臺灣博碩士論文加值系統

Oroxylin A (oro-A) 是中草藥黃芩中一種具有活性的成份,而過去黃芩已廣泛被使用在治療發炎。因此本研究的目的是為了探討oro-A對局部缺血再灌流所造成的腦部損傷是否具有保護效果。利用chloral hydrate (400 mg/kg，腹腔注射) 麻醉實驗動物。將大鼠的兩側總頸動脈和右側中大腦動脈的腦血流阻斷90分，利用雷射杜卜勒去監測大腦皮質血流，再使腦血流重新流通經過3天的再灌流，取出鼠腦，以TTC染色法評估大腦皮質的損傷體積。建立一個缺血再灌流的腦損傷動物模式。我們的結果顯示，在阻斷兩側總頸動脈和右側中大腦動脈的腦血流前30和60分鐘前靜脈注射方式給予oro-A 30 mg/kg能有效的降低中風的體積，同時，伴隨抑制了缺血再灌流所引起的環氧化酵素 2的表現。這個結果建議oro-A可能具有保護作用對抗腦中風。

Oroxylin A (oro-A), an active component of the Chinese herb Scutellariae radix (Huang Qin), has been used to treat inflammation. The aim of the present study was to investigate the possible protective role of oro-A on focal ischemia-reperfusion-induced brain injury in Sprague-Dawley (SD) rats. Animals were anesthetized with chloral hydrate (400 mg/kg/IP). Ischemia/reperfusion brain injury was induced by ligating bilateral common carotid arteries (CCA) and right middle cerebral artery (MCA) for 90 min, followed by reperfusion for 3 days. The cortical cerebral blood flow was monitored by a laser Doppler flowmeter. The injury was estimated by the infarction volume. The results indicated that oro-A (30 mg/kg) administered intravenously 60 min before the ligation of CCA and MCA significantly reduced the infarction volume. This was accompanied by inhibition of COX-2 expression induced by ischemic-reperfusion injury. These findings suggest that oro-A may be useful in protecting the brain against ischemic insult.

Abstract……………………………………….………………………………………1
中文摘要……………………………………………………………………………..2
1. Introduction
1.1 Clinical stroke…………………………………………………………3
1.2 Pathophysiology of acute stroke………………………………………3
1.3 Ischemic reperfusion injury…………………………………………...4
1.4 Cyclooxygenase (COX)……………………………………………….5
1.5 COX-2 in normal brain homeostasis…………………………………..6
1.6 COX-2 in brain ischemia……………………………………………....6
1.7 Oroxylin A: a polyphenolic compound………….……………………..8
2. Materials and methods
2.1 Experimental animals……………………………………………...….9
2.2 Drug treatment………………………………………………………...9
2.3 In Vivo Brain Ischemia/Reperfusion…………………………………..9
2.4 Triphenyltetrazolium chloride (TTC) staining…….………………….10
2.5 Cerebral blood flow (CBF)…………………………………………...10
2.6 Blood gas measurement……………………………………………….11
2.7 Western blotting for COX-2 protein production………………………11
2.8 Chemicals……………………………………………………………..12
2.9 Statistical analysis…………………………………………………….12
3. Results
3.1 Establishment of an animal model for stroke

3.1.1 Cerebral blood flow ……..………………………………….….13
3.1.2 Infarction volume………………………………………….........13
3.1.3 Effect of perfusion-reperfusion on physiological parameters…..13
3.2 Time course of COX-2 expression after ischemia reperfusion injury..14
3.3 Oro-A pretreatment prevented ischemia-reperfusion-induced brain injury………………………………………………………………………14
3.4 Effect of oro-A on COX-2 expression………………………………15
4. Discussion………………………………………………..………………………16
5. Reference……………………………………………………………...………….19
6. Appendix…………………………………………………………………………22

