中樞神經系統受傷後，膠質細胞活化釋放前發炎因子，星狀膠質細胞在活化後細胞增生，同時還伴隨細胞骨架蛋白大量表現，造成細胞肥大 (hypertrophy) ，進而形成膠質疤 (glial scar) 。目前發現在許多病理狀況下，例如大腦缺血、kainic acid所誘導的癲癇、頭部外傷等，會造成鋅離子從神經末稍大量的釋放，高量的鋅離子會增加活性氧分子，還會造成細胞內醣分解作用 (glycolysis) 下降，導致神經細胞死亡。此外PGE2也是受傷後大量產生的發炎分子之一，在中樞神經系統中PGE2的主要來源是小膠質細胞和星狀膠質細胞，但由於PGE2對星狀膠質細胞活化的影響不甚清楚。這些中樞神經系統受損後大量產生的分子對星狀膠質細胞的影響並不十分明確，所以本篇論文分為兩大部分探討PGE2對星狀膠質細胞的影響： (一) 以ZnCl2及PGE2刺激星狀膠質細胞，結果發現PGE2 後處理（posttreatment） 48小時對細胞產生加成性傷害，並且是透過降低細胞ATP的生成導致細胞能量供應不足而死亡。 (二) 使用前發炎因子 (TNFa+IFNg) 和PGE2共同處理星狀膠質細胞，模擬中樞神經受傷後的微環境，探討誘導型一氧化氮合成酶 (iNOS) 的表現，推測PGE2可能透過EP2 receptor增加iNOS的表現及NO的生成。

　　Astrocytes, the most abundant cells in the central nervous system (CNS), serve as supporting cells for neurons during CNS development and in the adult CNS. These cells can be reactivated by CNS injury and become immune cells via producing pro-inflammatory mediators such as TNF-, IL-1, iNOS, prostaglandins and free radicals. Astrocyte activation causes astrocytic hypertrophy with the high production of astrocytic skeleton proteins, glial fibrillary acidic protein (GFAP) and vimentin, causing the formation of glial scar that hinders axonal regeneration. It has been accepted that excess zinc can be released from presynpatic axonal terminal in the injured CNS, and induce neuroexcitotoxicity. In addition, zinc has been known to disrupt neuronal mitochondronial dysfunction. Protaglandin E2 (PGE2), one of protaglandins produced by reactivated astrocytes has been reported to play a contradictory role in neuronal survival. However, the effect of PGE2 on astrocytic function is unclear. Since PGE2 is involved in diverse biological activity in the normal and injured CNS, the E type PG receptor containing four subtypes EP1, EP2, EP3 and EP4, has been intensively studied. After activation, the EP1 and EP3 receptor increase the intracellular concentration of calcium. EP2 and EP4 activate adenylyl cyclase (AC) through the stimulatory G protein, and then increase intracellular cAMP to trigger the action of protein kinase A (PKA). It has been concluded that distinct EP receptor subtypes may be involved in the different responsiveness of CNS cells to PGE2. Thus, we attempted to understand whether PGE2 plays the regulatory role in astocytic function after CNS injury. We utilized an in vitro model of astrocytes treated with zinc, and examined whether PGE2 exerts detrimental or protective role in astrocyte survival. Our results indicated that PGE2 posttreatment for 48 hr induced synergetic cell death of zinc-treated astrocytes, which may be due to reduced production of ATP by PGE2. Furthermore, treatment of astrocyte with TNF-a and IFN-g were used as the model of astrocyte activation to study the involvement of PGE2 receptors in PGE2 regulation. By examining the production of iNOS that is a marker for astrocyte activation, we found that an agonist of EP2 receptor, increased the production of iNOS and NO in astrocytes treated with TNF-a and IFN-g. Accordingly, we suggest that PGE2 mediate astrocyte activation mainly through EP2-triggered intracellular transduction pathway that is known to link with cAMP-signaling.

Albina JE and Reichner JS. (1998) Role of nitric oxide in mediation of macrophage cytotoxicity and apoptosis. Cancer Metastasis Rev 17:39-53.

