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系統識別號 U0026-0812200914263821
論文名稱(中文) 探討 Minocycline 在薑黃素誘發的神經細胞凋亡過程中的保護作用
論文名稱(英文) Neuroprotective effect of Minocycline on neuronal apoptosis induced by a synthetic gingerdione compound
校院名稱 成功大學
系所名稱(中) 藥理學研究所
系所名稱(英) Department of Pharmacology
學年度 96
學期 2
出版年 97
研究生(中文) 余泱蓉
研究生(英文) Yang-Jung Yu
學號 s2695111
學位類別 碩士
語文別 中文
論文頁數 65頁
口試委員 口試委員-莊季瑛
指導教授-簡伯武
口試委員-黃阿敏
中文關鍵字 神經保護  凋亡 
英文關鍵字 Neuroprotection  Minocycline  Apoptosis 
學科別分類
中文摘要 薑除了當作辛辣成分外,在東方醫學上常被應用在改善發炎、風濕或者腸胃道不適等症狀。在2005年的Leukemia Research中指出薑辛辣成分Gingerdion衍生物,1-(3,4-dimethoxyphenyl)-3,5- dodecenedione (簡稱I6) 可使HL-60 cells造成G1 arrest及apoptosis,對人類leukemia cell有抗癌的作用。因此,藉由之前所知I6有抗癌作用,我們以對一個新藥開發的觀點,考慮對於其他正常細胞是否會產生傷害或威脅,因此,在本論文中尋找I6誘發神經細胞凋亡路徑,並藉此探討Minocycline對此的保護機制。目前實驗室已有針對I6對神經皮質細胞的作用進行研究,我們以17-18天大胚胎鼠的皮質神經細胞培養五天後,給予不同濃度I6處理24小時後,利用MTT assay偵測細胞存活率,結果發現隨著I6濃度增加,神經細胞存活率跟著減少。也利用Hoechst 33342, propidium iodide (PI) 及TUNEL staining確認I6誘發神經細胞死亡是屬於細胞凋亡(Apoptosis) (C. H. Lin et al., 2006)。
  因此本論文的研究目的,是利用I6所誘發神經細胞凋亡現象,探討神經保護試劑Minocycline的保護機制。實驗進行方式是前處理神經保護試劑Minocycline三十分鐘,而後加入I6作用18小時,分別利用MTT assay、西方點墨法、免疫細胞染色及ELISA方法分析探討。
  由實驗結果顯示,發現隨著Minocycline濃度增加,降低了I6所誘發的神經細胞死亡。更發現I6誘發神經細胞死亡會伴隨著ROS產生增加、caspase 3、caspase 9的活化,進而使PARP失去活性,造成DNA片段化,而造成Apoptosis。上述現象,在前處理Minocycline後,發現能抑制caspase的活化,並減少細胞核內非活化態的PARP,減少DNA片段化,達到神經保護作用。由以上結果,發現Minocycline藉由抑制內因性apoptosis的caspase活化路徑而對於I6所誘發的神經細胞死亡產生保護作用。更進一步地,我們也觀察I6會減少anti-apoptosis protein Bcl-2表現量,而Minocycline可使Bcl-2量回升。
英文摘要 Besides being used as a spice, ginger has been applied in oriental medicine to ameliorate symptoms such as inflammatory, rheumatic disorders and gastrointestinal discomforts. Present study indicated that the effect of 1-(3,4-dimethoxyphenyl)-3,5-dodecenedione(I6), a derivative of gingerdione, could induce HL-60 cell G1 arrest and apoptosis. Therefore, we would investigate if I6 affect neuron. In the present study, we further studied the mechanism of I6 on the cortical neurons. After a 5-day maturation period in vitro, cortical neurons were treated with I6 at different concentration for 24 h and cell viability was assessed by using MTT assay. We found that I6 induced neuronal death in a concentration-dependent manner. Furthermore, using Hoechst 33342, propidium iodide (PI) and TUNEL staining confirmed that the reduced cell viability induced by I6 was due to apoptosis.
