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系統識別號 U0007-1208201408553100
論文名稱(中文) Platonin 於創傷性腦損傷之保護作用
論文名稱(英文) The Protective Effects of Platonin on Traumatic Brain Injury
校院名稱 臺北醫學大學
系所名稱(中) 醫學科學研究所
系所名稱(英) Graduate Institute of Medical Sciences
學年度 102
學期 2
出版年 103
研究生(中文) 詹岱華
研究生(英文) Tai-Hua Chan
學號 M120102011
學位類別 碩士
語文別 中文
口試日期 2014-07-09
論文頁數 80頁
口試委員 指導教授-施純明
委員-葉添順
委員-謝榮鴻
中文關鍵字 Platonin  創傷性腦損傷  神經膠質星狀細胞  Glutamate  GSK-3β  Nrf2  細胞自噬  細胞凋亡 
英文關鍵字 Platonin  Traumatic brain injury  Astrocyte  Glutamate  GSK-3β  Nrf2  Autophagy  Apoptosis 
學科別分類
中文摘要 根據2013年行政院衛生福利部統計,事故傷害位居十大死因的第六位,其中創傷性腦損傷(traumatic brain injury, TBI)是意外傷害死亡最主要的原因,患者以15至24歲的青少年居多。根據美國Defense and Veterans Brain Injury Center 2013年統計,全球每年耗費於TBI治療之醫療支出高達765億美元,因此發展有效治療TBI的藥物以減少患者症狀並降低醫療成本,為當前十分重要的課題。TBI過程中,神經傳導物質glutamate會被大量釋放導致氧化壓力及發炎反應的產生,造成神經細胞損傷,終使病患癱瘓甚至死亡,其預後差且目前並無有效治療用藥。Platonin為臨床用藥,傳統上用於治療切創傷、潰瘍、燙傷及風濕性關節炎引起的急性發炎等。此外,文獻亦指出其具有抗氧化、抗發炎及免疫調節作用;然而目前並未有研究指出platonin是否能應用於TBI之治療。
本研究利用glutamate處理大鼠神經膠質星狀細胞 (CTX TNA2) 建立TBI細胞模式,並利用TBI 0310 Impactor撞擊Spraque-Dawley rat大腦皮質作為TBI動物模式,探討glutamate造成細胞傷害之途徑以及platonin之細胞保護機制。本實驗室過去已證實:glutamate能誘發活性氧化物 (reactive oxygen species, ROS)之產生,並透過活化下游glycogen synthase kinase-3β (GSK-3β),造成CTX TNA2細胞死亡。本論文續以MTT assay分析指出:以glutamate處理CTX TNA2後,給予300 nM platonin可見細胞存活率回升23.39 %;利用流式細胞儀偵測細胞自噬(autophagy)及細胞凋亡(apoptosis)的比例,結果顯示platonin能降低glutamate引起CTX TNA2之細胞自噬(21.51 ± 0.21 % 下降至13.88 ± 1.25 %) 及細胞凋亡(32.94 ± 3.94 % 下降至12.85 ± 0.62 %)。再者,西方墨漬法分析結果指出:glutamate可提高GSK-3β Tyr216位置磷酸化(p-tyr216-GSK-3β)、降低GSK-3β Ser9位置磷酸化(p-ser9-GSK-3β)程度,並減少細胞核內nuclear factor erythroid 2-related factor 2 (Nrf2)的累積,而若共同處理platonin則可恢復其表現。此外,利用GSK-3β inhibitor (SB216763)搭配glutamate處理細胞後,可見核內Nrf2量回升,顯示GSK-3β與Nrf2具上、下游關係。綜合以上數據顯示platonin具有保護神經膠質細胞之效果,其機轉可能透過抑制GSK-3β活化而使Nrf2得以累積於細胞核內,降低glutamate誘導之細胞傷害。
此外,利用西方墨漬法觀察TBI大鼠患部組織蛋白發現:TBI組別中p-ser9-GSK-3β磷酸化程度及pro-poly ADP ribose polymerase (pro-PARP)之表現量下降。若搭配腹腔注射platonin則能使p-ser9-GSK-3β磷酸化程度與pro-PARP表現回升,顯示platonin能抑制TBI後腦細胞之GSK-3β活化及細胞凋亡發生,於創傷性腦損傷具保護效果。本論文透過釐清platonin在細胞及大鼠TBI模式中對於細胞傷害之保護機制以及臨床應用的可能性,提供臨床開發治療TBI藥物的新方向。
英文摘要 The statistical results indicate that accident ranks the sixth leading cause of death in Taiwan. Furthermore, traumatic brain injury (TBI), the main reason of accidental death, is the most common among adolescents aged from 15 to 24. An estimation made by Defense and Veterans Brain Injury Center in 2013 indicates that the medical care and rehabilitation costs of TBI patients are approximately US$ 76.5 billion. Therefore, it is very important to develop new drugs for TBI therapy.
