微小膠質細胞在神經退化性疾病中所扮演的角色與機制，在目前許多研究中發現與氧化壓力和發炎反應有關。和厚朴酚 (honokiol)
[2-(4-hydroxy-3-prop-2-enylphenyl)-4-prop-2-enylphenol] 又稱異厚朴酚，為木蘭科植物厚朴 (Magnolia officinalis) 的主要活性成份之一，結構為具有雙丙烯基的多酚化合物 (C18H18O2,MW=266.33)，且具有抗氧化和抗發炎的作用。ST-24 化合物為和厚朴酚的衍生物，用和厚朴酚分子結構為主體修飾而成的化合物，本研究針對和厚朴酚與 ST-24 化合物在細菌性脂多醣刺激微小膠質細胞活化反應中的抗氧化與抗發炎能力作探討。實驗以 3-(4,5-dimethyl-2 thiazoyl)-2,5-diphenyl-tetrazolium bromide (MTT) 分析細胞粒線體的還原能力，藉此判斷細胞的存活率。吉爾任試劑 (Griess reagent) 用來測量藥物對活化後的微小膠質細胞產生活性氮自由基 (reactive nitrogen species, RNS) Nitric oxide (NO‧) 的影響。MTT 與吉爾任試劑分析結果顯示，和厚朴酚與 ST-24 在低濃度不會造成細胞死亡且具有減緩細菌性脂多醣刺激微小膠質細胞過度活化所產生的活性氮自由基。本論文的目標除了比較和厚朴酚與 ST-24 化合物的抗氧化及抗發炎能力外，也探討減緩微小膠質過度活化可能的分子機制。透過觀察細胞型態和西方墨點法，發現給予和厚朴酚與 ST-24 藥物可以減少微小膠質細胞因為 LPS 刺激所產生的偽足、iNOS 及 COX-2 蛋白質表現。而給予和厚朴酚與 ST-24 藥物，則會回復 iNOS 上游分子 PI3K/Akt 蛋白質表現。實驗結果顯示和厚朴酚與 ST-24 可能透過 PI3k/Akt 這個分子機制去調控微小膠質細胞過度活化，但仍需進一步的實驗確認。

The role and mechanisms of microglia cell in neurodegenerative diseases might be related to oxidative stress and inflammatory responses. Honokiol is one of the main constituents from Magnolia officinalis. The structure of honokiol possesses a biphenolic structure with two allyl groups (C18H18O2, MW=266.33) that has anti-oxidative and anti-inflammatory effects. In addition, the anti-oxidative and anti-inflammatory properties of honokiol and ST-24 compounds were explored as well as their inhibitory effect on over-activated microglial cells in this study. In the present study, the 3-(4,5-dimethyl-2 thiazoyl)-2,5-diphenyl-tetrazolium bromide (MTT) assay was used to assess mitochondrial activity, reflecting cell survival. The Griess reagent was used to detect the effects of (reactive nitrogen species, RNS) Nitric oxide (NO‧) in activated microglial cells with honokiol and ST-24.The results show that honokiol and ST-24 did not induce cell death in lower concentrations. Both biphenolic compounds attenuated nitric oxide free radical productions in LPS-stimulated over-activated microglial cells. Besides compared honokiol and ST-24 capability of anti-oxidation and anti-inflammation, this study also explored the biological molecular mechanisms of attenuating over-activated microglial cells. By observation of cell morphology and Western blot, we found that honokiol and ST-24 can reduce pseudopodia, and increases in iNOS and COX-2 protein expression induced by LPS in microglia .Honokiol and ST-24 could restore the upstream molecular PI3K/Akt protein expression. The results suggested that honokiol and ST-24 regulated excessive activation of microglia through PI3K/Akt pathway.

