，而餵食 EH201 則可改善 metVLDL 所導致的認知退化。相關蛋白 分子研究顯示 metVLDL 組其血液 TGF-β1、皮質區 BDNF 及 Aβ42 皆顯著 增加，而餵食 EH201 可改善 metVLDL 所導致的相關蛋白質表現。另外，膠 細胞染色分析顯示，長期注射 nVLDL 及 metVLDL 皆使小鼠之前中額葉皮 質 (medial prefrontal cortex, mPFC) 與海馬迴腦區之小膠細胞表現量增加，其 中又以 metVLDL 增加最為顯著。 MetVLDL 也使海馬迴 CA1 及 CA3 腦區 的星狀膠細胞表現量明顯增加。而餵食 EH201 能減少 metVLDL 所引起的 膠細胞增生。綜合上述，不同陰電性 VLDL 誘發不同程度中樞神經發炎並影 響中年小鼠認知功能，而餵食 EH201 能改善 metVLDL 所引起的神經副作用。

Previous study indicated that very low density lipoprotein (VLDL) with negatively charged propriety in metabolic syndrome subjects, proven as a vascular risk factor. However, its effect to neuronal function is still unknown. We hypothesize that negative charged VLDL might damage the neuronal cells and impaired the cognitive function. The animal behavior model and related brain analysis were performed to investigate its effects and mechanism. First, we isolated the VLDL from the normal (nVLDL) and metabolic syndrome (metVLDL) subjects. Second, to verify the long-term effects of different negative charged VLDL in central nervous system. 10-month-old mice were tail vein injected with nVLDL (15 μg/g BW) or metVLDL (15 μg/g BW) for 6 weeks. Moreover, the compounds extracted from Radix Polygoni multiflori (EH201) was fed to the mice injected with metVLDL in the last two weeks (6-8 week). In locomotor activity test, long term (6 weeks) injected of nVLDL or metVLDL increased of travel distance in central or peripheral zone respectively. In Y-Maze test, metVLDL group showed early cognitive dysfunction due to a lower exploration rate of novel arm at the 3rd week. At the 6th week, both nVLDL and metVLDL groups exhibited a significant declined in novel arm exploration. Treatment with EH201 protect the metVLDL-induced cognitive impairment. In proteomic molecular analysis, long term metVLDL induced significant elevation of TGF-β1 in plasma, BDNF and Aβ42 expression in cortex. EH201 treatment improved these molecular expression induced by metVLDL. We further analyzed the degrees of glia cell expression in cognition related brain region. In both nVLDL and metVLDL groups, microglial expression significantly increased in medial prefrontal cortex (mPFC) and hippocampus. Moreover, significant increasing of astroglia in hippocampus CA3 and CA1 was found in metVLDL group. Combination of EH201 with suppressed the overexpression of glia cell in brain. Taken together, our findings suggest that long term exposure of different negative charged VLDL induced different degree of neuronal inflammation and contribute to cognitive dysfunction, combination of EH201 treatment may attenuate the impact of metVLDL.

目 錄
頁次
縮寫對照表 I 中文摘要 II Abstract III
第一章 緒論
研究背景 1
第一節 認知功能退化與失智症 2
第二節 血脂異常或 MetS 為認知功能退化與失智症的危險因子 3
第三節 血脂異常或 MetS 造成認知功能退化之病理生理機轉 4
一、長期血脂異常造成不正常的蛋白質堆積4
二、長期血脂異常與神經元軸突病變的關係 4
三、長期血脂異常造成週邊及中樞系統發炎反應 5
第四節 血脂異常與極低密度脂蛋白6
一、VLDL 在血液中的角色及運輸代謝6
二、VLDL 與認知功能退化的關係7
三、陰電性 VLDL 8
第五節 神經膠細胞與中樞神經發炎反應 8
一、小膠細胞 (microglia)8
二、星狀膠細胞 (astrocyte) 10
第六節 認知功能相關腦區 13
第七節 紅血球生成素誘發劑 EH-201 14
第二章 研究目的與假說 16
第三章 材料與方法
第一節 實驗方法 17
第二節 實驗材料 26
第四章 實驗結果
第一節 長期注射不同陰電性 VLDL 小鼠之血脂分布與體重變化 31
第二節 長期暴露於不同陰電性 VLDL 導致小鼠活動度增加 32
第三節 長期暴露於不同陰電性 VLDL 導致小鼠認知能力下降 34
第四節 不同陰電性 VLDL 對於血液蛋白質表現之影響 36
第五節 不同陰電性 VLDL 對於認知腦區蛋白質表現之影響 37
第六節 乙型類澱粉蛋白-42 (amyloid β-42, Aβ-42) 的表現量 38 第七節 不同陰電性 VLDL 導致腦中不同程度之 microglia 活化
第八節 不同陰電性 VLDL 導致腦中不同程度之 astrocyte 活化 41 第五章 討論
第一節 VLDL 血脂異常造成小鼠活動度及認知行為改變 43
第二節 長期暴露於 met VLDL 造成小鼠血液之 TGF-β1 增加並且 未使認 知相關腦區的發炎相關因子產生變化 45
第三節 長期暴露於 metVLDL 使小鼠前額葉 BDNF 表現上升46
第四節 不同陰電性 VLDL 造成小鼠神經膠細胞表現不同程度之活47
第五節 metVLDL 組小鼠的皮質區 Aβ-42 表現量增加49
第六節 藥物 EH201 改善 metVLDL 造成的血脂異常、腦組織膠細胞活化及認知障礙 50
第六章 結論 52
附表 53
附圖 61
參考文獻 78

Reference
Adiels M, Olofsson SO, Taskinen MR, & Boren J (2008). Overproduction of very low-density lipoproteins is the hallmark of the dyslipidemia in the metabolic syndrome. Arterioscler Thromb Vasc Biol 28: 1225-1236.