Allan SM, Rothwell NJ (2001) Cytokines and acute neurodegeneration. Nat Rev Neurosci 2:734-744.
Breder CD, Dewitt D, Kraig RP (1995) Characterization of inducible cyclooxygenase in rat brain. J Comp Neurol 355:296-315.
Busija DW, Thore C, Beasley T, Bari F (1996) Induction of cyclooxygenase-2 following anoxic stress in piglet cerebral arteries. Microcirculation 3:379-386.
Chen J, Weinstein PR, Graham SH (1995) Attenuation of postischemic brain hypoperfusion and reperfusion injury by the cyclooxygenase-lipoxygenase inhibitor BW755C. J Neurosurg 83:99-104.
Chen ST, Hsu CY, Hogan EL, Maricq H, Balentine JD (1986) A model of focal ischemic stroke in the rat: reproducible extensive cortical infarction. Stroke 17:738-743.
Chen Y, Yang L, Lee TJ (2000) Oroxylin A inhibition of lipopolysaccharide-induced iNOS and COX-2 gene expression via suppression of nuclear factor-kappaB activation. Biochem Pharmacol 59:1445-1457.

Chen YC, Shen SC, Chen LG, Lee TJ, Yang LL (2001) Wogonin, baicalin, and baicalein inhibition of inducible nitric oxide synthase and cyclooxygenase-2 gene expressions induced by nitric oxide synthase inhibitors and lipopolysaccharide. Biochem Pharmacol 61:1417-1427.
Cho J, Lee HK (2004) Wogonin inhibits ischemic brain injury in a rat model of permanent middle cerebral artery occlusion. Biol Pharm Bull 27:1561-1564.
Clemens JA, Ho PP, Panetta JA (1991) LY178002 reduces rat brain damage after transient global forebrain ischemia. Stroke 22:1048-1052.
Cole DJ, Patel PM, Reynolds L, Drummond JC, Marcantonio S (1993) Temporary focal cerebral ischemia in spontaneously hypertensive rats: the effect of ibuprofen on infarct volume. J Pharmacol Exp Ther 266:1713-1717.
Collaco-Moraes Y, Aspey B, Harrison M, de Belleroche J (1996) Cyclo-oxygenase-2 messenger RNA induction in focal cerebral ischemia. J Cereb Blood Flow Metab 16:1366-1372.
Hall ED, Andrus PK, Althaus JS, VonVoigtlander PF (1993) Hydroxyl radical production and lipid peroxidation parallels selective post-ischemic vulnerability in gerbil brain. J Neurosci Res 34:107-112.
Hallenbeck JM, Furlow TW, Jr. (1979) Prostaglandin I2 and indomethacin prevent impairment of post-ischemic brain reperfusion in the dog. Stroke 10:629-637.
Herschman HR (1994) Regulation of prostaglandin synthase-1 and prostaglandin synthase-2. Cancer Metastasis Rev 13:241-256.
Hossmann KA (1994) Viability thresholds and the penumbra of focal ischemia. Ann Neurol 36:557-565.
Kang JS, Jeon YJ, Kim HM, Han SH, Yang KH (2002) Inhibition of inducible nitric-oxide synthase expression by silymarin in lipopolysaccharide-stimulated macrophages. J Pharmacol Exp Ther 302:138-144.
Kinouchi H, Huang H, Arai S, Mizoi K, Yoshimoto T (1999) Induction of cyclooxygenase-2 messenger RNA after transient and permanent middle cerebral artery occlusion in rats: comparison with c-fos messenger RNA by using in situ hybridization. J Neurosurg 91:1005-1012.
Koistinaho J, Koponen S, Chan PH (1999) Expression of cyclooxygenase-2 mRNA after global ischemia is regulated by AMPA receptors and glucocorticoids. Stroke 30:1900-1905; discussion 1905-1906.
Kondo F, Kondo Y, Gomez-Vargas M, Ogawa N (1998) Indomethacin inhibits delayed DNA fragmentation of hippocampal CA1 pyramidal neurons after transient forebrain ischemia in gerbils. Brain Res 791:352-356.
Lee H, Kim YO, Kim H, Kim SY, Noh HS, Kang SS, Cho GJ, Choi WS, Suk K (2003) Flavonoid wogonin from medicinal herb is neuroprotective by inhibiting inflammatory activation of microglia. Faseb J 17:1943-1944.
Lin CC, Shieh DE (1996) The anti-inflammatory activity of Scutellaria rivularis extracts and its active components, baicalin, baicalein and wogonin. Am J Chin Med 24:31-36.
Lin TN, He YY, Wu G, Khan M, Hsu CY (1993) Effect of brain edema on infarct volume in a focal cerebral ischemia model in rats. Stroke 24:117-121.
Madrigal JL, Moro MA, Lizasoain I, Lorenzo P, Fernandez AP, Rodrigo J, Bosca L, Leza JC (2003) Induction of cyclooxygenase-2 accounts for restraint stress-induced oxidative status in rat brain. Neuropsychopharmacology 28:1579-1588.
Nakagomi T, Sasaki T, Kirino T, Tamura A, Noguchi M, Saito I, Takakura K (1989) Effect of cyclooxygenase and lipoxygenase inhibitors on delayed neuronal death in the gerbil hippocampus. Stroke 20:925-929.
Nakayama M, Uchimura K, Zhu RL, Nagayama T, Rose ME, Stetler RA, Isakson PC, Chen J, Graham SH (1998) Cyclooxygenase-2 inhibition prevents delayed death of CA1 hippocampal neurons following global ischemia. Proc Natl Acad Sci U S A 95:10954-10959.
Niwa K, Araki E, Morham SG, Ross ME, Iadecola C (2000) Cyclooxygenase-2 contributes to functional hyperemia in whisker-barrel cortex. J Neurosci 20:763-770.
Nogawa S, Zhang F, Ross ME, Iadecola C (1997) Cyclo-oxygenase-2 gene expression in neurons contributes to ischemic brain damage. J Neurosci 17:2746-2755.
O'Neill GP, Ford-Hutchinson AW (1993) Expression of mRNA for cyclooxygenase-1 and cyclooxygenase-2 in human tissues. FEBS Lett 330:156-160.