After TBI, over-released glutamate often leads to the responses of oxidative stress and inflammation, which subsequently causes damage of nerve cells. In clinics, TBI patients exhibit poor prognosis and lack of efficient therapies. Platonin, a clinical drug with anti-oxidative and anti-inflammatory functions, is used to treat incised wounds, ulcers, burns and acute inflammation in rheumatoid arthritis. However, the effects of platonin on TBI are unclear.
In this study, glutamate-treated CTX TNA2 astrocytes were used as a cell model to mimic TBI. As for animal models, TBI was induced at the left brain cortex of Sprague-Dawley rats using a TBI 0310 Impactor. In our previous report, glutamate could induce ROS production, GSK-3β activation, and CTX TNA2 cell death. Using MTT assay, we demonstrated that 300 nM platonin could reduce glutamate-induced cytotoxicity significantly. In addition, we investigated that platonin was able to reduce glutamate-induced cell autophagy (from 21.51 ± 0.21 % to 13.88 ± 1.25 %) and apoptosis (from 32.94 ± 3.94 % to 12.85 ± 0.62 %) using flow cytometry. Immunoblotting assay demonstrated that the increase of p-tyr216-GSK-3β, the decrease of p-ser9-GSK-3β and the decrease of nuclear Nrf2 level could be reversed after co-treatment with platonin. Using GSK-3β inhibitor (SB216763), we also demonstrated that GSK-3β could be the upstream regulator of Nrf2 in the glutamate-mediated signaling pathway. These results suggested that platonin may protect astrocytes from glutamate-induced cytotoxicity through inactivating GSK-3β and maintaining the nuclear Nrf2 level.
Following the line, we further investigated whether platonin could induce GSK-3β inactivation and PARP cleavage in vivo. Immunoblotting assay showed that TBI could lead to decrease of p-ser9-GSK-3β and pro-PARP level. In contrast, this phenomenon could be reversed by platonin treatment. Taken together, the results suggested that platonin may increase cell survival by suppressing GSK-3β activation and Nrf2 translocation. This study provides the evidence that platonin could be a potential therapeutic agent for TBI treatment.
論文目次 章節目錄---------------------------------- 1
縮寫表------------------------------------ 2
中文摘要---------------------------------- 4
英文摘要---------------------------------- 7
圖表目次---------------------------------- 10
緒論------------------------------------- 12
實驗材料與方法----------------------------- 22
結果------------------------------------- 30
討論------------------------------------- 43
未來實驗方向與展望-------------------------- 50
參考文獻---------------------------------- 53
圖表------------------------------------- 64
附圖------------------------------------- 80
參考文獻 Anderson CM, Swanson RA. Astrocyte glutamate transport: Review of properties, regulation, and physiological functions. Glia. 2000; 32: 1−14.
Ansari MA, Roberts KN, Scheff SW. Oxidative stress and modification of synaptic proteins in hippocampus after traumatic brain injury. Free Radic Biol Med. 2008; 45: 443-452.
Atlante A, Calissano P, Bobba A, Giannattasio S, Marra E, Passarella S. Glutamate neurotoxicity, oxidative stress and mitochondria. FEBS Lett. 2001; 497: 1-5.
Attwell D, Buchan AM, Charpak S, Lauritzen M, Macvicar BA, Newman EA. Glial and neuronal control of brain blood flow. Nature. 2010; 468: 232-243.
Boulares AH, Yakovlev AG, Ivanova V, Stoica BA, Wang G, Iyer S, Smulson M. Role of poly(ADP-ribose) polymerase (PARP) cleavage in apoptosis. Caspase 3-resistant PARP mutant increases rates of apoptosis in transfected cells. J Biol Chem. 1999; 274: 22932-22940.
Blommaart EF, Luiken JJ, Meijer AJ. Autophagic proteolysis: control and specificity. Histochem J. 1997; 29: 365-385.