Here we want to study the neuroprotective mechanism of Minocycline by I6 inducing neuronal apoptosis. We treated Minocycline prior to I6 to cortical neurons, and after 18 hours we were assessed by MTT assay, Western blot, Immuocytochemistry and ELISA.
Here, we show that pre-treatment of cell with Minocycline prevented cell death in a concentration-dependent manner. Constitutive expression of the 32-kD a pro-caspase-3 protein was detected in controls. After treatment with I6, the expression of cleaved caspase-3、reactive oxygen species (ROS)、cleaved caspase-9 and cleaved PARP gradually increased. Pre-treatment of cell with Minocycline prevented the increase of cleaved fragment of those caspases. Furthermore, we revealed that DNA fragmentation was made by I6 treatment and this effect was blocked by Minocycline treatment. Therefore, these results suggest that Minocycline may have the protective effects on I6-induced neuronal apoptosis via inhibition of caspase activation. Additionally, We investigate I6 cause Bcl-2 level decrease and Minocycline could reverse it。
論文目次 中 文 摘 要 1
英 文 摘 要 3
縮 寫 檢 索 表 6
第 一 章 緒 論 8
1-1 Apoptosis與中樞神經疾病 9
1-2 關於薑黃素(I6) 10
1-3 Minocycline與神經保護作用 11
1-4 Tetracycline、Doxycycline與神經保護作用 12
1-5 研究目的 13
第 二 章 實驗材料與研究方法 14
2-1 實驗動物 15
2-2 實驗藥品與試劑來源 15
2-3 神經細胞元培養方法(Primary cortical neuron culture) 16
2-4 製備薑黃素(I6) 17
2-5 製備Minocycline、Tetracycline、Doxycycline 17
2-6 神經細胞存活率分析(Neuronal viability assay) 17
2-7 免疫螢光染色 (Immunocytochemistry) 18
2-8 Primary neurons sample 的收集: 18
2-9 西方點墨法(Western blot) 19
2-10 DNA fragmentation 23
2-11 萃取細胞核 (Nucleus Extraction) 25
2-12 ROS detection 26
2-13 CellR live cell imaging 26
2-14 統計分析 26
第 三 章 結 果 29
3-1 Minocycline對於I6所誘發的皮質神經細胞死亡具有保護作用 30
3-2 Minocycline相較於Tetracycline及Doxycycline而言,其對於I6誘發之細胞死亡有較好的保護作用 30
3-3 PI3K抑制劑不能阻斷Minocycline的保護作用 31
3-4 Minocycline能降低I6所導致caspase-3活化態的增加 32
3-5 I6造成PARP不活化態增加,使DNA片段無法修復,而Minocycline可回復此情形。 32
3-6 Minocycline能降低I6所導致的ROS增加 33
3-7 Vitamin E能阻斷I6造成的細胞傷害 33
3-8 Minocycline能降低I6所導致caspase-9活化態的增加 34
3-9 Minocycline能回復I6所造成Bcl-2降低 34
第 四 章 討 論 35
參 考 文 獻 39
圖 表 索 引 47
參考文獻 Allen JC (1976) Minocycline. Ann Intern Med 85:482-487.
Arvin KL, Han BH, Du Y, Lin SZ, Paul SM, Holtzman DM (2002) Minocycline markedly protects the neonatal brain against hypoxic-ischemic injury. Ann Neurol 52:54-61.
Ashkenazi A (2002) Targeting death and decoy receptors of the tumour-necrosis factor superfamily. Nat Rev Cancer 2:420-430.
Berger NA, Petzold SJ (1985) Identification of minimal size requirements of DNA for activation of poly(ADP-ribose) polymerase. Biochemistry 24:4352-4355.
Chan PH (2004) Mitochondria and neuronal death/survival signaling pathways in cerebral ischemia. Neurochem Res 29:1943-1949.
Chen M, Ona VO, Li M, Ferrante RJ, Fink KB, Zhu S, Bian J, Guo L, Farrell LA, Hersch SM, Hobbs W, Vonsattel JP, Cha JH, Friedlander RM (2000) Minocycline inhibits caspase-1 and caspase-3 expression and delays mortality in a transgenic mouse model of Huntington disease. Nat Med 6:797-801.
