巴金森氏症是常見的漸進性神經退化性疾病，許多相關研究認為氧化壓力在造成巴金森氏症上，扮演極重要的角色。MPTP（1-methyl- 4-phenyl-1,2,3,6-tetrahydropyridine）是一種選擇性破壞多巴胺神經細胞的神經毒素，目前被廣泛應用於建立巴金森氏症實驗動物模式。Resveratrol是一種存在於紅葡萄酒的植物性動情素，具有神經保護的效果。本實驗是採用MPTP細胞模式來探討MPTP對細胞傷害及處理resveratrol的保護效果。實驗材料是以retinoic acid誘導SK-N-SH神經瘤細胞分化之SK-N-SH神經細胞。實驗結果發現在10 mM MPTP 處理SK-N-SH細胞24小時只有70%的存活率；MPTP對SK-N-SH細胞的氧化性傷害並非呈現在提高細胞內的ROS量，細胞亦不會有提高HSP70量的反應。以40~50 nM resveratrol處理細胞3小時會顯著的降低細胞的存活率，低於30 nM resveratrol處理對細胞是沒有毒性的。如以10~30 nM resveratrol先處理細胞3小時再共同處理10 mM MPTP 24小時，則有使細胞的存活率提高約為對照組的1.08倍的效果。resveratrol 處理細胞27小時組比其對應的處理3小時組，細胞內ROS量都有下降的趨勢，以10~40 nM resveratrol先處理細胞3小時再共同處理10 mM MPTP 24小時，則細胞的ROS量均顯著的下降。以20nM resveratrol處理SK-N-SH細胞3小時或27小時，均會讓細胞HSP70量上升，經AG490（JAK抑制劑）處理證實細胞是藉由JAK/STAT pathway 的訊息傳遞路徑誘導HSP70產生。根據以上結果推測resveratrol先處理對細胞是一種壓力，低劑量時細胞HSP70增加，可以用以抵抗的MPTP的傷害，而細胞內HSP70的產生並不一定藉由細胞內ROS量升高引起的。高濃度40~50 nM resveratrol的處理，對細胞造成的壓力大，所以會顯著的傷害細胞存活、上升ROS量；如又同時與MPTP共同處理細胞就會加成的傷害細胞及增加ROS的產生量。本研究探討resveratrol對神經細胞保護效果的劑量及機制，祈能在人類巴金森氏症的預防及治療運用上能有所助益。

Large numbers of experimental evidences supported that oxidative stress played an important role in the mediation of nerve cell death in Parkinson’s disease (PD), a common progressively neurodegenerative disease. 1-Methyl-4-phenyl-1,2,3,6-tetra- hydropyridine (MPTP)-induced neurotoxicity is the most common model to study the pathogenesis of PD. Resveratrol, a phytoestrogen in red wine extracts, had been shown to exhibit neuroprotective effects in several experimental models. In this study, the protective effects of resveratrol on MPTP-induced cell death in differentiated SK-N-SH neuronal cell were investigated. The data indicated that cells treated with 10 mM MPTP for 24 h, the viability was near 70%, MPTP damage on SK-N-SH cells was not by the process of higher ROS leveling and the MPTP-treated cells did not exhibited the response to rise their cellular HSP70 proteins. SK-N-SH cells treated with 40~50 nM resveratrol for 3 h, their cell viability were significantly decreased. Concentrations below 30 nM resveratrol for 24h treatment were not exhibited cytotoxicity. Cells pretreated with resveratrol at 10~30 nM for 3 h and then cotreated with 10 mM MPTP cells for 24 h, the viabilities rise up to near 1.08 fold. The ROS quantities in resveratrol/27 h-treated cells were lower than that in resveratrol/3 h-treated cells respectively. In cells pretreated with 10~40 nM resveratrol for 3 h and then cotreated with 10 mM MPTP for 3 h, the ROS quantities were decreased significantly. The quantities of HSP 70 proteins were increased in SK-N-SH cells treated with 20 nM resveratrol for 3 or 27 h. The induction of HSP 70 proteins through signal transduction of JAK/STAT pathway were proofed by AG490 inhibition.
