臺灣博碩士論文加值系統

甲基安非他命是一種精神興奮劑，但在世界各地廣泛被濫用，會使神經系統受到影響，但其對人體的危害不只限於中樞神經系統，在過去的文獻指出，使用過量的甲基安非他命會造成心律不整導致心臟衰竭或死亡。本研究期望能找出相對應之細胞保護機制，在未來針對甲基安非他命而造成心肌損傷之病患可利用此機制採取治療方式。
　　在本研究中發現，甲基安非他命會使H9c2心肌細胞活性氧化物 (Reactive Oxygen Species, ROS) 增加，進而造成細胞凋亡。本研究進一步發現，處理甲基安非他命24小時後，第一型血基質氧化酶 (Heme oxygenase-1, HO-1) 蛋白質表現量顯著上升，預處理HO-1抑制劑tin protoporphyrin (SnPP) 會使甲基安非他命所誘發之H9c2心肌細胞凋亡顯著增加，說明HO-1扮演抗細胞凋亡之角色。為了進一步釐清HO-1被誘發的來源是否由ROS而來，因此分別預處理抗氧化劑Vitamin C、N-acetyl cysteine (NAC) 之實驗組別中也發現，甲基安非他命會使ROS增加，在預處理NAC及Vitamin C後能阻擋甲基安非他命所誘發之HO-1蛋白表現量。本研究也發現，NADPH oxidase抑制劑apocynin無法抑制甲基安非他命所誘發之HO-1，暗示NADPH oxidase可能不參與在甲基安非他命所誘發之ROS產生。
　　而根據文獻指出，PI3K/Akt路徑也具有抗細胞凋亡之作用，且在實驗室過去的研究中發現，甲基安非他命在SH-SY5Y神經細胞中可透過PI3K/Akt路徑活化 HO-1的蛋白質表現。研究結果顯示，處理甲基安非他命於ve心肌細胞後，確實可在1小時內明顯活化Akt，接著利用PI3K抑制劑wortmannin的情況下，釐清PI3K/Akt路徑具有細胞保護作用。而在預處理Akt抑制劑API-2的實驗組別中也更加驗證Akt能對抗甲基安非他命所誘發之H9c2心肌細胞凋亡。本研究也進一步探討Akt是否能透過ROS所活化，因此在預處理抗氧化劑Vitamin C及NAC後能阻擋甲基安非他命所誘發之Akt活化，可得知甲基安非他命誘發Akt之活化會透過ROS而來。最後，利用預處理PI3K抑制劑wortmannin及轉染Akt的方式觀察Akt對HO-1的調控情形，研究結果顯示，PI3K/Akt的活化能調控HO-1之蛋白質表現量並達到抗細胞凋亡的效果。
　　總結以上的結果發現，處理甲基安非他命除了會使H9c2心肌細胞之ROS增加造成細胞凋亡外，也會啟動另一條保護路徑來對抗本身造成的傷害，而甲基安非他命會透過ROS的活化啟動PI3K/Akt路徑以誘發HO-1蛋白質表現，並達到抗細胞凋亡之保護作用。