Alirezaei M, Mordelet E, Rouach N, Nairn AC, Glowinski J and Premont J. (2002) Zinc-induced inhibition of protein synthesis and reduction of connexin-43 expression and intercellular communication in mouse cortical astrocytes. Eur J Neurosci 16:1037-1044.

Aloisi F, Penna G, Cerase J, Menendez Iglesias B and Adorini L. (1997) IL-12 production by central nervous system microglia is inhibited by astrocytes. J Immunol 159:1604-1612.

Azmitia EC and Whitaker PM. (1983) Formation of a glial scar following microinjection of fetal neurons into the hippocampus or midbrain of the adult rat: an immunocytochemical study. Neurosci Lett 38:145-150.

Bennett MV, Contreras JE, Bukauskas FF and Saez JC. (2003) New roles for astrocytes: gap junction hemichannels have something to communicate. Trends Neurosci 26:610-617.

Bezzi P, Carmignoto G, Pasti L, Vesce S, Rossi D, Rizzini BL, Pozzan T and Volterra A. (1998) Prostaglandins stimulate calcium-dependent glutamate release in astrocytes. Nature 391:281-285.

Bhat NR, Feinstein DL, Shen Q and Bhat AN. (2002) p38 MAPK-mediated transcriptional activation of inducible nitric-oxide synthase in glial cells. Roles of nuclear factors, nuclear factor kappa B, cAMP response element-binding protein, CCAAT/enhancer-binding protein-beta and activating transcription factor-2. J Biol Chem 277:29584-29592.

Boucher JL, Moali C and Tenu JP. (1999) Nitric oxide biosynthesis, nitric oxide synthase inhibitors and arginase competition for L-arginine utilization. Cell Mol Life Sci 55:1015-1028.

Broillet MC. (1999) S-nitrosylation of proteins. Cell Mol Life Sci 55:1036-1042.

Brosnan CF, Battistini L, Raine CS, Dickson DW, Casadevall A and Lee SC. (1994) Reactive nitrogen intermediates in human neuropathology: an overview. Dev Neurosci 16:152-161.

Bune AJ, Shergill JK, Cammack R and Cook HT. (1995) L-arginine depletion by arginase reduces nitric oxide production in endotoxic shock: an electron paramagnetic resonance study. FEBS Lett 366:127-130.

Cheng C and Reynolds IJ. (1998) Calcium-sensitive fluorescent dyes can report increases in intracellular free zinc concentration in cultured forebrain neurons. J Neurochem 71:2401-2410.

Chu SC, Marks-Konczalik J, Wu HP, Banks TC and Moss J. (1998) Analysis of the cytokine-stimulated human inducible nitric oxide synthase (iNOS) gene: characterization of differences between human and mouse iNOS promoters. Biochem Biophys Res Commun 248:871-878.

Da Silva J, Pierrat B, Mary JL and Lesslauer W. (1997) Blockade of p38 mitogen-activated protein kinase pathway inhibits inducible nitric-oxide synthase expression in mouse astrocytes. J Biol Chem 272:28373-28380.

Darnell JE, Jr., Kerr IM and Stark GR. (1994) Jak-STAT pathways and transcriptional activation in response to IFNs and other extracellular signaling proteins. Science 264:1415-1421.

Dawson VL and Dawson TM. (1998) Nitric oxide in neurodegeneration. Prog Brain Res 118:215-229.

Dineley KE, Scanlon JM, Kress GJ, Stout AK and Reynolds IJ. (2000) Astrocytes are more resistant than neurons to the cytotoxic effects of increased [Zn(2+)](i). Neurobiol Dis 7:310-320.

Eddleston M and Mucke L. (1993) Molecular profile of reactive astrocytes--implications for their role in neurologic disease. Neuroscience 54:15-36.

Feinstein DL, Galea E, Roberts S, Berquist H, Wang H and Reis DJ. (1994) Induction of nitric oxide synthase in rat C6 glioma cells. J Neurochem 62:315-321.

Gotoh T and Mori M. (1999) Arginase II downregulates nitric oxide (NO) production and prevents NO-mediated apoptosis in murine macrophage-derived RAW 264.7 cells. J Cell Biol 144:427-434.