Based on above data, we speculated that resveratrol pretreatment was a kind of cell stress. In cells treated at lower concentrations, resveratrol enhanced HSP 70 production and increased in resistance toward MPTP damage and it might indicate that cellular HSP 70 production was not only mediated through rising ROS quantities. SK-N-SH cells pretreated in 40~50 nM resveratrol resulted in sever cell damage and higher ROS production, and additional effect of MPTP co-treatment to enhance cell damage and ROS production. This study may provide some information of dosage effect and protection mechanism of resveratrol for prevention and curing of PD.

賴茲漢、賴業超。1994。食品科技辭典。富林出版社。台中。
顏文妍。2004。肌肽之生體外免疫調節活性評估。國立海洋大學食品科學系碩士論文。基隆。
Adams JD, Chang ML, Klaidman L (2001) Parkinson’s disease-redox mechanisms. Curr. Med. Chem. 8: 809-814.
Alarcon de la Lastra C, Villegas I (2005) Resveratrol as an anti- inflammatory and anti-aging agent: Mechanisms and clinical implications. Mol. Nutr. Food Res. 49: 405-430.
Ames BN, Shigenaga MK, Hagen TM (1993) Oxidants, antioxidants, and the degenerative diseases of aging. Proc. Natl. Acad. Sci. USA, 90: 7915-7922.
Barbosa ER, Limongi JCP, Cummings JL (1997) Parkinson’s disease. Psych. Clin. North. Am. 20: 769-790.
Becker J, Craig EA (1994) Heat-shock proteins as molecular chaperones. Eur. J. Biochem. 219: 11-23.
Betarbet R, Sherer TB, MacKenzie G, Garcia-Osuna M, Panov AV, Greenamyre JT (2000) Chronic systemic pesticide exposure reproduces features of Parkinson's disease. Nat. Neurosci. 3: 1301-1306.
Brown IR (1994) Induction of heat shock genes in mammalian brain by hyperthermia and tissue injury. IN: Heat Shock Protein in the Nervous System (Mayer J. and Brown I. eds), pp.31-53. Academic press，New York.
Bukau B, Horwich AL (1998) The hsp70 and hsp60 chaperone machines. Cell 92: 351-366.
Buttke TM, Sandstrom PA (1994) Oxidative stress as a mediator of apoptosis. Immunol. Today 5: 7-10.
Calabrese V, Bates TE, Stella AM (2000a) NO synthase and NO- dependent signal pathways in brain aging and neurodegenerative disorders: the role of oxidant/antioxidant balance. Neurochem. Res. 25: 1315-1341.
Calabrese V, Copani A, Testa D, Ravagna A, Spadaro F, Tendi E, et al, (2000b) Nitric oxide synthase induction in astroglial cell cultures: effect on heat shock protein 70 synthesis and oxidant/antioxidant balance. J. Neurosci. Res. 60: 613-622.
Calabrese V, Scapagnini G, Colombrita C, Lavagna A, Pennini G, Giuffrida Stella AM, et al. (2003) Redox regulation of heat shock protein expression in aging and neurodegenerative disorders associated with oxidative stress: a nutritional approach. Amino Acids 25: 144-437.
Calabrese V, Testa G, Ravagna A, Bates TE, Stella AM (2000c) HSP70 induction in the brain following ethanol administration in the rat: regulation by glutathione redox state. Biochem. Biophys. Res. Commun. 269: 397-400.
Chambery D, de Galle´B, Babajko S (1998) Retinoic acid stimulates IGF binding protein (IGFBP)-6 and depresses IGFBP-2 and IGFBP-4 in SK-N-SH human neuroblastoma cells. J. Endocr. 159: 227-232.
Chan WH, Chang YJ (2006) Dosage effect of resveratrol on ethanol- induced cell death in the human K562 cell line. Toxicol. Lett. 161: 1-9.