Acute adverse effects of methamphetamine (METH) on human health have been mainly attributed to the insults of nervous system. To date, very limited information regarding detrimental effects of acute METH exposure on the cardiovascular system is available. Nevertheless, the molecular and cellular mechanisms by which METH induces cardiotoxicity remain unclear. This study was thus aimed to uncover the endogenous mechanisms and find potentially effective protection approach against the METH-induced cell damages in H9c2 cardiomyocytes.
　　In this study, METH caused a rapid increase of reactive oxygen species (ROS) and resulted in apoptosis in H9c2 cardiomyocytes. The level of HO-1 expression was dose-dependently elevated by METH. Moreover, METH-induced decline in cell viability and increase in cleaved caspase-3 level were enhanced by pretreatment with the HO-1 inhibitor tin-protoporphyrin (SnPP). These results suggest a protective role for HO-1 in the METH-induced apoptotic death of H9c2 cardiomyocytes. Following pretreatment with antioxidants of Vitamin C and N-acetyl cysteine (NAC) to decrease the level of HO-1, significantly reduce in HO-1 levels were observed in METH treated H9c2 cells. However, METH-induced decrease in the cell survival rate could not be attenuated by apocynin, an inhibitor of NADPH oxidase (NOX) which plays a major role in ROS production in cardiomyocytes.
　　PI3K/Akt pathway was demonstrated to play protective roles in cardoimyocytes. Previous studies in our lab found that METH induced HO-1 expression via activation of PI3K/Akt pathway in SH-SY5Y neuronal cells. The results showed that Akt was significant trend for activated after 1-hour exposure to METH. As was expected , METH-induced apoptosis of H9c2 cardiomyocytes was inhibited by wortmannin, a PI3K inhibitor, suggesting the involvement of PI3K/Akt signaling pathway in METH-induced apoptosis. The Akt inhibitor, API-2, further confirmed the anti-apoptotic role of Akt after METH exposure. Moreover, the increase of ROS under METH treatment may activate Akt which was proven by the addition to Vitamin C and NAC pretreatment followed by METH stimulation. Last, HO-1 protein levels were also regulated by pretreatment of wortmannin. Overexpression of Akt significantly increased the HO-1 level accompanied by a decrease in the activation of caspase-3 in H9c2 cardiomyocytes.
　　In conclusion, these results suggest that METH-induced ROS resulted in apoptosis could be attenuated by induction of HO-1 via PI3K/Akt signaling in H9c2 cardiomyocytes. 

致謝........ i
目錄...... ii
中文摘要.............................................. …iii
Abstract ..... v
研究背景................................................ 1
一、甲基安非他命對心肌細胞的毒性作用 .................... 1
二、 活性氧化物 對心肌細 胞的影響 ........................ .. 2
三、細胞自我保護機制 3
四、 HO -1對心肌細胞的保護作用 對心肌細胞的保護作用 ... 4
五、 PI3K/AktPI3K/AktPI3K/Akt 對心肌細胞的調控功能及抗凋亡機制 對心肌細胞的調控功能及抗凋亡機制 .............. ..... 5
研究目的.......................................... 7
材料與方法.......................................... 8
研究結果...................................... 17
討論................................ 23
結論........... 33
參考文獻........................... 28
附圖........ 37

林欽昱：甲基安非他命透過活化PI3K/Akt路徑誘發第一型血基質氧化酶表現之研究。台北，陽明大學藥理學研究所，碩士論文，2007

Ajjimaporn A, Shavali S, Ebadi M, Govitrapong P (2008). Zinc rescues dopaminergic SK-N-SH cell lines from methamphetamine-induced toxicity. Brain Res Bull 77(6): 361-366.

Bishop A, Cashman NR (2003). Induced adaptive resistance to oxidative stress in the CNS: a discussion on possible mechanisms and their therapeutic potential. Curr Drug Metab 4(2): 171-184.

Burgering BM, Coffer PJ (1995). Protein kinase B (c-Akt) in phosphatidylinositol-3-OH kinase signal transduction. Nature 376(6541): 599-602.

Cosentino F, Sill JC, Katusic ZS (1994). Role of superoxide anions in the mediation of endothelium-dependent contractions. Hypertension 23(2): 229-235.

Cruickshank CC, Dyer KR (2009). A review of the clinical pharmacology of methamphetamine. Addiction 104(7): 1085-1099.

Das S, Fraga CG, Das DK (2006). Cardioprotective effect of resveratrol via HO-1 expression involves p38 map kinase and PI-3-kinase signaling, but does not involve NFkappaB. Free Radic Res 40(10): 1066-1075.

El Jamali A, Freund C, Rechner C, Scheidereit C, Dietz R, Bergmann MW (2004). Reoxygenation after severe hypoxia induces cardiomyocyte hypertrophy in vitro: activation of CREB downstream of GSK3beta. FASEB J 18(10): 1096-1098.

Fortuno A, San Jose G, Moreno MU, Beloqui O, Diez J, Zalba G (2006). Phagocytic NADPH oxidase overactivity underlies oxidative stress in metabolic syndrome. Diabetes 55(1): 209-215.

Franke TF, Kaplan DR, Cantley LC, Toker A (1997). Direct regulation of the Akt proto-oncogene product by phosphatidylinositol-3,4-bisphosphate. Science 275(5300): 665-668.

