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研究生:程錦宜
研究生(外文):Chin-Yi Cheng
論文名稱:阿魏酸對腦缺血-再灌流損傷大鼠之抗炎症、抗氧化作用及其機轉之探討
論文名稱(外文):The Anti-Inflammatory and Antioxidative Effects and Mechanisms of Ferulic acid on Cerebral Ischemia-Reperfusion Injury in Rats
指導教授:謝慶良謝慶良引用關係
學位類別:博士
校院名稱:中國醫藥大學
系所名稱:中國醫學研究所博士班
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2008
畢業學年度:97
語文別:中文
論文頁數:101
中文關鍵詞:阿魏酸細胞間黏附因子-1(ICAM-1)核因子-κB(NF-κB)4-羥烯酸(4-HNE)細胞凋亡
外文關鍵詞:Ferulic acidIntercellular adhesion molecule-1Nuclear factor-κB8-hydroxy- 2′- deoxyguanosine (8-OHdG)4-hydroxy-2-nonenal (4-HNE)apoptosis
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當歸與川芎於傳統中醫藥用於治療腦中風已有數個世紀之久,而阿魏酸為當歸、川芎之主要成分之一,具有神經保護的效用。本實驗最主要目的在於探討阿魏酸對於腦缺血-再灌流損傷大鼠之抗炎症及抗氧化損傷效用及其機轉。老鼠於中大腦動脈梗塞90分鐘後分別進行再灌流2、10、24及36小時。第一階段於再灌流24小時進行腦梗塞面積及神經學缺陷評估;另外再灌流2小時進行超氧陰離子偵測,再灌流2及24小時進行細胞間黏附因子-1(ICAM-1)、過氧化骨髓酶(MPO)及核因子-κB (NF-κB)等免疫陽性細胞(血管)之評估。第二階段於再灌流2小時偵測ICAM-1 及巨噬细胞-1抗原(Mac-1) mRNA之表現;再灌流2、10、24 及36小時偵測Mac-1、 8-氫氧2''-去氧鳥糞核糖(8-OHdG)、4-羥烯酸( 4-HNE)、 active caspase 3、神經元核抗原(NeuN) 及 TUNEL 陽性細胞。結果顯示於中大腦動脈梗塞的同時給予阿魏酸劑量80、100 mg/kg bw,再灌流24小時可有效地降低梗塞面積及神經學缺陷分數。阿魏酸 (100 mg/kg bw)於再灌流2小時可有效地抑制超氧陰離子於腦實質梗塞區的表現且可顯著抑制紋狀體梗塞區ICAM-1免疫陽性血管及ICAM-1、Mac-1 mRNA之表現;再灌流24小時可降低ICAM-1 及 NF-κB於皮質和紋狀體梗塞區之表現,同時亦可於皮質梗塞區調降MPO免疫陽性細胞;再灌流10、24及36小時於半陰影區及梗塞區可顯著抑制Mac-1、 4-HNE 及 8-OHdG陽性細胞之表現,再灌流10小時半陰影及再灌流24、36小時半陰影區及梗塞區可顯著抑制TUNEL陽性細胞之表現;另外可於再灌流10小時半陰影區降低active caspase 3的表現及再灌流36小時於半陰影區及梗塞區顯著提升NeuN陽性細胞的表現。
結論為阿魏酸於再灌流24小時具有降低腦梗塞面積及神經學缺陷之療效,而其神經保護機轉包括再灌流早期超氧陰離子及ICAM-1 mRNA抑制作用,而此抗氧化及抗炎症效用可於再灌流晚期進一步抑制NF-κB、活化微膠細胞/巨噬細胞、氧化損傷及氧化損傷相關之細胞凋亡。
Both Angelica sinensis (Oliv.) Diels (AS) and Ligusticum chuanxiong Hort., (LC) have been used to treat stroke in Traditional Chinese Medicine for centuries. Ferulic acid (4-hydroxy-3-methoxycinnamic acid, FA), a component of both AS and LC, plays a role in neuroprotection. The purpose of this study was to investigate the anti-inflammatory and antioxidative effects and mechnaisms of FA on cerebral ischemia-reperfusion injury in rats. Rats underwent 2, 10, 24 and 36h of reperfusion after 90 