Chiou WF, Lee WS, Yeh PH (2006) Tetrandrine selectively protects against amyloid-beta protein - but not against MPTP-induced cytotoxicity in SK-N-SH neuroblastoma cells. Planta Med. 72(14): 1300-1304.
Clement MV, Hirpara JL, Chawdhury SH, Pervaiz S (1998) Chemopreventive agent resveratrol, a natural product derived from grapes, triggers CD95 signaling-dependent apoptosis in human tumor cells. Blood 92: 996-1002.
Di-Monte DA, Lavasani M, Manning-Bog AB (2002) Environmental factors in Parkinson’s disease. Neurotoxicology 23: 487–502.
Edwards SW (1995) Cell signaling by integrins and immunoglobulin receptors in primed neutrophils. Trends Biochem. Sci. 20: 362-367.
Ellis RJ, van der Vies SM (1991) Molecular chaperones. Annu. Rev. Biochem. 60: 321-347.
Fall CP, Bennett Jp, Jr. (1999) Characterization and time course of MPP+-induced apoptosis in human SH-SY5Y neuroblastoma cells. J. Neurosci. Res. 55: 620-628.
Feige U, Polla BS (1994) Hsp70-a multi-gene, multi-structure, multi-function family with potential clinical application. Experientia 50: 979-86.
Floyd RA (1999) Antioxidants, oxidative stress, and degenerative neurological disorders. Proc. Soc. Exp. Biol. Med. 222:236-245.
Fremont L (2000) Biological effects of resveratrol. Life Sci. 66: 663-673.
Fremont L, Belguendouz L, Delpal S (1999) Antioxidant activity of resveratrol and alcohol-free wine polyphenols related to LDL oxidation and polyunsaturated fatty acids. Life Sci. 64: 2511-2521.
Fulda S, Debatin KM (2004) Sensitization for anticancer drug-induced apoptosis by the chemopreventive agent resveratrol. Oncogene 23: 6702-6711.
Gamaley IA, Klyubin IV (1999) Roles of reactive oxygen species: signaling and regulation of cellular functions. Int. Rev. Cytol. 188: 203-255.
Gao HM, Jiang J, Wilson B, Zhang W, Hong JS, Liu B (2002) Microglial activation-mediated delayed and progressive degeneration of rat nigral dopaminergic neurons: relevance to Parkinson's disease. J. Neurochem. 81: 1285-1297.
Garnier P, Demougeot C, Bertrand N, Prigent-Tessier A, Marie C, Beley A (2001) Stress response to hypoxia in gerbil brain: HO-1 and Mn SOD expression and glial activation. Brain Res. 893: 301-309.
Gelinas S, Martinoli MG (2002) Neuroprotective effect of estradiol and phytoestrogens on MPP+-induced cytotoxicity in neuronal PC12 cells. J. Neurosci. Res. 70: 90-96.
Gerlier D, Thomasset N (1986) Use of MTT colorimetric assay to measure cell activation. J. Immunol. Methods 94(1-2): 57-63.
Goralski KB, Renton KW (2003) Brain inflammation enhances 1-methyl-4- phenylpyridinium-evoked neurotoxicity in rats. Toxicol. Appl. Pharmacol. 196: 381-389.
Hantraye P, Brouillet E, Ferrante R, Palfi S, Dolan R, Matthews RT, Beal MF (1996) Inhibition of neuronal nitric oxide synthase prevents MPTP-induced parkinsonism in baboons. Nat. Med. 2: 1017-1021.
Hightower LE (1991) Heat shock, stress protein, chaperones and proteotoxicity. Cell 66: 191-197.
Hsieh TC, Burfeind P, Laud K, Backer JM, Traganos F, Darzynkiewicz Z, Wu JM (1999) Cell cycle effects and control of gene expression by resveratrol in human breast carcinoma cell lines with different metastatic potentials. Int. J. Oncol. 15: 245-252.