Fujio Y, Nguyen T, Wencker D, Kitsis RN, Walsh K (2000). Akt promotes survival of cardiomyocytes in vitro and protects against ischemia-reperfusion injury in mouse heart. Circulation 101(6): 660-667.

Fumagalli F, Gainetdinov RR, Wang YM, Valenzano KJ, Miller GW, Caron MG (1999). Increased methamphetamine neurotoxicity in heterozygous vesicular monoamine transporter 2 knock-out mice. J Neurosci 19(7): 2424-2431.

Funakoshi-Hirose I, Aki T, Unuma K, Funakoshi T, Noritake K, Uemura K (2013a). Distinct effects of methamphetamine on autophagy-lysosome and ubiquitin-proteasome systems in HL-1 cultured mouse atrial cardiomyocytes. Toxicology 312: 74-82.

Funakoshi-Hirose I, Unuma K, Funakoshi T, Noritake K, Uemura K (2013b). Distinct effects of methamphetamine on autophagy-lysosome and ubiquitin-proteasome systems in HL-1 cultured mouse atrial cardiomyocytes. Toxicology 312C: 74-82.

Halliwell B (2007). Biochemistry of oxidative stress. Biochem Soc Trans 35(Pt 5): 1147-1150.

Harrison DG (1997). Cellular and molecular mechanisms of endothelial cell dysfunction. J Clin Invest 100(9): 2153-2157.

He SY, Matoba R, Fujitani N, Sodesaki K, Onishi S (1996). Cardiac muscle lesions associated with chronic administration of methamphetamine in rats. Am J Forensic Med Pathol 17(2): 155-162.

Islam MN, Jesmine K, Kong Sn Molh A, Hasnan J (2009). Histopathological studies of cardiac lesions after long term administration of methamphetamine in high dosage--Part II. Leg Med (Tokyo) 11 Suppl 1: S147-150.

Klongpanichapak S, Phansuwan-Pujito P, Ebadi M, Govitrapong P (2008). Melatonin inhibits amphetamine-induced increase in alpha-synuclein and decrease in phosphorylated tyrosine hydroxylase in SK-N-SH cells. Neurosci Lett 436(3): 309-313.

Kruger AL, Peterson S, Turkseven S, Kaminski PM, Zhang FF, Quan S, et al. (2005). D-4F induces heme oxygenase-1 and extracellular superoxide dismutase, decreases endothelial cell sloughing, and improves vascular reactivity in rat model of diabetes. Circulation 111(23): 3126-3134.

L'Abbate A, Neglia D, Vecoli C, Novelli M, Ottaviano V, Baldi S, et al. (2007). Beneficial effect of heme oxygenase-1 expression on myocardial ischemia-reperfusion involves an increase in adiponectin in mildly diabetic rats. Am J Physiol Heart Circ Physiol 293(6): H3532-3541.

Lakshmi SV, Padmaja G, Kuppusamy P, Kutala VK (2009). Oxidative stress in cardiovascular disease. Indian J Biochem Biophys 46(6): 421-440.

LaVoie MJ, Card JP, Hastings TG (2004). Microglial activation precedes dopamine terminal pathology in methamphetamine-induced neurotoxicity. Exp Neurol 187(1): 47-57.

Lee YJ, Jeong HY, Kim YB, Lee YJ, Won SY, Shim JH, et al. (2012). Reactive oxygen species and PI3K/Akt signaling play key roles in the induction of Nrf2-driven heme oxygenase-1 expression in sulforaphane-treated human mesothelioma MSTO-211H cells. Food Chem Toxicol 50(2): 116-123.

Leusen JH, Verhoeven AJ, Roos D (1996). Interactions between the components of the human NADPH oxidase: intrigues in the phox family. J Lab Clin Med 128(5): 461-476.

Liu SX, Zhang Y, Wang YF, Li XC, Xiang MX, Bian C, et al. (2012). Upregulation of heme oxygenase-1 expression by hydroxysafflor yellow A conferring protection from anoxia/reoxygenation-induced apoptosis in H9c2 cardiomyocytes. Int J Cardiol 160(2): 95-101.

Looi YH, Grieve DJ, Siva A, Walker SJ, Anilkumar N, Cave AC, et al. (2008). Involvement of Nox2 NADPH oxidase in adverse cardiac remodeling after myocardial infarction. Hypertension 51(2): 319-325.