Clarke PG (1990) Developmental cell death: morphological diversity and multiple mechanisms. Anat Embryol (Berl) 181:195-213.
Cregan SP, MacLaurin JG, Craig CG, Robertson GS, Nicholson DW, Park DS, Slack RS (1999) Bax-dependent caspase-3 activation is a key determinant in p53-induced apoptosis in neurons. J Neurosci 19:7860-7869.
Culmsee C, Mattson MP (2005) p53 in neuronal apoptosis. Biochem Biophys Res Commun 331:761-777.
Dejda A, Jolivel V, Bourgault S, Seaborn T, Fournier A, Vaudry H, Vaudry D (2008) Inhibitory Effect of PACAP on Caspase Activity in Neuronal Apoptosis: A Better Understanding Towards Therapeutic Applications in Neurodegenerative Diseases. J Mol Neurosci.
Ding R, Pommier Y, Kang VH, Smulson M (1992) Depletion of poly(ADP-ribose) polymerase by antisense RNA expression results in a delay in DNA strand break rejoining. J Biol Chem 267:12804-12812.
Du Y, Ma Z, Lin S, Dodel RC, Gao F, Bales KR, Triarhou LC, Chernet E, Perry KW, Nelson DL, Luecke S, Phebus LA, Bymaster FP, Paul SM (2001) Minocycline prevents nigrostriatal dopaminergic neurodegeneration in the MPTP model of Parkinson's disease. Proc Natl Acad Sci U S A 98:14669-14674.
Emerit J, Edeas M, Bricaire F (2004) Neurodegenerative diseases and oxidative stress. Biomed Pharmacother 58:39-46.
Freeman K (1989) Therapeutic focus. Minocycline in the treatment of acne. Br J Clin Pract 43:112-115.
Ghobrial IM, Witzig TE, Adjei AA (2005) Targeting apoptosis pathways in cancer therapy. CA Cancer J Clin 55:178-194.
Hsu MH, Kuo SC, Chen CJ, Chung JG, Lai YY, Huang LJ (2005) 1-(3,4-dimethoxyphenyl)-3,5-dodecenedione (I6) induces G1 arrest and apoptosis in human promyelocytic leukemia HL-60 cells. Leuk Res 29:1399-1406.
Jayanthi S, Deng X, Noailles PA, Ladenheim B, Cadet JL (2004) Methamphetamine induces neuronal apoptosis via cross-talks between endoplasmic reticulum and mitochondria-dependent death cascades. FASEB J 18:238-251.
Kanduc D, Mittelman A, Serpico R, Sinigaglia E, Sinha AA, Natale C, Santacroce R, Di Corcia MG, Lucchese A, Dini L, Pani P, Santacroce S, Simone S, Bucci R, Farber E (2002) Cell death: apoptosis versus necrosis (review). Int J Oncol 21:165-170.
Kim SO, Kundu JK, Shin YK, Park JH, Cho MH, Kim TY, Surh YJ (2005) [6]-Gingerol inhibits COX-2 expression by blocking the activation of p38 MAP kinase and NF-kappaB in phorbol ester-stimulated mouse skin. Oncogene 24:2558-2567.
Klein NC, Cunha BA (1995) Tetracyclines. Med Clin North Am 79:789-801.
Kuo PC, Damu AG, Cherng CY, Jeng JF, Teng CM, Lee EJ, Wu TS (2005) Isolation of a natural antioxidant, dehydrozingerone from Zingiber officinale and synthesis of its analogues for recognition of effective antioxidant and antityrosinase agents. Arch Pharm Res 28:518-528.
Lavrik IN, Golks A, Krammer PH (2005) Caspases: pharmacological manipulation of cell death. J Clin Invest 115:2665-2672.
Le Rhun Y, Kirkland JB, Shah GM (1998) Cellular responses to DNA damage in the absence of Poly(ADP-ribose) polymerase. Biochem Biophys Res Commun 245:1-10.