Hung LM, Chen JK, Huang SS, Lee RS, Su MJ (2000) Cardioprotective effect of resveratrol, a natural antioxidant derived from grapes. Cardiovasc. Res. 47: 549-555.
Itano Y, Kitamura Y, Nomura Y (1994) 1-Methyl-4-phenylpyridinium (MPP+)-induced cell death in PC12 cell: inhibitory effects of several drugs. Neurochem. Int. 25: 419-424.
Janero DR (1990) Malondialdehyde and thiobarbituric acid-reactivity as diagnostic indices of lipid peroxidation and peroxidative tissue injury. Free Radi. Biol. Med. 9: 515-540.
Jang JH, Surh YJ (2001) Protective effects of resveratrol on hydrogen peroxide-induced apoptosis in rat pheochromocytoma (PC12) cell. Mutat. Res. 496: 181-190.
Kim WG, Mohney RP, Wilson B, Jeohn GH, Liu B, Hong JS (2000) Regional difference in susceptibility to lipopolysaccharide-induced neurotoxicity in the rat brain: role of microglia. J. Neurosci. 20: 6309-6316.
Kim YH, Park EJ, Han ST, Park JW, Kwon TK (2005) Arsenic trioxide induces Hsp70 expression via reactive oxygen species and JNK pathway in MDA231 cells. Life Sci. 77: 2783-2793.
Kruger R, Vieira-Saecker AM, Kuhn W, Berg D, Muller T, Kuhnl N, Fuchs GA, Storch A, Hungs M, Woitalla D, Przuntek H, Epplen JT, Schols L, Riess O (1999) Increased susceptibility to sporadic Parkinson's disease by a certain combined alpha-synuclein /apolipoprotein E genotype. Ann. Neurol. 45: 611-617.
Kurkowska-Jastrzebska I, Babiuch M, Joniec I, Przybylkowski A, Czlonkowski A, Czlonkowska A (2002) Indomethacin protects against neurodegeneration caused by MPTP intoxication in mice. Int. Immunopharmacol. 2: 1213-1218.
Kurkowska-Jastrzebska I, Litwin T, Joniec I, Ciesielska A, Przybylkowski A, Czlonkowski A, Czlonkowska A (2004) Dexamethasone protects against dopaminergic neurons damage in a mouse model of Parkinson's disease. Int. Immunopharmacol. 4: 1307-1318.
Langston JW, Ballard P, Tetrud JW, Irwin I (1983) Chronic Parkinsonism in humans due to a product of meperidine-analog synthesis. Science 219: 979–980.
Langston JW, Irwin I, Langston EB, Forno LS (1984) 1-Methyl-4- phenylpyridinium ion (MPP+): identification of a metabolite of MPTP, a toxin selective to the substantia nigra. Neurosci. Lett. 48: 87-92.
LeBel CP, Ischiropoulos H, Bondy SC (1992) Evaluation of the probe 2’, 7’-dichlorofluorescin as an indicator of reactive oxygen species formation and oxidative stress. Chem. Res. Toxicol. 5: 227-231.
Lee EJ, Min HY, Joo Park H, Chung HJ, Kim S, Nam Han Y, Lee SK (2004) G2/M cell cycle arrest and induction of apoptosis by a stilbenoid, 3,4,5-trimethoxy-4'-bromo-cis-stilbene, in human lung cancer cells. Life Sci. 75: 2829-2839.
Lombet A, Zujovic V (2001) Resistance to induced apoptosis in the human neuroblastoma cell line SK-N-SH in relation to neuronal differentiation: Role of Bcl-2 protein family. J. Biochem. 268(5): 1352-1362.
Lu X, Bing G, Hagg T (2000) Naloxone prevents microglia-induced degeneration of dopaminergic substantia nigra neurons in adult rats. Neuroscience 97: 285-291.
Madamanchi NR, Li S, Patterson C, Runge MS (2001) Reactive oxygen species regulate heat-shock protein 70 via the JAK/STAT pathway. Arterioscler. Thromb. Vasc. Boil. 21: 321-326.