Lord KC, Shenouda SK, McIlwain E, Charalampidis D, Lucchesi PA, Varner KJ (2010). Oxidative stress contributes to methamphetamine-induced left ventricular dysfunction. Cardiovasc Res 87(1): 111-118.

Maines MD, Trakshel GM, Kutty RK (1986). Characterization of two constitutive forms of rat liver microsomal heme oxygenase. Only one molecular species of the enzyme is inducible. J Biol Chem 261(1): 411-419.

Maldonado C, Cea P, Adasme T, Collao A, Diaz-Araya G, Chiong M, et al. (2005). IGF-1 protects cardiac myocytes from hyperosmotic stress-induced apoptosis via CREB. Biochem Biophys Res Commun 336(4): 1112-1118.

Manning BD, Cantley LC (2007). AKT/PKB signaling: navigating downstream. Cell 129(7): 1261-1274.

Matsui T, Li L, del Monte F, Fukui Y, Franke TF, Hajjar RJ, et al. (1999). Adenoviral gene transfer of activated phosphatidylinositol 3'-kinase and Akt inhibits apoptosis of hypoxic cardiomyocytes in vitro. Circulation 100(23): 2373-2379.

Meier F, Schittek B, Busch S, Garbe C, Smalley K, Satyamoorthy K, et al. (2005). The RAS/RAF/MEK/ERK and PI3K/AKT signaling pathways present molecular targets for the effective treatment of advanced melanoma. Front Biosci 10: 2986-3001.

Milroy CM, Clark JC, Forrest AR (1996). Pathology of deaths associated with "ecstasy" and "eve" misuse. J Clin Pathol 49(2): 149-153.

Mitra A, Ray A, Datta R, Sengupta S, Sarkar S (2014). Cardioprotective Role of P38 MAPK During Myocardial Infarction Via Parallel Activation of alpha-Crystallin B and Nrf2. J Cell Physiol 229(9): 1272-1282.

Mullonkal CJ, Toledo-Pereyra LH (2007). Akt in ischemia and reperfusion. J Invest Surg 20(3): 195-203.

Pachori AS, Smith A, McDonald P, Zhang L, Dzau VJ, Melo LG (2007). Heme-oxygenase-1-induced protection against hypoxia/reoxygenation is dependent on biliverdin reductase and its interaction with PI3K/Akt pathway. J Mol Cell Cardiol 43(5): 580-592.

Padiya R, Chowdhury D, Borkar R, Srinivas R, Pal Bhadra M, Banerjee SK (2014). Garlic Attenuates Cardiac Oxidative Stress via Activation of PI3K/AKT/Nrf2-Keap1 Pathway in Fructose-Fed Diabetic Rat. PLoS One 9(5): e94228.

Qin F, Simeone M, Patel R (2007). Inhibition of NADPH oxidase reduces myocardial oxidative stress and apoptosis and improves cardiac function in heart failure after myocardial infarction. Free Radic Biol Med 43(2): 271-281.

Rahmi Garcia RM, Rezende PC, Hueb W (2014). Impact of hypoglycemic agents on myocardial ischemic preconditioning. World J Diabetes 5(3): 258-266.

Riddle EL, Fleckenstein AE, Hanson GR (2006). Mechanisms of methamphetamine-induced dopaminergic neurotoxicity. AAPS J 8(2): E413-418.

Robinson SR, Dang TN, Dringen R, Bishop GM (2009). Hemin toxicity: a preventable source of brain damage following hemorrhagic stroke. Redox Rep 14(6): 228-235.

Ryter SW, Otterbein LE, Morse D, Choi AM (2002). Heme oxygenase/carbon monoxide signaling pathways: regulation and functional significance. Mol Cell Biochem 234-235(1-2): 249-263.

Sarbassov DD, Guertin DA, Ali SM, Sabatini DM (2005). Phosphorylation and regulation of Akt/PKB by the rictor-mTOR complex. Science 307(5712): 1098-1101.

Shah A, Kumar S, Simon SD, Singh DP, Kumar A (2013). HIV gp120- and methamphetamine-mediated oxidative stress induces astrocyte apoptosis via cytochrome P450 2E1. Cell Death Dis 4: e850.