Camilla L. Cellular and molecular responses to traumatic brain injury. 2014.
Chan JY, Han XL, Kan YW. Isolation of cDNA encoding the human NF-E2 protein. Proc Natl Acad Sci U S A. 1993; 90: 11366-11370.
Chen YC, Mao H, Yang KH, Abel T, Meaney DF. A modified controlled cortical impact technique to model mild traumatic brain injury mechanics in mice. Front. Neurol. 2014.
Chen YJ, Huang WP, Yang YC, Lin CP, Chen SH, Hsu ML, Tseng YJ, Shieh HR, Chen YY, Lee JJ. Platonin induces autophagy-associated cell death in human leukemia cells. Autophagy. 2009; 5: 173-183.
Chen Y, Swanson RA. Astrocytes and brain injury. J Cereb Blood Flow Metab. 2003; 23: 137-149.
Chen Y, Chan PH, Swanson RA. Astrocytes overexpressing Cu, Zn superoxide dismutase have increased resistance to oxidative injury. Glia. 2001; 33: 343−347.
Choi AM, Ryter SW, Levine B. Autophagy in human health and disease. N Engl J Med. 2013; 368: 651-662.
Choi DW. Glutamate neurotoxicity and diseases of the nervous system. Neuron. 1988; 1: 623-634.
Chu HN, Tsai PS, Wang TY, Huang CJ. Platonin mitigates acute lung injury in haemorrhagic shock rats. Resuscitation. 2011; 82: 97-104.
Cohn JJ. Apoptosis. Immunology Today 1997, 14:120.
Cuervo AM. Autophagy and aging - when ”all you can eat” is yourself. Sci. Aging Knowledge Environ. 2003; 36: 25.
Cuervo AM. Autophagy: in sickness and in health. Trends Cell Biol. 2004; 14: 70-77.
Dal-Cim T, Molz S, Egea J, Parada E, Romero A, Budni J, Martin de Saavedra MD, del Barrio L, Tasca CI, Lopez MG. Guanosine protects human neuroblastoma SH-SY5Y cells against mitochondrial oxidative stress by inducing heme oxigenase-1 via PI3K/Akt/GSK-3beta pathway. Neurochem Int. 2012; 61: 397-404.
Elmore SP, Qian T, Grissom SF, Lemasters JJ. The mitochondrial permeability transition initiates autophagy in rat hepatocytes. FASEB J. 2001; 15: 2286-2287.
e Pinto EA, Alves JG. The causes of death of hospitalized children in Angola. Trop Doct. 2008; 38:66-67.
Forde JE, Dale TC. Glycogen synthase kinase 3: a key regulator of cellular fate. Cell Mol Life Sci. 2007; 64: 1930-1944.
Gozuacik D. and Kimchi A. Autophagy as a cell death and tumor suppressor mechanism. Oncogene. 2004; 23: 2891-2906.
Grimes CA, Jope RS. The multifaceted roles of glycogen synthase kinase 3beta in cellular signaling. Prog Neurobiol. 2001; 65: 391-426.
Halatsch ME, Gehrke EE, Vougioukas VI, Botefur IC, A-Borhani F, Efferth T, Gebhart E, Domhof S, Schmidt U, Buchfelder M. Inverse correlation of epidermal growth factor receptor messenger RNA induction and suppression of anchorage-independent growth by OSI-774, an epidermal growth factor receptor tyrosine kinase inhibitor, in glioblastoma multiforme cell lines. J Neurosurg. 2004; 100: 523-533.
Hellewell SC, Morganti-Kossmann MC. Guilty molecules, guilty minds? The conflicting roles of the innate immune response to traumatic brain injury. Mediators Inflamm. 2012; 2012: 356494.
Hsiao G, Lee JJ, Chou DS, Fong TH, Shen MY, Lin CH, Sheu JR. Platonin, a photosensitizing dye, improves circulatory failure and mortality in rat models of endotoxemia. Biol Pharm Bull. 2002; 25: 995-999.
Innamorato NG1, Rojo AI, Garcia-Yague AJ, Yamamoto M, de Ceballos ML, Cuadrado A. The transcription factor Nrf2 is a therapeutic target against brain inflammation. J Immunol. 2008 ; 181:680-689.
Ishihara M, Kadoma Y, Fujisawa S. Kinetic radical-scavenging activity of platonin, a cyanine photosensitizing dye. In Vivo. 2006; 20: 845-848.