Marcucilli C, Miller RJ (1994) CNS stress response: too hot to handle? Trends Neurosci. 17: 135-137.
Martin ER, Scott WK, Nance MA, Watts RL, Hubble JP, Koller WC, Lyons K, Pahwa R, Stern MB, Colcher A, Hiner BC, Jankovic J, Ondo WG, Allen FH Jr, Goetz CG, Small GW, Masterman D, Mastaglia F, Laing NG, Stajich JM, Ribble RC, Booze MW, Rogala A, Hauser MA, Zhang F, Gibson RA, Middleton LT, Roses AD, Haines JL, Scott BL, Pericak-Vance MA, Vance JM (2001) Association of single-nucleotide polymorphisms of the tau gene with late-onset Parkinson disease. JAMA. 286: 2245-2250.
Massey V (1994) Activation of molecular oxygen by flavins and flavoproteins. J. Biol. Chem. 269: 22459-22462.
Morimoto RI, Santoro MG (1998) Stress-inducible responses and heat shock proteins: new pharmacologic targets for cytoprotection. Nat. Biotechnol. 16: 833-838.
Mutoh M, Takahashi M, Fukuda K, Matsushima-Hibiya Y, Mutoh H, Sugimura T, Wakabayashi K (2000) Suppression of cyclooxygenase -2 promoter-dependent transcriptional activity in colon cancer cells by chemopreventive agents with a resorcin-type structure. Carcinogenesis 21: 959-963.
Nicolini C, Rigolio R, Miloso M, Bertelli AA, TRedici G (2001) Anti- apoptotic effect of trans-resveratrol on paclitaxel-induced apoptosis in the human neuroblastoma SH-SY5Y cell line. Neurosci. Lett. 302: 41-44.
Nishikawa M, Kakemizu N, Ito T, Kudo M, Kaneto T, Suzuki M, Udaka N, Ikeda H, Okubo T (1999) Superoxide mediates cigarette smoke- induced infiltration of neutrophils into the airways through nuclear factor-kappa B activation and IL-8 mRNA expression in guinea pigs in vivo. Am. J. Resp. Cell Mol. Bio. 20: 189-198.
Nowak TS, Suga S, Saito N (1994) The heat shock response and gene expression in brain after ischemia. In: Heat Shock Proteins in the Nervous System (Mayer, J., Brown, T., eds), pp. 55-81. Academic press, New York.
Ohtsuka K, Suzuki T (2000) Roles of molecular chaperones in the nervous system. Brain Res. Bull. 53(2): 141-146.
Pervaiz S (2003) Resveratrol: from grapevines to mammalian biology. FASEB J. 17: 1975-1985.
Przedborski S, Ischiropoulos H (2005) Reactive oxygen and nitrogen species: weapons of neuronal destruction in models of Parkinson’s disease. Antioxid Redox Signal 7: 685-693.
Przedborski S, Kostic V, Jackson-Lewis V, Naini AB, Simonetti S, Fahn S, Carlson E, Epstein CJ, Cadet JL (1992) Transgenic mice with increased Cu/Zn-superoxide dismutase activity are resistant to N- methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced neurotoxicity. J. Neurosci. 12: 1658-1667.
Przedborski S, Vila M (2001) MPTP: a review of its mechanisms of neurotoxicity. Clin. Neurosci. 3: 81-88.
Rembish SJ, Trush MA (1994) Further evidence that lucigenin-derived chemiluminescence monitors mitochondrial superoxide generation in rat alveolar macrophages. Free Radi. Biol. Med. 17: 117-126.
Rimando AM, Nagmani R, Feller DR, Yokoyama W (2005) Pterostilbene, a new agonist for the peroxisome proliferator-activated receptor alpha-isoform, lowers plasma lipoproteins and cholesterol in hypercholesterolemic hamsters. J. Agric. Food Chem. 53: 3403-3407.