Lee E, Surh YJ (1998) Induction of apoptosis in HL-60 cells by pungent vanilloids, [6]-gingerol and [6]-paradol. Cancer Lett 134:163-168.
Lee SM, Yune TY, Kim SJ, Park DW, Lee YK, Kim YC, Oh YJ, Markelonis GJ, Oh TH (2003) Minocycline reduces cell death and improves functional recovery after traumatic spinal cord injury in the rat. J Neurotrauma 20:1017-1027.
Lin CH, Kuo SC, Huang LJ, Gean PW (2006) Neuroprotective effect of N-acetylcysteine on neuronal apoptosis induced by a synthetic gingerdione compound: involvement of ERK and p38 phosphorylation. J Neurosci Res 84:1485-1494.
Lin S, Zhang Y, Dodel R, Farlow MR, Paul SM, Du Y (2001) Minocycline blocks nitric oxide-induced neurotoxicity by inhibition p38 MAP kinase in rat cerebellar granule neurons. Neurosci Lett 315:61-64.
Lin S, Wei X, Xu Y, Yan C, Dodel R, Zhang Y, Liu J, Klaunig JE, Farlow M, Du Y (2003) Minocycline blocks 6-hydroxydopamine-induced neurotoxicity and free radical production in rat cerebellar granule neurons. Life Sci 72:1635-1641.
Martin LJ (2001) Neuronal cell death in nervous system development, disease, and injury (Review). Int J Mol Med 7:455-478.
Masada Y, Inoue T, Hashimoto K, Fujioka M, Shiraki K (1973) [Studies on the pungent principles of ginger (Zingiber officinale Roscoe) by GC-MS]. Yakugaku Zasshi 93:318-321.
Morrison RS, Kinoshita Y, Johnson MD, Guo W, Garden GA (2003) p53-dependent cell death signaling in neurons. Neurochem Res 28:15-27.
Nicholson DW, Ali A, Thornberry NA, Vaillancourt JP, Ding CK, Gallant M, Gareau Y, Griffin PR, Labelle M, Lazebnik YA, et al. (1995) Identification and inhibition of the ICE/CED-3 protease necessary for mammalian apoptosis. Nature 376:37-43.
Pollack M, Leeuwenburgh C (2001) Apoptosis and aging: role of the mitochondria. J Gerontol A Biol Sci Med Sci 56:B475-482.
Popovic N, Schubart A, Goetz BD, Zhang SC, Linington C, Duncan ID (2002) Inhibition of autoimmune encephalomyelitis by a tetracycline. Ann Neurol 51:215-223.
Power C, Henry S, Del Bigio MR, Larsen PH, Corbett D, Imai Y, Yong VW, Peeling J (2003) Intracerebral hemorrhage induces macrophage activation and matrix metalloproteinases. Ann Neurol 53:731-742.
Ryan ME, Greenwald RA, Golub LM (1996) Potential of tetracyclines to modify cartilage breakdown in osteoarthritis. Curr Opin Rheumatol 8:238-247.
Sanchez Mejia RO, Ona VO, Li M, Friedlander RM (2001) Minocycline reduces traumatic brain injury-mediated caspase-1 activation, tissue damage, and neurological dysfunction. Neurosurgery 48:1393-1399; discussion 1399-1401.
Teng YD, Choi H, Onario RC, Zhu S, Desilets FC, Lan S, Woodard EJ, Snyder EY, Eichler ME, Friedlander RM (2004) Minocycline inhibits contusion-triggered mitochondrial cytochrome c release and mitigates functional deficits after spinal cord injury. Proc Natl Acad Sci U S A 101:3071-3076.
Tewari M, Quan LT, O'Rourke K, Desnoyers S, Zeng Z, Beidler DR, Poirier GG, Salvesen GS, Dixit VM (1995) Yama/CPP32 beta, a mammalian homolog of CED-3, is a CrmA-inhibitable protease that cleaves the death substrate poly(ADP-ribose) polymerase. Cell 81:801-809.