謝誌 I
中文摘要 II
ABSTRACT IV
目錄 VI
表次 VIII
圖次 IX
縮寫對照表 XI
中英文名詞對照表 XII
第一章 緒論及文獻回顧 1
第一節、前言 1
第二節、自由基的簡介及發炎反應 4
第三節、微小膠質細胞的簡介 10
第四節、雙酚類化合物之簡介 14
第五節、實驗目的與假說 17
第二章 實驗材料與實驗方法 18
第一節、實驗材料 18
第二節、實驗方法 20
第三節、實驗數據分析 24
第三章 實驗設計 25
目標(一) 建立內毒素 (脂多醣) 對微小膠質細胞活化之模式 25
目標(二) 評估內毒素 (脂多醣) 與藥物所引起微小膠質細胞過度活化及細胞凋亡 26
目標(三) 評估雙酚類化合物對內毒素引起微小膠質細胞型態改變之影響 27
目標(四) 評估觀察同時給予內毒素 (脂多醣) 和不同藥物後對內毒素 (脂多醣) 所引起微小膠質細胞過度活化之作用 27
目標(五) 確認HONOKIOL及ST-24號化合物是否是透過 PI3K/AKT 路徑改善內毒素 (脂多醣) 所引起微小膠質細胞過度活化之分子機制 28
第四章 實驗結果 29
一、內毒素 (脂多醣) 對微小膠質細胞存活之影響 29
二、雙酚類化合物對內毒素引起微小膠質細胞活化及細胞死亡之影響 32
三、雙酚類化合物對內毒素引起微小膠質細胞型態改變之影響 35
四、雙酚類化合物對內毒素引起微小膠質細胞發炎反應之影響 45
五、雙酚類化合物抑制內毒素引起微小膠質細胞活化反應之分子機制 48
第五章 討論 51
第六章 結論 56
第七章 參考文獻 57
1.期刊論文 57
2.書籍論文 67
3.網路資源 67
附錄 68

1.期刊論文
Aarum, J., et al. (2003). "Migration and differentiation of neural precursor cells can be directed by microglia." Proc Natl Acad Sci U S A 100(26): 15983-15988.
abd-el-Basset, E. and S. Fedoroff (1995). "Effect of bacterial wall lipopolysaccharide (LPS) on morphology, motility, and cytoskeletal organization of microglia in cultures." J Neurosci Res 41(2): 222-237.
Akira, S., et al. (2006). "Pathogen recognition and innate immunity." Cell 124(4): 783-801.
Alexopoulou, L., et al. (2001). "Recognition of double-stranded RNA and activation of NF-kappaB by Toll-like receptor 3." Nature 413(6857): 732-738.
Bai, X., et al. (2003). "Honokiol, a small molecular weight natural product, inhibits angiogenesis in vitro and tumor growth in vivo." J Biol Chem 278(37): 35501-35507.
Ballard, R. A., et al. (2006). "Inhaled nitric oxide in preterm infants undergoing mechanical ventilation." N Engl J Med 355(4): 343-353.
Barron, E. S. (1954). "The role of free radicals and oxygen in reactions produced by ionizing radiations." Radiat Res 1(1): 109-124.
Battle, T. E., et al. (2005). "The natural product honokiol induces caspase-dependent apoptosis in B-cell chronic lymphocytic leukemia (B-CLL) cells." Blood 106(2): 690-697.
Block, M. L., et al. (2007). "Microglia-mediated neurotoxicity: uncovering the molecular mechanisms." Nat Rev Neurosci 8(1): 57-69.
Bredt, D. S., et al. (1990). "Localization of nitric oxide synthase indicating a neural role for nitric oxide." Nature 347(6295): 768-770.
Cantley, L. C. (2002). "The phosphoinositide 3-kinase pathway." Science 296(5573): 1655-1657.
Chen, C. M., et al. (2007). "Honokiol, a neuroprotectant against mouse cerebral ischaemia, mediated by preserving Na+, K+-ATPase activity and mitochondrial functions." Basic Clin Pharmacol Toxicol 101(2): 108-116.
Cho, H. J., et al. (1992). "Calmodulin is a subunit of nitric oxide synthase from macrophages." J Exp Med 176(2): 599-604.
Choi, J. H., et al. (2002). "Costunolide triggers apoptosis in human leukemia U937 cells by depleting intracellular thiols." Jpn J Cancer Res 93(12): 1327-1333.
Colton, C. and D. M. Wilcock (2010). "Assessing activation states in microglia." CNS Neurol Disord Drug Targets 9(2): 174-191.