min middle cerebral artery occlusion (MCAo). First, the cerebral infarct and neurological deficits were measured at 24 h of reperfusion. Furthermore, the expression of superoxide radicals, intercellular adhesion molecule-1 (ICAM-1), myeloperoxidase (MPO), nuclear factor-κB (NF-κB) immunoreactive cells (vessels) were assessed at 2 and 24 h of reperfusion. Second, ICAM-1 and macrophage-1 antigen (Mac-1) mRNA were detected at 2 h of reperfusion; Mac-1, 8-hydroxy- 2′- deoxyguanosine (8-OHdG), 4-hydroxy-2-nonenal (4-HNE), active caspase 3, NeuN and TUNEL positive cells were measured at 2, 10, 24 and 36 h of reperfusion. Administration of 80 and 100 mg/kg of FA at the beginning of MCAo significantly reduced cerebral infarct and neurological deficit-score. FA treatment (100 mg/kg i.v.) effectively suppressed superoxide radicals production in the parenchyma lesion, and the expression of ICAM-1 immunoreactivity, ICAM-1 and Mac-1 mRNA in the ischemic striatum at 2 h of reperfusion; FA reduced the expression of ICAM-1 and NF-κB in the ischemic cortex and striatum, also down-regulated MPO immunoreactive cells in the ischemic cortex at 24 h of reperfusion. Furthermore, FA reduced the expression of Mac-1, 4-HNE and 8-OHdG positive cells in the ischemic rim and core at 10, 24 and 36 h of reperfusion. FA also decreased TUNEL positive cells in the penumbra at 10 h, and in the ischemic boundary and core at 24 and 36 h of reperfusion. FA curtailed active caspase 3 enhancement in the penumbra at 10 h and restored NeuN-labeled neurons in the penumbra and ischemic core at 36 h of reperfusion.
FA reduces cerebral infarct area and neurological deficit-score at 24 h of reperfusion, and the neuroprotective mechanism may involve suppression of superoxide radicals production and ICAM-1 mRNA expression in the early reperfusion period, and the antioxidative and anti-inflammatory effects could further down-regulate NF-κB, activated microglia/macrophages, oxidative stress and oxidative stress-related apoptosis during the late reperfusion period.
目錄
第一章 前言 1