Akiyama H, Barger S, Barnum S, Bradt B, Bauer J, Cole GM, et al. (2000). Inflammation and Alzheimer''s disease. Neurobiol Aging 21: 383-421.
Aloisi F (2001). Immune function of microglia. Glia 36: 165-179.
Angelucci F, Spalletta G, di Iulio F, Ciaramella A, Salani F, Colantoni L, et al. (2010). Alzheimer''s disease (AD) and Mild Cognitive Impairment (MCI) patients are characterized by increased BDNF serum levels. Curr Alzheimer Res 7: 15-20.
Argaw AT, Asp L, Zhang J, Navrazhina K, Pham T, Mariani JN, et al. (2012). Astrocyte-derived VEGF-A drives blood-brain barrier disruption in CNS inflammatory disease. J Clin Invest 122: 2454-2468.
Brambilla R, Bracchi-Ricard V, Hu WH, Frydel B, Bramwell A, Karmally S, et al. (2005). Inhibition of astroglial nuclear factor kappaB reduces inflammation and improves functional recovery after spinal cord injury. J Exp Med 202: 145-156.
Brambilla R, Persaud T, Hu X, Karmally S, Shestopalov VI, Dvoriantchikova G, et al. (2009). Transgenic inhibition of astroglial NF-kappa B improves functional outcome in experimental autoimmune encephalomyelitis by suppressing chronic central nervous system inflammation. J Immunol 182: 2628-2640.
Buchman AS, Yu L, Boyle PA, Schneider JA, De Jager PL, & Bennett DA (2016). Higher brain BDNF gene expression is associated with slower cognitive decline in older adults. Neurology 86: 735-741.
Bush TG, Puvanachandra N, Horner CH, Polito A, Ostenfeld T, Svendsen CN, et al. (1999). Leukocyte infiltration, neuronal degeneration, and neurite outgrowth after ablation of scar-forming, reactive astrocytes in adult transgenic mice. Neuron 23: 297-308.
Chaldakov GN, Fiore M, Stankulov IS, Hristova M, Antonelli A, Manni L, et al. (2001). NGF, BDNF, leptin, and mast cells in human coronary atherosclerosis and metabolic syndrome. Arch Physiol Biochem 109: 357-360.
Chen CH, Lu J, Chen SH, Huang RY, Yilmaz HR, Dong J, et al. (2012). Effects of electronegative VLDL on endothelium damage in metabolic syndrome. Diabetes Care 35: 648-653.
Chen JH, Ke KF, Lu JH, Qiu YH, & Peng YP (2015). Protection of TGF-beta1 against neuroinflammation and neurodegeneration in Abeta1-42-induced Alzheimer''s disease model rats. PLoS One 10: e0116549.
Chen SJ, Wang YL, Lo WT, Wu CC, Hsieh CW, Huang CF, et al. (2010).
79

Erythropoietin enhances endogenous haem oxygenase-1 and represses immune responses to ameliorate experimental autoimmune encephalomyelitis. Clin Exp Immunol 162: 210-223.
Chen TB, Yiao SY, Sun Y, Lee HJ, Yang SC, Chiu MJ, et al. (2017). Comorbidity and dementia: A nationwide survey in Taiwan. PLoS One 12: e0175475.
Chen X, Gawryluk JW, Wagener JF, Ghribi O, & Geiger JD (2008). Caffeine blocks disruption of blood brain barrier in a rabbit model of Alzheimer''s disease. J Neuroinflammation 5: 12.