Ebert, S., et al. (2005). "Dose-dependent activation of microglial cells by Toll-like receptor agonists alone and in combination." J Neuroimmunol 159(1-2): 87-96.
el-Remessy, A. B., et al. (2005). "Oxidative stress inactivates VEGF survival signaling in retinal endothelial cells via PI 3-kinase tyrosine nitration." J Cell Sci 118(Pt 1): 243-252.
Facchinetti, F., et al. (1999). "Lack of involvement of neuronal nitric oxide synthase in the pathogenesis of a transgenic mouse model of familial amyotrophic lateral sclerosis." Neuroscience 90(4): 1483-1492.
Fernandez-Lizarbe, S., et al. (2009). "Critical role of TLR4 response in the activation of microglia induced by ethanol." J Immunol 183(7): 4733-4744.
Feron, O., et al. (1996). "Endothelial nitric oxide synthase targeting to caveolae. Specific interactions with caveolin isoforms in cardiac myocytes and endothelial cells." J Biol Chem 271(37): 22810-22814.
Florence, T. M. (1995). "The role of free radicals in disease." Aust N Z J Ophthalmol 23(1): 3-7.
Fujita, M., et al. (1973). "[Studies on the components of Magnolia obovata Thunb. 3. Occurrence of magnolol and honokiol in M. obovata and other allied plants]." Yakugaku Zasshi 93(4): 429-434.
Goodnough, J., et al. (1980). "The effect of dimethyl sulfoxide on gray matter injury in experimental spinal cord trauma." Surg Neurol 13(4): 273-276.
Halliwell, B. and J. M. Gutteridge (1990). "Role of free radicals and catalytic metal ions in human disease: an overview." Methods Enzymol 186: 1-85.
Hanisch, U. K., et al. (2008). "Toll-like receptors: roles in neuroprotection?" Trends Neurosci 31(4): 176-182.
Hansson, G. K. and K. Edfeldt (2005). "Toll to be paid at the gateway to the vessel wall." Arteriosclerosis Thrombosis and Vascular Biology 25(6): 1085-1087.
Harry, G. J., et al. (2004). "Trimethyltin-induced neurogenesis in the murine hippocampus." Neurotox Res 5(8): 623-627.
Hebert, L. E., et al. (2001). "Annual incidence of Alzheimer disease in the United States projected to the years 2000 through 2050." Alzheimer Dis Assoc Disord 15(4): 169-173.
Hellberg, C. B., et al. (1998). "Phosphatidylinositol 3-kinase is a target for protein tyrosine nitration." Biochem Biophys Res Commun 252(2): 313-317.
Hibbs, J. B., Jr., et al. (1987). "Macrophage cytotoxicity: role for L-arginine deiminase and imino nitrogen oxidation to nitrite." Science 235(4787): 473-476.
Inoue, M., et al. (2003). "Mitochondrial generation of reactive oxygen species and its role in aerobic life." Curr Med Chem 10(23): 2495-2505.
Ischiropoulos, H., et al. (1992). "Peroxynitrite formation from macrophage-derived nitric oxide." Arch Biochem Biophys 298(2): 446-451.
Jack, C. S., et al. (2005). "TLR signaling tailors innate immune responses in human microglia and astrocytes." J Immunol 175(7): 4320-4330.
Jacob, S. W. and J. C. de la Torre (2009). "Pharmacology of dimethyl sulfoxide in cardiac and CNS damage." Pharmacol Rep 61(2): 225-235.
Kang, J. S., et al. (2008). "Antiinflammatory activity of methanol extract isolated from stem bark of Magnolia kobus." Phytother Res 22(7): 883-888.
Kang, K. W., et al. (2002). "Peroxynitrite activates NF-E2-related factor 2/antioxidant response element through the pathway of phosphatidylinositol 3-kinase: the role of nitric oxide synthase in rat glutathione S-transferase A2 induction." Nitric Oxide 7(4): 244-253.
Kettenmann, H., et al. (2011). "Physiology of microglia." Physiol Rev 91(2): 461-553.