Tenhunen R, Marver HS, Schmid R (1969). Microsomal heme oxygenase. Characterization of the enzyme. J Biol Chem 244(23): 6388-6394.

Tian C, Murrin LC, Zheng JC (2009). Mitochondrial fragmentation is involved in methamphetamine-induced cell death in rat hippocampal neural progenitor cells. PLoS One 4(5): e5546.

Touyz RM, Briones AM, Sedeek M, Burger D, Montezano AC (2011). NOX isoforms and reactive oxygen species in vascular health. Mol Interv 11(1): 27-35.

Tsai HY, Huang PH, Lin FY, Chen JS, Lin SJ, Chen JW (2013). Ginkgo biloba extract reduces high-glucose-induced endothelial reactive oxygen species generation and cell adhesion molecule expression by enhancing HO-1 expression via Akt/eNOS and p38 MAP kinase pathways. Eur J Pharm Sci 48(4-5): 803-811.

Wang G, Hamid T, Keith RJ, Zhou G, Partridge CR, Xiang X, et al. (2010a). Cardioprotective and antiapoptotic effects of heme oxygenase-1 in the failing heart. Circulation 121(17): 1912-1925.

Wang J, Yang H, Hu X, Fu W, Xie J, Zhou X, et al. (2013). Dobutamine-mediated heme oxygenase-1 induction via PI3K and p38 MAPK inhibits high mobility group box 1 protein release and attenuates rat myocardial ischemia/reperfusion injury in vivo. J Surg Res 183(2): 509-516.

Wang L, Chen Y, Sternberg P, Cai J (2008). Essential roles of the PI3 kinase/Akt pathway in regulating Nrf2-dependent antioxidant functions in the RPE. Invest Ophthalmol Vis Sci 49(4): 1671-1678.

Wang M, Zhang J, Walker SJ, Dworakowski R, Lakatta EG, Shah AM (2010b). Involvement of NADPH oxidase in age-associated cardiac remodeling. J Mol Cell Cardiol 48(4): 765-772.

Wang X, McCullough KD, Franke TF, Holbrook NJ (2000). Epidermal growth factor receptor-dependent Akt activation by oxidative stress enhances cell survival. J Biol Chem 275(19): 14624-14631.

Wang X, Yuan B, Dong W, Yang B, Yang Y, Lin X, et al. (2014). Humid heat exposure induced oxidative stress and apoptosis in cardiomyocytes through the angiotensin II signaling pathway. Heart Vessels.

Wijetunga M, Seto T, Lindsay J, Schatz I (2003). Crystal methamphetamine-associated cardiomyopathy: tip of the iceberg? J Toxicol Clin Toxicol 41(7): 981-986.

Wolkoff DA (1997). Methamphetamine abuse: an overview for health care professionals. Hawaii Med J 56(2): 34-36, 44.

Wu CW, Ping YH, Yen JC, Chang CY, Wang SF, Yeh CL, et al. (2007). Enhanced oxidative stress and aberrant mitochondrial biogenesis in human neuroblastoma SH-SY5Y cells during methamphetamine induced apoptosis. Toxicol Appl Pharmacol 220(3): 243-251.

Wu ML, Ho YC, Yet SF (2011). A central role of heme oxygenase-1 in cardiovascular protection. Antioxid Redox Signal 15(7): 1835-1846.

Xu J, Qian J, Xie X, Lin L, Zou Y, Fu M, et al. (2012). High Density Lipoprotein Protects Mesenchymal Stem Cells from Oxidative Stress-Induced Apoptosis via Activation of the PI3K/Akt Pathway and Suppression of Reactive Oxygen Species. Int J Mol Sci 13(12): 17104-17120.

Yoshida T, Kikuchi G (1978). Purification and properties of heme oxygenase from pig spleen microsomes. J Biol Chem 253(12): 4224-4229.

Zalba G, San Jose G, Moreno MU, Fortuno MA, Fortuno A, Beaumont FJ, et al. (2001). Oxidative stress in arterial hypertension: role of NAD(P)H oxidase. Hypertension 38(6): 1395-1399.

Zhang M, Shah AM (2014). ROS signalling between endothelial cells and cardiac cells. Cardiovasc Res 102(2): 249-257.