Chiu MJ, Chen TF, Yip PK, Hua MS, & Tang LY (2006). Behavioral and psychologic symptoms in different types of dementia. J Formos Med Assoc 105: 556-562.
Colombo E, & Farina C (2016). Astrocytes: Key Regulators of Neuroinflammation. Trends Immunol 37: 608-620.
Corder EH, Saunders AM, Strittmatter WJ, Schmechel DE, Gaskell PC, Small GW, et al. (1993). Gene dose of apolipoprotein E type 4 allele and the risk of Alzheimer''s disease in late onset families. Science 261: 921-923.
Culley DJ, Snayd M, Baxter MG, Xie Z, Lee IH, Rudolph J, et al. (2014). Systemic inflammation impairs attention and cognitive flexibility but not associative learning in aged rats: possible implications for delirium. Front Aging Neurosci 6: 107.
Dallinga-Thie GM, Franssen R, Mooij HL, Visser ME, Hassing HC, Peelman F, et al. (2010). The metabolism of triglyceride-rich lipoproteins revisited: new players, new insight. Atherosclerosis 211: 1-8.
Dik MG, Jonker C, Comijs HC, Deeg DJ, Kok A, Yaffe K, et al. (2007). Contribution of metabolic syndrome components to cognition in older individuals. Diabetes Care 30: 2655-2660.
Diniz LP, Tortelli V, Matias I, Morgado J, Bergamo Araujo AP, Melo HM, et al. (2017). Astrocyte Transforming Growth Factor Beta 1 Protects Synapses against Abeta Oligomers in Alzheimer''s Disease Model. J Neurosci 37: 6797-6809.
Ehrenreich H, Hinze-Selch D, Stawicki S, Aust C, Knolle-Veentjer S, Wilms S, et al. (2007). Improvement of cognitive functions in chronic schizophrenic patients by recombinant human erythropoietin. Mol Psychiatry 12: 206-220.
Ehrenreich H, Weissenborn K, Prange H, Schneider D, Weimar C, Wartenberg K, et al. (2009). Recombinant human erythropoietin in the treatment of acute ischemic stroke. Stroke 40: e647-656.
Expert Panel on Detection E, & Treatment of High Blood Cholesterol in A (2001). Executive Summary of The Third Report of The National Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation, And Treatment of High
80

Blood Cholesterol In Adults (Adult Treatment Panel III). JAMA 285: 2486-2497.
Faria MC, Goncalves GS, Rocha NP, Moraes EN, Bicalho MA, Gualberto Cintra MT, et al. (2014). Increased plasma levels of BDNF and inflammatory markers in Alzheimer''s disease. J Psychiatr Res 53: 166-172.
Farina C, Aloisi F, & Meinl E (2007). Astrocytes are active players in cerebral innate immunity. Trends Immunol 28: 138-145.
Gehrmann J, Matsumoto Y, & Kreutzberg GW (1995). Microglia: intrinsic immuneffector cell of the brain. Brain Res Brain Res Rev 20: 269-287.
Gibbons GF, Wiggins D, Brown AM, & Hebbachi AM (2004). Synthesis and function of hepatic very-low-density lipoprotein. Biochem Soc Trans 32: 59-64.
Goldwasser E (1975). Erythropoietin and the differentiation of red blood cells. Fed Proc 34: 2285-2292.
Gordon GR, Mulligan SJ, & MacVicar BA (2007). Astrocyte control of the cerebrovasculature. Glia 55: 1214-1221.
Gruter T, Wiescholleck V, Dubovyk V, Aliane V, & Manahan-Vaughan D (2015). Altered neuronal excitability underlies impaired hippocampal function in an animal model of psychosis. Front Behav Neurosci 9: 117.
Guillemot-Legris O, Masquelier J, Everard A, Cani PD, Alhouayek M, & Muccioli GG (2016). High-fat diet feeding differentially affects the development of inflammation in the central nervous system. J Neuroinflammation 13: 206.
Hamley IW (2012). The amyloid beta peptide: a chemist''s perspective. Role in Alzheimer''s and fibrillization. Chem Rev 112: 5147-5192.
Heneka MT, Carson MJ, El Khoury J, Landreth GE, Brosseron F, Feinstein DL, et al. (2015). Neuroinflammation in Alzheimer''s disease. Lancet Neurol 14: 388-405.
Heneka MT, Golenbock DT, & Latz E (2015). Innate immunity in Alzheimer''s disease. Nat Immunol 16: 229-236.