Kim, Y. S. and T. H. Joh (2006). "Microglia, major player in the brain inflammation: their roles in the pathogenesis of Parkinson's disease." Exp Mol Med 38(4): 333-347.
Kong, C. W., et al. (2000). "Magnolol attenuates peroxidative damage and improves survival of rats with sepsis." Shock 13(1): 24-28.
Koppula, S., et al. (2012). "Reactive oxygen species and inhibitors of inflammatory enzymes, NADPH oxidase, and iNOS in experimental models of Parkinson's disease." Mediators Inflamm 2012: 823902.
Lee, P., et al. (2001). "NO as an autocrine mediator in the apoptosis of activated microglial cells: correlation between activation and apoptosis of microglial cells." Brain Res 892(2): 380-385.
Lee, Y. J., et al. (2011). "Therapeutic applications of compounds in the Magnolia family." Pharmacol Ther 130(2): 157-176.
Lemaitre, B., et al. (1996). "The dorsoventral regulatory gene cassette spatzle/Toll/cactus controls the potent antifungal response in Drosophila adults." Cell 86(6): 973-983.
Lin, Y. R., et al. (2006). "Neuroprotective activity of honokiol and magnolol in cerebellar granule cell damage." Eur J Pharmacol 537(1-3): 64-69.
Lo, Y. C., et al. (1994). "Magnolol and honokiol isolated from Magnolia officinalis protect rat heart mitochondria against lipid peroxidation." Biochem Pharmacol 47(3): 549-553.
Lynch, M. A. (2009). "The multifaceted profile of activated microglia." Mol Neurobiol 40(2): 139-156.
Martindale, J. L. and N. J. Holbrook (2002). "Cellular response to oxidative stress: signaling for suicide and survival." J Cell Physiol 192(1): 1-15.
McGeer, P. L., et al. (2006). "Inflammation, anti-inflammatory agents and Alzheimer disease: the last 12 years." J Alzheimers Dis 9(3 Suppl): 271-276.
Morgan, S. C., et al. (2004). "Microglia release activators of neuronal proliferation mediated by activation of mitogen-activated protein kinase, phosphatidylinositol-3-kinase/Akt and delta-Notch signalling cascades." J Neurochem 90(1): 89-101.
Muller, J. M., et al. (1993). "Nuclear factor kappa B, a mediator of lipopolysaccharide effects." Immunobiology 187(3-5): 233-256.
Munroe, M. E., et al. (2007). "Honokiol, a natural plant product, inhibits inflammatory signals and alleviates inflammatory arthritis." J Immunol 179(2): 753-763.
Nathan, C. and Q. W. Xie (1994). "Nitric oxide synthases: roles, tolls, and controls." Cell 78(6): 915-918.
Nomiyama, T., et al. (2004). "Reduction of insulin-stimulated glucose uptake by peroxynitrite is concurrent with tyrosine nitration of insulin receptor substrate-1." Biochem Biophys Res Commun 320(3): 639-647.
Okun, E., et al. (2009). "Toll-like receptors in neurodegeneration." Brain Res Rev 59(2): 278-292.
Pacher, P., et al. (2007). "Nitric oxide and peroxynitrite in health and disease." Physiol Rev 87(1): 315-424.
Park, E. J., et al. (2006). "Honokiol reduces oxidative stress, c-jun-NH2-terminal kinase phosphorylation and protects against glycochenodeoxycholic acid-induced apoptosis in primary cultured rat hepatocytes." Planta Med 72(7): 661-664.
Pelicano, H., et al. (2004). "ROS stress in cancer cells and therapeutic implications." Drug Resist Updat 7(2): 97-110.
Radi, R., et al. (1994). "Inhibition of mitochondrial electron transport by peroxynitrite." Arch Biochem Biophys 308(1): 89-95.
Regoli, F. and G. W. Winston (1999). "Quantification of total oxidant scavenging capacity of antioxidants for peroxynitrite, peroxyl radicals, and hydroxyl radicals." Toxicol Appl Pharmacol 156(2): 96-105.
Rezaie, P., et al. (2002). "Motility and ramification of human fetal microglia in culture: an investigation using time-lapse video microscopy and image analysis." Exp Cell Res 274(1): 68-82.