Gross SS and Wolin MS. (1995) Nitric oxide: pathophysiological mechanisms. Annu Rev Physiol 57:737-769.

Harrison BC and Mobley PL. (1991) Phorbol myristate acetate and 8-bromo-cyclic AMP-induced phosphorylation of glial fibrillary acidic protein and vimentin in astrocytes: comparison of phosphorylation sites. J Neurochem 56:1723-1730.

Hayaishi O, Matsumura H and Urade Y. (1993) Prostaglandin D synthase is the key enzyme in the promotion of physiological sleep. J Lipid Mediat 6:429-431.

Hooper DC, Scott GS, Zborek A, Mikheeva T, Kean RB, Koprowski H and Spitsin SV. (2000) Uric acid, a peroxynitrite scavenger, inhibits CNS inflammation, blood-CNS barrier permeability changes and tissue damage in a mouse model of multiple sclerosis. FASEB J 14:691-698.

Horner PJ and Palmer TD. (2003) New roles for astrocytes: the nightlife of an 'astrocyte'. La vida loca! Trends Neurosci 26:597-603.

Ignarro LJ. (1996) Physiology and pathophysiology of nitric oxide. Kidney Int Suppl 55:S2-5.

Jana M anderson JA, Saha RN, Liu X and Pahan K. (2005) Regulation of inducible nitric oxide synthase in proinflammatory cytokine-stimulated human primary astrocytes. Free Radic Biol Med 38:655-664.

Katsuki H and Okuda S. (1995) Arachidonic acid as a neurotoxic and neurotrophic substance. Prog Neurobiol 46:607-636.

Kennedy I, Coleman RA, Humphrey PP, Levy GP and Lumley P. (1982) Studies on the characterisation of prostanoid receptors: a proposed classification. Prostaglandins 24:667-689.

Kim EJ, Kwon KJ, Park JY, Lee SH, Moon CH and Baik EJ. (2002) Neuroprotective effects of prostaglandin E2 or cAMP against microglial and neuronal free radical mediated toxicity associated with inflammation. J Neurosci Res 70:97-107.

Kim EY, Koh JY, Kim YH, Sohn S, Joe E and Gwag BJ. (1999) Zn2+ entry produces oxidative neuronal necrosis in cortical cell cultures. Eur J Neurosci 11:327-334.

Knowles RG and Moncada S. (1994) Nitric oxide synthases in mammals. Biochem J 298 (Pt 2):249-258.

Koh JY and Choi DW. (1994) Zinc toxicity on cultured cortical neurons: involvement of N-methyl-D-aspartate receptors. Neuroscience 60:1049-1057.

Kress Y, Gaskin F, Brosnan CF and Levine S. (1981) Effects of zinc on the cytoskeletal proteins in the central nervous system of the rat. Brain Res 220:139-149.

Kristof AS, Marks-Konczalik J and Moss J. (2001) Mitogen-activated protein kinases mediate activator protein-1-dependent human inducible nitric-oxide synthase promoter activation. J Biol Chem 276:8445-8452.

Kroncke KD, Fehsel K and Kolb-Bachofen V. (1997) Nitric oxide: cytotoxicity versus cytoprotection--how, why, when and where? Nitric Oxide 1:107-120.

Lee EO, Shin YJ and Chong YH. (2004) Mechanisms involved in prostaglandin E2-mediated neuroprotection against TNF-alpha: possible involvement of multiple signal transduction and beta-catenin/T-cell factor. J Neuroimmunol 155:21-31.

Linn SC, Morelli PJ, Edry I, Cottongim SE, Szabo C and Salzman AL. (1997) Transcriptional regulation of human inducible nitric oxide synthase gene in an intestinal epithelial cell line. Am J Physiol 272:G1499-1508.

Lipton SA. (1999) Neuronal protection and destruction by NO. Cell Death Differ 6:943-951.

Liu L and Stamler JS. (1999) NO: an inhibitor of cell death. Cell Death Differ 6:937-942.

Liu X, Jana M, Dasgupta S, Koka S, He J, Wood C and Pahan K. (2002) Human immunodeficiency virus type 1 (HIV-1) tat induces nitric-oxide synthase in human astroglia. J Biol Chem 277:39312-39319.