Heneka MT, Kummer MP, Stutz A, Delekate A, Schwartz S, Vieira-Saecker A, et al. (2013). NLRP3 is activated in Alzheimer''s disease and contributes to pathology in APP/PS1 mice. Nature 493: 674-678.
Holmes C, Cunningham C, Zotova E, Woolford J, Dean C, Kerr S, et al. (2009). Systemic inflammation and disease progression in Alzheimer disease. Neurology 73: 768-774.
Hoogland IC, Houbolt C, van Westerloo DJ, van Gool WA, & van de Beek D (2015). Systemic inflammation and microglial activation: systematic review of animal experiments. J Neuroinflammation 12: 114.
81


Horng LY, Hsu PL, Chen LW, Tseng WZ, Hsu KT, Wu CL, et al. (2015). Activating mitochondrial function and haemoglobin expression with EH-201, an inducer of erythropoietin in neuronal cells, reverses memory impairment. Br J Pharmacol 172: 4741-4756.
Koistinaho M, Lin S, Wu X, Esterman M, Koger D, Hanson J, et al. (2004). Apolipoprotein E promotes astrocyte colocalization and degradation of deposited amyloid-beta peptides. Nat Med 10: 719-726.
Lee HC, Chen CC, Tsai WC, Lin HT, Shiao YL, Sheu SH, et al. (2017). Very-Low-Density Lipoprotein of Metabolic Syndrome Modulates Gap Junctions and Slows Cardiac Conduction. Sci Rep 7: 12050.
Lee HC, Lin HT, Ke LY, Wei C, Hsiao YL, Chu CS, et al. (2016). VLDL from Metabolic Syndrome Individuals Enhanced Lipid Accumulation in Atria with Association of Susceptibility to Atrial Fibrillation. Int J Mol Sci 17.
Liu QL, Xiao JH, Ma R, Ban Y, & Wang JL (2007). Effect of 2,3,5,4''-tetrahydroxystilbene-2-O-beta-D-glucoside on lipoprotein oxidation and proliferation of coronary arterial smooth cells. J Asian Nat Prod Res 9: 689-697.
Lund A, Lundby C, & Olsen NV (2014). High-dose erythropoietin for tissue protection. Eur J Clin Invest 44: 1230-1238.
Maldonado-Ruiz R, Fuentes-Mera L, & Camacho A (2017). Central Modulation of Neuroinflammation by Neuropeptides and Energy-Sensing Hormones during Obesity. Biomed Res Int 2017: 7949582.
Mauro C, De Rosa V, Marelli-Berg F, & Solito E (2014). Metabolic syndrome and the immunological affair with the blood-brain barrier. Front Immunol 5: 677.
McGraw J, Hiebert GW, & Steeves JD (2001). Modulating astrogliosis after neurotrauma. J Neurosci Res 63: 109-115.
Meyer A, Wang W, Qu J, Croft L, Degen JL, Coller BS, et al. (2012). Platelet TGF-beta1 contributions to plasma TGF-beta1, cardiac fibrosis, and systolic dysfunction in a mouse model of pressure overload. Blood 119: 1064-1074.
Min KJ, Yang MS, Kim SU, Jou I, & Joe EH (2006). Astrocytes induce hemeoxygenase-1 expression in microglia: a feasible mechanism for preventing excessive brain inflammation. J Neurosci 26: 1880-1887.
Norden DM, Trojanowski PJ, Walker FR, & Godbout JP (2016). Insensitivity of astrocytes to interleukin 10 signaling following peripheral immune challenge results in prolonged microglial activation in the aged brain. Neurobiol Aging 44: 22-41.
Okada S, Nakamura M, Katoh H, Miyao T, Shimazaki T, Ishii K, et al. (2006). Conditional ablation of Stat3 or Socs3 discloses a dual role for reactive astrocytes
82

after spinal cord injury. Nat Med 12: 829-834.
Peng S, Wuu J, Mufson EJ, & Fahnestock M (2005). Precursor form of brain-derived neurotrophic factor and mature brain-derived neurotrophic factor are decreased in the pre-clinical stages of Alzheimer''s disease. J Neurochem 93: 1412-1421.
Perry VH, Newman TA, & Cunningham C (2003). The impact of systemic infection on the progression of neurodegenerative disease. Nat Rev Neurosci 4: 103-112.
Petersen RC (2016). Mild Cognitive Impairment. Continuum (Minneap Minn) 22: 404-418.
Petersen RC, Roberts RO, Knopman DS, Boeve BF, Geda YE, Ivnik RJ, et al. (2009). Mild cognitive impairment: ten years later. Arch Neurol 66: 1447-1455.
Pfrieger FW (2003). Cholesterol homeostasis and function in neurons of the central nervous system. Cell Mol Life Sci 60: 1158-1171.