Sase, K. and T. Michel (1997). "Expression and regulation of endothelial nitric oxide synthase." Trends Cardiovasc Med 7(1): 28-37.
Schroeder, P., et al. (2001). "Epicatechin selectively prevents nitration but not oxidation reactions of peroxynitrite." Biochem Biophys Res Commun 285(3): 782-787.
Simmonds, R. E. and B. M. Foxwell (2008). "Signalling, inflammation and arthritis: NF-kappaB and its relevance to arthritis and inflammation." Rheumatology (Oxford) 47(5): 584-590.
Sims, J. E. and D. E. Smith (2010). "The IL-1 family: regulators of immunity." Nat Rev Immunol 10(2): 89-102.
Smith, W. L., et al. (1996). "Prostaglandin endoperoxide H synthases (cyclooxygenases)-1 and -2." J Biol Chem 271(52): 33157-33160.
Spear, N., et al. (1997). "Nerve growth factor protects PC12 cells against peroxynitrite-induced apoptosis via a mechanism dependent on phosphatidylinositol 3-kinase." J Neurochem 69(1): 53-59.
Stence, N., et al. (2001). "Dynamics of microglial activation: a confocal time-lapse analysis in hippocampal slices." Glia 33(3): 256-266.
Stiles, B. L. (2009). "PI-3-K and AKT: Onto the mitochondria." Adv Drug Deliv Rev 61(14): 1276-1282.
Streit, W. J. and Q. S. Xue (2009). "Life and death of microglia." J Neuroimmune Pharmacol 4(4): 371-379.
Stuehr, D. J. and C. F. Nathan (1989). "Nitric oxide. A macrophage product responsible for cytostasis and respiratory inhibition in tumor target cells." J Exp Med 169(5): 1543-1555.
Teng, C. M., et al. (1990). "EDRF-release and Ca+(+)-channel blockade by magnolol, an antiplatelet agent isolated from Chinese herb Magnolia officinalis, in rat thoracic aorta." Life Sci 47(13): 1153-1161.
Walton, N. M., et al. (2006). "Microglia instruct subventricular zone neurogenesis." Glia 54(8): 815-825.
Wang, H. J., et al. (2013). "Honokiol blocks store operated calcium entry in CHO cells expressing the M3 muscarinic receptor: honokiol and muscarinic signaling." J Biomed Sci 20: 11.
Wang, Y., et al. (2013). "Honokiol protects rat hearts against myocardial ischemia reperfusion injury by reducing oxidative stress and inflammation." Exp Ther Med 5(1): 315-319.
Weeks, B. S. (2009). "Formulations of dietary supplements and herbal extracts for relaxation and anxiolytic action: Relarian." Med Sci Monit 15(11): RA256-262.
Wymann, M. P. and R. Marone (2005). "Phosphoinositide 3-kinase in disease: timing, location, and scaffolding." Curr Opin Cell Biol 17(2): 141-149.
Xu, Q., et al. (2008). "Antidepressant-like effects of the mixture of honokiol and magnolol from the barks of Magnolia officinalis in stressed rodents." Prog Neuropsychopharmacol Biol Psychiatry 32(3): 715-725.
Zou, M. H., et al. (2002). "Modulation by peroxynitrite of Akt- and AMP-activated kinase-dependent Ser1179 phosphorylation of endothelial nitric oxide synthase." J Biol Chem 277(36): 32552-32557.





2.書籍論文
顏正華, “中藥學(上)”, 知音出版社, 民國八十三年二月初版二刷, 320頁。
莊朝欽。(2009)。和厚朴酚抑制巨噬細胞中細菌內毒素所誘發之細胞激素表現。國立台灣海洋大學食品科學系碩士論文。
卓俊宏。(2010)。芳香烴受體在微小膠細胞活化過程中所扮演的角色。台北醫學大學 醫學院 醫學科學研究所碩士論文。
楊雅婷。(2005)。細胞自然凋亡與發炎反應參與在脂多醣刺激腦微膠細胞移動及聚集現象。國立陽明大學解剖暨細胞生物學研究所碩士論文。
3.網路資源
中藥知識學習網