Ohtsuki T, Kitagawa K, Yamagata K, Mandai K, Mabuchi T, Matsushita K, Yanagihara T, Matsumoto M (1996) Induction of cyclooxygenase-2 mRNA in gerbil hippocampal neurons after transient forebrain ischemia. Brain Res 736:353-356.
Pasinetti GM (1998) Cyclooxygenase and inflammation in Alzheimer's disease: experimental approaches and clinical interventions. J Neurosci Res 54:1-6.
Pasinetti GM, Aisen PS (1998) Cyclooxygenase-2 expression is increased in frontal cortex of Alzheimer's disease brain. Neuroscience 87:319-324.
Planas AM, Soriano MA, Justicia C, Rodriguez-Farre E (1999) Induction of cyclooxygenase-2 in the rat brain after a mild episode of focal ischemia without tissue inflammation or neural cell damage. Neurosci Lett 275:141-144.
Smith WL, Dewitt DL (1996) Prostaglandin endoperoxide H synthases-1 and -2. Adv Immunol 62:167-215.
Wang Y, Lin SZ, Chiou AL, Williams LR, Hoffer BJ (1997) Glial cell line-derived neurotrophic factor protects against ischemia-induced injury in the cerebral cortex. J Neurosci 17:4341-4348.
Wang YH, Wang WY, Chang CC, Liou KT, Sung YJ, Liao JF, Chen CF, Chang S, Hou YC, Chou YC, Shen YC (2006) Taxifolin ameliorates cerebral ischemia-reperfusion injury in rats through its anti-oxidative effect and modulation of NF-kappa B activation. J Biomed Sci 13:127-141.
Yamagata K, Andreasson KI, Kaufmann WE, Barnes CA, Worley PF (1993) Expression of a mitogen-inducible cyclooxygenase in brain neurons: regulation by synaptic activity and glucocorticoids. Neuron 11:371-386.
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.