Thornberry NA, Lazebnik Y (1998) Caspases: enemies within. Science 281:1312-1316.
Tikka T, Fiebich BL, Goldsteins G, Keinanen R, Koistinaho J (2001) Minocycline, a tetracycline derivative, is neuroprotective against excitotoxicity by inhibiting activation and proliferation of microglia. J Neurosci 21:2580-2588.
Tikka TM, Koistinaho JE (2001) Minocycline provides neuroprotection against N-methyl-D-aspartate neurotoxicity by inhibiting microglia. J Immunol 166:7527-7533.
Tikka TM, Vartiainen NE, Goldsteins G, Oja SS, Andersen PM, Marklund SL, Koistinaho J (2002) Minocycline prevents neurotoxicity induced by cerebrospinal fluid from patients with motor neurone disease. Brain 125:722-731.
Vandekerckhove BN, Quirynen M, van Steenberghe D (1998) The use of locally delivered minocycline in the treatment of chronic periodontitis. A review of the literature. J Clin Periodontol 25:964-968; discussion 978-969.
Vila M, Przedborski S (2003) Targeting programmed cell death in neurodegenerative diseases. Nat Rev Neurosci 4:365-375.
Wang J, Wei Q, Wang CY, Hill WD, Hess DC, Dong Z (2004) Minocycline up-regulates Bcl-2 and protects against cell death in mitochondria. J Biol Chem 279:19948-19954.
Wang JY, Shum AY, Ho YJ (2003) Oxidative neurotoxicity in rat cerebral cortex neurons: synergistic effects of H2O2 and NO on apoptosis involving activation of p38 mitogen-activated protein kinase and caspase-3. J Neurosci Res 72:508-519.
Wang X, Zhu S, Drozda M, Zhang W, Stavrovskaya IG, Cattaneo E, Ferrante RJ, Kristal BS, Friedlander RM (2003) Minocycline inhibits caspase-independent and -dependent mitochondrial cell death pathways in models of Huntington's disease. Proc Natl Acad Sci U S A 100:10483-10487.
Wu DC, Jackson-Lewis V, Vila M, Tieu K, Teismann P, Vadseth C, Choi DK, Ischiropoulos H, Przedborski S (2002) Blockade of microglial activation is neuroprotective in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine mouse model of Parkinson disease. J Neurosci 22:1763-1771.
Wyllie AH, Kerr JF, Currie AR (1980) Cell death: the significance of apoptosis. Int Rev Cytol 68:251-306.
Xiang H, Kinoshita Y, Knudson CM, Korsmeyer SJ, Schwartzkroin PA, Morrison RS (1998) Bax involvement in p53-mediated neuronal cell death. J Neurosci 18:1363-1373.
Yang J, Liu X, Bhalla K, Kim CN, Ibrado AM, Cai J, Peng TI, Jones DP, Wang X (1997) Prevention of apoptosis by Bcl-2: release of cytochrome c from mitochondria blocked. Science 275:1129-1132.
Yrjanheikki J, Keinanen R, Pellikka M, Hokfelt T, Koistinaho J (1998) Tetracyclines inhibit microglial activation and are neuroprotective in global brain ischemia. Proc Natl Acad Sci U S A 95:15769-15774.
Yrjanheikki J, Tikka T, Keinanen R, Goldsteins G, Chan PH, Koistinaho J (1999) A tetracycline derivative, minocycline, reduces inflammation and protects against focal cerebral ischemia with a wide therapeutic window. Proc Natl Acad Sci U S A 96:13496-13500.
Zemke D, Majid A (2004) The potential of minocycline for neuroprotection in human neurologic disease. Clin Neuropharmacol 27:293-298.
Zhu S, Stavrovskaya IG, Drozda M, Kim BY, Ona V, Li M, Sarang S, Liu AS, Hartley DM, Wu DC, Gullans S, Ferrante RJ, Przedborski S, Kristal BS, Friedlander RM (2002) Minocycline inhibits cytochrome c release and delays progression of amyotrophic lateral sclerosis in mice. Nature 417:74-78.
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