Ishihara M, Fujisawa S. Review. Photooxygenation, photodegradation and antioxidative activity of platonin, a cyanine photosensitizing dye. In Vivo. 2007; 21: 163-173.
Itoh K, Mimura J, Yamamoto M. Discovery of the negative regulator of Nrf2, Keap1: a historical overview. Antioxid Redox Signal. 2010; 13: 1665-1678.
Jain AK, Jaiswal AK. GSK-3beta acts upstream of Fyn kinase in regulation of nuclear export and degradation of NF-E2 related factor 2. J Biol Chem. 2007; 282: 16502-16510.
Jin W, Wang H, Yan W, Zhu L, Hu Z, Ding Y, Tang K. Role of Nrf2 in protection against traumatic brain injury in mice. J Neurotrauma. 2009; 26: 131-139.
Jope RS, Johnson GV. The glamour and gloom of glycogen synthase kinase-3. Trends Biochem Sci. 2004; 29: 95-102.
Juillerat-Jeanneret L. The targeted delivery of cancer drugs across the blood-brain barrier: chemical modifications of drugs or drug-nanoparticles? Drug Discov Today. 2008; 13: 1099-1106.
Kanninen K, White AR, Koistinaho J, Malm T. Targeting Glycogen Synthase Kinase-3β for Therapeutic Benefit against Oxidative Stress in Alzheimer's Disease: Involvement of the Nrf2-ARE Pathway. Int J Alzheimers Dis. 2011; 2011: 985085.
Kensler TW, Wakabayashi N, Biswal S. Cell survival responses to environmental stresses via the Keap1-Nrf2-ARE pathway. Annu Rev Pharmacol Toxicol. 2007; 47: 89-116.
Kondo N, Motoyoshi F, Ozawa T, Orii T. A case report of a 9-year old boy with polyarteritis nodosa in which a combination therapy of corticosteroids and a photosensitive dye platonin was effective. Biotherapy. 1991; 3: 261-264.
Kraft AD, Johnson DA, Johnson JA. Nuclear factor E2-related factor 2-dependent antioxidant response element activation by tertbutylhydroquinone and sulforaphane occurring preferentially in astrocytes conditions neurons against oxidative insult. J Neurosci. 2004; 24: 1101–1112.
Keller JN. Autophagy and cell fate. Int. J. Biochem. Cell Biol. 2004; 36: 2363.
Laird MD, Vender JR, Dhandapani KM. Opposing roles for reactive astrocytes following traumatic brain injury. Neurosignals. 2008; 16: 154-164.
Lawen A. Apoptosis-an introduction. Bioessays. 2003; 25: 888-896.
Lee JM, Johnson JA. An important role of Nrf2-ARE pathway in the cellular defense mechanism. J Biochem Mol Biol. 2004; 37: 139-143.
Lemasters JJ, Nieminen AL, Qian T, Trost LC, Elmore SP, Nishimura Y, Crowe RA, Cascio WE, Bradham CA, Brenner DA, Herman B. The mitochondrial permeability transition in cell death: a common mechanism in necrosis, apoptosis and autophagy. Bichim. Biophys. Acta-Bioenerg. 1998; 1366: 177-196.
Lin CJ, Chen TH, Yang LY, Shih CM. Resveratrol protects astrocytes against traumatic brain injury through inhibiting apoptotic and autophagic cell death. Cell Death Dis. 2014; 5: e1147.
Lockshin RA, Zakeri Z. Apoptosis, autophagy, and more. Int J Biochem Cell Biol. 2004; 36: 2405-2419.
Lopez NE, Krzyzaniak MJ, Costantini TW, Putnam J, Hageny AM, Eliceiri B, Coimbra R, Bansal V. Vagal nerve stimulation decreases blood-brain barrier disruption after traumatic brain injury. J Trauma Acute Care Surg. 2012; 72: 1562-1566.
Matute C, Domercq M, Sanchez-Gomez MV. Glutamate-mediated glial injury: mechanisms and clinical importance. Glia. 2006; 53: 212-224.
Meldrum BS. Glutamate as a neurotransmitter in the brain: review of physiology and pathology. J Nutr. 2000; 130: 1007S-1015S.
Moi P, Chan K, Asunis I, Cao A, Kan YW. Isolation of NF-E2-related factor 2 (Nrf2), a NF-E2-like basic leucine zipper transcriptional activator that binds to the tandem NF-E2/AP1 repeat of the beta-globin locus control region. Proc Natl Acad Sci U S A. 1994; 91: 9926-9930.