Power MC, Rawlings A, Sharrett AR, Bandeen-Roche K, Coresh J, Ballantyne CM, et al. (2018). Association of midlife lipids with 20-year cognitive change: A cohort study. Alzheimers Dement 14: 167-177.
Reaven GM, Chen YD, Jeppesen J, Maheux P, & Krauss RM (1993). Insulin resistance and hyperinsulinemia in individuals with small, dense low density lipoprotein particles. J Clin Invest 92: 141-146.
Refolo LM, Pappolla MA, LaFrancois J, Malester B, Schmidt SD, Thomas-Bryant T, et al. (2001). A cholesterol-lowering drug reduces beta-amyloid pathology in a transgenic mouse model of Alzheimer''s disease. Neurobiol Dis 8: 890-899.
Reitz C, Tang MX, Luchsinger J, & Mayeux R (2004). Relation of plasma lipids to Alzheimer disease and vascular dementia. Arch Neurol 61: 705-714.
Selkoe DJ (2011). Alzheimer''s disease. Cold Spring Harb Perspect Biol 3.
Sofroniew MV (2009). Molecular dissection of reactive astrogliosis and glial scar formation. Trends Neurosci 32: 638-647.
Sofroniew MV (2015). Astrocyte barriers to neurotoxic inflammation. Nat Rev Neurosci 16: 249-263.
Sparks JD, Sparks CE, & Adeli K (2012). Selective hepatic insulin resistance, VLDL overproduction, and hypertriglyceridemia. Arterioscler Thromb Vasc Biol 32: 2104-2112.
Strickland DK, Gonias SL, & Argraves WS (2002). Diverse roles for the LDL receptor family. Trends Endocrinol Metab 13: 66-74.
83


Sun FL, Zhang L, Zhang RY, & Li L (2011). Tetrahydroxystilbene glucoside protects human neuroblastoma SH-SY5Y cells against MPP+-induced cytotoxicity. Eur J Pharmacol 660: 283-290.
Taylor VH, & MacQueen GM (2007). Cognitive dysfunction associated with metabolic syndrome. Obes Rev 8: 409-418.
Trousson A, Bernard S, Petit PX, Liere P, Pianos A, El Hadri K, et al. (2009). 25-hydroxycholesterol provokes oligodendrocyte cell line apoptosis and stimulates the secreted phospholipase A2 type IIA via LXR beta and PXR. J Neurochem 109: 945-958.
Vanhanen M, Koivisto K, Moilanen L, Helkala EL, Hanninen T, Soininen H, et al. (2006). Association of metabolic syndrome with Alzheimer disease: a population-based study. Neurology 67: 843-847.
Vinik AI (2005). The metabolic basis of atherogenic dyslipidemia. Clin Cornerstone 7: 27-35.
Wang H, & Eckel RH (2014). What are lipoproteins doing in the brain? Trends Endocrinol Metab 25: 8-14.
Wang R, Tang Y, Feng B, Ye C, Fang L, Zhang L, et al. (2007). Changes in hippocampal synapses and learning-memory abilities in age-increasing rats and effects of tetrahydroxystilbene glucoside in aged rats. Neuroscience 149: 739-746.
Wang WY, Tan MS, Yu JT, & Tan L (2015). Role of pro-inflammatory cytokines released from microglia in Alzheimer''s disease. Ann Transl Med 3: 136.
Wimo A, Guerchet M, Ali GC, Wu YT, Prina AM, Winblad B, et al. (2017). The worldwide costs of dementia 2015 and comparisons with 2010. Alzheimers Dement 13: 1-7.
Yadav H, Quijano C, Kamaraju AK, Gavrilova O, Malek R, Chen W, et al. (2011). Protection from obesity and diabetes by blockade of TGF-beta/Smad3 signaling. Cell Metab 14: 67-79.
Yang HT, Sheen YJ, Kao CD, Chang CA, Hu YC, & Lin JL (2013). Association between the characteristics of metabolic syndrome and Alzheimer''s disease. Metab Brain Dis 28: 597-604.
Yang XP, Liu TY, Qin XY, & Yu LC (2014). Potential protection of 2,3,5,4''-tetrahydroxystilbene-2-O-beta-D-glucoside against staurosporine-induced toxicity on cultured rat hippocampus neurons. Neurosci Lett 576: 79-83.
Zhang L, Xing Y, Ye CF, Ai HX, Wei HF, & Li L (2006). Learning-memory deficit with aging in APP transgenic mice of Alzheimer''s disease and intervention by using tetrahydroxystilbene glucoside. Behav Brain Res 173: 246-254.