Lowenstein CJ, Dinerman JL and Snyder SH. (1994) Nitric oxide: a physiologic messenger. Ann Intern Med 120:227-237.

Marin P, Israel M, Glowinski J and Premont J. (2000) Routes of zinc entry in mouse cortical neurons: role in zinc-induced neurotoxicity. Eur J Neurosci 12:8-18.

Marrif H and Juurlink BH. (1999) Astrocytes respond to hypoxia by increasing glycolytic capacity. J Neurosci Res 57:255-260.

Merrill JE, Ignarro LJ, Sherman MP, Melinek J and Lane TE. (1993) Microglial cell cytotoxicity of oligodendrocytes is mediated through nitric oxide. J Immunol 151:2132-2141.

Miller RH, Abney ER, David S, Ffrench-Constant C, Lindsay R, Patel R, Stone J and Raff MC. (1986) Is reactive gliosis a property of a distinct subpopulation of astrocytes? J Neurosci 6:22-29.

Minghetti L, Nicolini A, Polazzi E, Creminon C, Maclouf J and Levi G. (1997) Prostaglandin E2 downregulates inducible nitric oxide synthase expression in microglia by increasing cAMP levels. Adv Exp Med Biol 433:181-184.

Mitrovic B, Ignarro LJ, Montestruque S, Smoll A and Merrill JE. (1994) Nitric oxide as a potential pathological mechanism in demyelination: its differential effects on primary glial cells in vitro. Neuroscience 61:575-585.

Nathan C. (1995) Natural resistance and nitric oxide. Cell 82:873-876.

Newman EA. (2003) New roles for astrocytes: regulation of synaptic transmission. Trends Neurosci 26:536-542.

Nishigaki N, Negishi M and Ichikawa A. (1996) Two Gs-coupled prostaglandin E receptor subtypes, EP2 and EP4, differ in desensitization and sensitivity to the metabolic inactivation of the agonist. Mol Pharmacol 50:1031-1037.

Noh KM and Koh JY. (2000) Induction and activation by zinc of NADPH oxidase in cultured cortical neurons and astrocytes. J Neurosci 20:RC111.

Okuda S, Saito H and Katsuki H. (1994) Arachidonic acid: toxic and trophic effects on cultured hippocampal neurons. Neuroscience 63:691-699.

Pahan K, Sheikh FG, Namboodiri AM and Singh I. (1997) Lovastatin and phenylacetate inhibit the induction of nitric oxide synthase and cytokines in rat primary astrocytes, microglia and macrophages. J Clin Invest 100:2671-2679.

Pahan K, Sheikh FG, Namboodiri AM and Singh I. (1998) Inhibitors of protein phosphatase 1 and 2A differentially regulate the expression of inducible nitric-oxide synthase in rat astrocytes and macrophages. J Biol Chem 273:12219-12226.

Pahan K, Namboodiri AM, Sheikh FG, Smith BT and Singh I. (1997) Increasing cAMP attenuates induction of inducible nitric-oxide synthase in rat primary astrocytes. J Biol Chem 272:7786-7791.

Pahan K, Liu X, McKinney MJ, Wood C, Sheikh FG and Raymond JR. (2000) Expression of a dominant-negative mutant of p21(ras) inhibits induction of nitric oxide synthase and activation of nuclear factor-kappaB in primary astrocytes. J Neurochem 74:2288-2295.

Pahan K, Sheikh FG, Liu X, Hilger S, McKinney M and Petro TM. (2001) Induction of nitric-oxide synthase and activation of NF-kappaB by interleukin-12 p40 in microglial cells. J Biol Chem 276:7899-7905.

Pahan K, Jana M, Liu X, Taylor BS, Wood C and Fischer SM. (2002) Gemfibrozil, a lipid-lowering drug, inhibits the induction of nitric-oxide synthase in human astrocytes. J Biol Chem 277:45984-45991.

Petrova TV, Akama KT and Van Eldik LJ. (1999) Selective modulation of BV-2 microglial activation by prostaglandin E(2). Differential effects on endotoxin-stimulated cytokine induction. J Biol Chem 274:28823-28827.