Motoyoshi F, Kondo N, Ono H, Orii T. The effect of photosensitive dye platonin on juvenile rheumatoid arthritis. Biotherapy. 1991; 3: 241-244.
Niture SK, Jaiswal AK. Nrf2 protein up-regulates antiapoptotic protein Bcl-2 and prevents cellular apoptosis. J Biol Chem. 2012; 287: 9873-9886.
Otterbein LE, Bach FH, Alam J, Soares M, Tao Lu H, Wysk M, Davis RJ, Flavell RA, Choi AM. Carbon monoxide has anti-inflammatory effects involving the mitogen-activated protein kinase pathway. Nat Med. 2000; 6: 422-428.
Pardridge WM. The blood-brain barrier: bottleneck in brain drug development. NeuroRx. 2005; 2: 3-14.
Perea G, Navarrete M, Araque A. Tripartite synapses: astrocytes process and control synaptic information. Trends Neurosci. 2009; 32: 421-431.
Reagan-Shaw S, Nihal M, Ahmad N. Dose translation from animal to human studies revisited. FASEB J. 2008; 22: 659-661.
Rojo AI, Sagarra MR, Cuadrado A. GSK-3beta down-regulates the transcription factor Nrf2 after oxidant damage: relevance to exposure of neuronal cells to oxidative stress. J Neurochem. 2008; 105: 192-202.
Rosenfeld JV, Maas AI, Bragge P, Morganti-Kossmann MC, Manley GT, Gruen RL. Early management of severe traumatic brain injury. Lancet. 2012; 380: 1088-1098.
Ryter SW, Choi AM. Cytoprotective and anti-inflammatory actions of carbon monoxide in organ injury and sepsis models. Novartis Found Symp. 2007; 280: 165-175; discussion 175-181.
Shao DZ, Lin M. Platonin inhibits LPS-induced NF-kappaB by preventing activation of Akt and IKKbeta in human PBMC. Inflamm Res. 2008; 57: 601-606.
Shih AY, Johnson DA, Wong G, Kraft AD, Jiang L, Erb H, Johnson JA, Murphy TH. Coordinate regulation of glutathione biosynthesis and release by Nrf2-expressing glia potently protects neurons from oxidative stress. J Neurosci. 2003; 23:3394 –3406.
Song H, Stevens CF, Gage FH. Astroglia induce neurogenesis from adult neural stem cells. Nature. 2002; 417: 39-44.
Sullivan PG, Bruce-Keller AJ, Rabchevsky AG, Christakos S, Clair DK, Mattson MP, Scheff SW. Exacerbation of damage and altered NF-kappaB activation in mice lacking tumor necrosis factor receptors after traumatic brain injury. J Neurosci. 1999; 19: 6248-6256.
Tsai CC, Lin MT, Yang CC, Liao JF, Lee JJ. Platonin, a cyanine photosensitizing dye, is effective for attenuation of heatstroke in rats. Shock. 2006; 26: 601-607.
Turner DA, Adamson DC. Neuronal-astrocyte metabolic interactions: understanding the transition into abnormal astrocytoma metabolism. J Neuropathol Exp Neurol. 2011; 70: 167-176.
Vargas MR, Johnson DA, Sirkis DW, Messing A, Johnson JA. Nrf2 activation in astrocytes protects against neurodegeneration in mouse models of familial amyotrophic lateral sclerosis. J Neurosci. 2008; 28: 13574-13581.
Vladimir P and Alexei V. Astrocytes revisited: concise historic outlook on glutamate homeostasis and signaling. Croat Med J. 2012; 53: 518–528.
Wang DD, Bordey A. The astrocyte odyssey. Prog Neurobiol. 2008; 86: 342-367.
Werner C, Engelhard K. Pathophysiology of traumatic brain injury. Br J Anaesth. 2007; 99: 4-9.
Yi JH, Hazell AS. Excitotoxic mechanisms and the role of astrocytic glutamate transporters in traumatic brain injury. Neurochem Int. 2006; 48: 394-403.
Ziebell JM, Morganti-Kossmann MC. Involvement of pro- and anti-inflammatory cytokines and chemokines in the pathophysiology of traumatic brain injury. Neurotherapeutics. 2010; 7: 22-30.
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