Sairam K, Saravanan KS, Banerjee R, Mohanakumar KP (2003) Non-steroidal anti-inflammatory drug sodium salicylate, but not diclofenac or celecoxib, protects against 1-methyl-4-phenyl pyridinium-induced dopaminergic neurotoxicity in rats. Brain Res. 966: 245-252.
Schulz JB, Matthews RT, Jenkins BG, Ferrante RJ, Siwek D, Henshaw DR, Cipolloni PB, Mecocci P, Kowall NW, Rosen BR (1995) Blockade of neuronal nitric oxide synthase protects against excitotoxicity in vivo. J. Neurosci. 15: 8419-8429.
Sen CK, Packer L (1996) Antioxidant and redox regulation of gene transcription. FASEB J. 10: 709-720.
Shigematsu S, Ishida S, Hara M, Takahashi N, Yoshimatsu H, Sakata T, Korthuis RJ (2003) Resveratrol, a red wine constituent polyphenol, prevents superoxide-dependent inflammatory responses induced by ischemia/reperfusion, platelet-activating factor, or oxidants. Free Radic. Biol. Med. 34: 810-817.
Sinha K, Chaudhary G, Gupta YK (2002) Protective effect of resveratrol against oxidative stress in middle cerebral artery occlusion model of stroke in rats. Life Sci. 71: 655-665.
Stojanovic S, Sprinz H, Brede O (2001) Efficiency and mechanism of the antioxidant action of trans-resveratrol and its analogues in the radical liposome oxidation. Arch. Biochem. Biophys. 391: 79-89.
Su JL, Lin MT, Hong CC, Chang CC, SHiah SG, Wu CW, Chen ST, Chau YP, Kuo ML (2004) Resveratrol induces FasL-related apoptosis through Cdc42 activation of ASK1/JNK-dependent signaling pathway in human leukemia HL-60 cells. Carcinogenesis 26: 1-10.
Subbaramaiah K, Michalurat P, Chung WJ, Tanabe T, Telang N, Dannenberg AJ (1998) Resveratrol inhibits cyclooxygenase-2 transcription in human mammary epithelial cells. Ann. N.Y. Acad. Sci. 899: 214-223.
Teismann P, Schwaninger M, Weih F, Ferger B (2001) Nuclear factor- kappa B activation is not involved in a MPTP model of Parkinson's disease. Neuroreport 12: 1049-1053.
Thiffault C, Aumont N, Quirion R, Poirier J (1995) Effect of MPTP and L-deprenyl on antioxidant enzymes and lipid peroxidation levels in mouse brain. J. Neurochem. 65: 2725-2733.
Tillerson JL, Caudle WM, Reveron ME, Miller GW (2002) Detection of behavioral impairments correlated to neurochemical deficits in mice treated with moderate doses of 1-methyl-4-phenyl-1,2,3,6- Tetrahydropyridine. Exp. Neurol. 178: 80-90.
Tillerson JL, Miller GW (2003) Grid performance test to measure behavioral impairment in the MPTP-treated-mouse model of parkinsonism. J. Neurosci. 123: 189-200.
Tomas-Camardiel M, Rite I, Herrera AJ, de Pablos RM, Cano J, Machado A, Venero JL (2004) Minocycline reduces the lipopolysaccharide- induced inflammatory reaction, peroxynitrite-mediated nitration of proteins, disruption of the blood-brain barrier, and damage in the nigral dopaminergic system. Neurobiol. Dis. 16: 190-201.
Wainwright LJ, Lasorella A, Iavarone A (2001) Distinct mechanisms of cell cycle arrest control the decision between differentiation and senescence in human neuroblastoma cells. Proc. Natl. Acad. Sci. USA, 98(16):9396-9400.
Wang JF, Komarov P, Groot H (1993) Luminol-chemiluminescence in rat macrophages and granulocytes: the role of NO, O2-/H2O2 and HOCl. Arch. Biochem. Biophys. 304: 189-196.