Pitchford S and Levine JD. (1991) Prostaglandins sensitize nociceptors in cell culture. Neurosci Lett 132:105-108.

Raff MC, Abney ER and Miller RH. (1984) Two glial cell lineages diverge prenatally in rat optic nerve. Dev Biol 106:53-60.

Raff MC, Abney ER, Cohen J, Lindsay R and Noble M. (1983) Two types of astrocytes in cultures of developing rat white matter: differences in morphology, surface gangliosides and growth characteristics. J Neurosci 3:1289-1300.

Ranjan P, Sodhi A and Singh SM. (1998) Murine peritoneal macrophages treated with cisplatin and interferon-gamma undergo NO-mediated apoptosis via activation of an endonuclease. Anticancer Drugs 9:333-341.

Ransom B, Behar T and Nedergaard M. (2003) New roles for astrocytes (stars at last). Trends Neurosci 26:520-522.

Reichenbach A, Siegel A, Senitz D and Smith TG, Jr. (1992) A comparative fractal analysis of various mammalian astroglial cell types. Neuroimage 1:69-77.

Rothwell NJ. (1992) Eicosanoids, thermogenesis and thermoregulation. Prostaglandins Leukot Essent Fatty Acids 46:1-7.

Sawada M, Suzumura A, Ohno K and Marunouchi T. (1993) Regulation of astrocyte proliferation by prostaglandin E2 and the alpha subtype of protein kinase C. Brain Res 613:67-73.

Segovia J, Lawless GM, Tillakaratne NJ, Brenner M and Tobin AJ. (1994) Cyclic AMP decreases the expression of a neuronal marker (GAD67) and increases the expression of an astroglial marker (GFAP) in C6 cells. J Neurochem 63:1218-1225.

Sensi SL, Canzoniero LM, Yu SP, Ying HS, Koh JY, Kerchner GA and Choi DW. (1997) Measurement of intracellular free zinc in living cortical neurons: routes of entry. J Neurosci 17:9554-9564.

Sheline CT, Behrens MM and Choi DW. (2000) Zinc-induced cortical neuronal death: contribution of energy failure attributable to loss of NAD(+) and inhibition of glycolysis. J Neurosci 20:3139-3146.

Sheline CT, Ying HS, Ling CS, Canzoniero LM and Choi DW. (2002) Depolarization-induced 65zinc influx into cultured cortical neurons. Neurobiol Dis 10:41-53.

Slezak M and Pfrieger FW. (2003) New roles for astrocytes: regulation of CNS synaptogenesis. Trends Neurosci 26:531-535.

Spitsin SV, Koprowski H and Michaels FH. (1996) Characterization and functional analysis of the human inducible nitric oxide synthase gene promoter. Mol Med 2:226-235.

Sugimoto Y, Negishi M, Hayashi Y, Namba T, Honda A, Watabe A, Hirata M, Narumiya S and Ichikawa A. (1993) Two isoforms of the EP3 receptor with different carboxyl-terminal domains. Identical ligand binding properties and different coupling properties with Gi proteins. J Biol Chem 268:2712-2718.

Thery C, Dobbertin A and Mallat M. (1994) Downregulation of in vitro neurotoxicity of brain macrophages by prostaglandin E2 and a beta-adrenergic agonist. Glia 11:383-386.

Toyomoto M, Ohta M, Okumura K, Yano H, Matsumoto K, Inoue S, Hayashi K and Ikeda K. (2004) Prostaglandins are powerful inducers of NGF and BDNF production in mouse astrocyte cultures. FEBS Lett 562:211-215.

Unger JW. (1998) Glial reaction in aging and Alzheimer's disease. Microsc Res Tech 43:24-28.

Willert K and Nusse R. (1998) Beta-catenin: a key mediator of Wnt signaling. Curr Opin Genet Dev 8:95-102.

Wise H and Jones RL. (1994) Characterization of prostanoid receptors on rat neutrophils. Br J Pharmacol 113:581-587.

Xie QW, Kashiwabara Y and Nathan C. (1994) Role of transcription factor NF-kappa B/Rel in induction of nitric oxide synthase. J Biol Chem 269:4705-4708.