第二章 文獻探討 5
2-1. 傳統醫學對缺血性腦中風之論述 5
2-1-1.病因病機之歷史沿革 5
(1).唐宋以前時期 5
(2).金元時期 6
(3).明清時期 6
2-1-2.治則 7
2-2. 缺血性腦中風之現代研究 7
2-2-1.臨床表現 7
2-2-2.病理機轉 8
2-2-3.臨床治療 9
2-3. 阿魏酸之現代研究 10
2-3-1.前言 10
2-3-2.阿魏酸之理化性質 10
2-3-3.阿魏酸及其衍生物之體內代謝 11
2-3-4.阿魏酸藥理作用 12
(1).抗氧化及清除自由基 12
(2).中樞神經保護作用 14
(3).心血管保護作用 16
(4).肝臟保護作用 17
(5).胃腸消化道保護作用 18
(6).抗癌病變作用 18
(7).治療糖尿病作用 19
(8).抗細胞凋亡作用 20
(9).抗內皮素-1作用 21

第三章 材料與方法 22
3-1.實驗動物 22
3-2.實驗室藥品與儀器 22
3-3.中大腦動脈梗塞模型 23
3-4.實驗步驟 23
3-4-1.第一階段又分為實驗A、B和C 23
3-4-1-A.實驗A 23
(1).動物分組 23
(2).生理指標偵測 24
(3).圖示中大腦動脈梗塞模型 24
(4).神經學狀態評估 25
(5).圖示神經學狀態評估 25
(6).評估腦梗塞面積 26
(7).實驗A流程圖 26
3-4-1-B.實驗B 26
(1).分組 26
(2). Hydroethidium原位染色及Hoechst染色 27
(3). Hydroethidium原位染色及Hoechst染色示意圖 27
(4). ICAM-1、MPO 及 NF-κB (p50 )免疫組織化學分析染色 28
3-4-1-C.實驗C 28
(1).分組 28
(2). ICAM-1、MPO 及 NF-κB (p50 )免疫組織化學分析染色 29
(3). ICAM-1、MPO 及 NF-κB (p50 )免疫組織化學分析染色示意圖 29
(4).實驗B、C流程圖 30
3-4-2.第二階段 30
(1).分組 30
(2).生理指標
(3). RNA萃取 30
31
(4).半定量反轉錄-聚合酶鏈式反應 31
(5).半定量反轉錄-聚合酶鏈式反應示意圖 34
(6).免疫組織化學分析染色 34
(7).免疫組織化學分析染色示意圖 35
(8).細胞凋亡染色分析 36
(9).細胞凋亡染色分析示意圖 36
(10).免疫組織化學分析雙重染色 37
(11).免疫組織化學分析雙重染色示意圖 37
(12).第二階段實驗流程圖 38
3-5.統計分析 38

第四章 結果 39
4-1. 第一階段實驗結果 39
4-1-1. 生理指標 39
4-1-2. FA對再灌流24小時後腦梗塞面積之影響 40
4-1-3. FA對再灌流24小時後神經學行為缺陷之影響 42
4-1-4. FA對再灌流2小時後oxidized hydroethidine螢光染色陽性細胞表現之影響 42
4-1-5. FA對再灌流2小時後ICAM-1、MPO及NF-κB(p50)陽性細胞表現之影響 44
4-1-6. FA對再灌流24小時後ICAM-1、MPO及NF-κB(p50)陽性細胞表現之影響 45
4-2. 第二階段實驗結果 49
4-2-1. 生理指標 49
4-2-2. FA對再灌流2小時後ICAM-1及Mac-1 mRNA表現之影響 51
4-2-3. FA對再灌流2、10、24及36小時後Mac-1、4-HNE及8-OHdG陽性細胞表現之影響
53
4-2-4. FA對再灌流2、10、24及36小時後TUNEL陽性細胞表現之影響 54
4-2-5. FA對再灌流10小時後active caspase 3及active caspase 3-NeuN陽性細胞表現之影響
60
4-2-6. FA對再灌流36小時後NeuN陽性細胞表現之影響 60

第五章 討論 67

第六章 結論 74

參考文獻 75

英文摘要 96

附錄 (1) 腦缺血-再灌流損傷之機轉圖 98
附錄 (2) 第一階段討論之機轉圖 99
附錄 (3) 第二階段討論之機轉圖 100
附錄 (4) 第一、二階段之綜合機轉圖 101



圖目錄
Figure 4-1. Effect of ferulic acid on cerebral infarct in ischemia- reperfusion injured rats 41
Figure 4-2. Representative photograph showed the brain coronal section located on the posterior bregma 0.92 mm position 43
Figure 4-3. Effect of ferulic acid on oxidized hydroethidine fluorescent stain positive cells at 2 h of reperfusion 44
Figure 4-4. Effect of ferulic acid on the intercellular adhesion molecule-1 (ICAM-1) immunoreative vessels at 2 h and 24 h of reperfusion 45
Figure 4-5. Effect of ferulic acid on the nuclear factor-κB (NF-κB) and myeloperoxidase (MPO) cells at 24 h of reperfusion 47
Figure 4-6. The expression of ICAM-1 and Mac-1 mRNA in the ischemic areas of the cortex and striatum were detected at 2 h of reperfusion 52
Figure 4-7. Representative photograph showed the brain coronal section located on the posterior bregma 0.92 mm position 55
Figure 4-8. Representative photographs depicted the expression of Mac-1 in the penumbra and ischemic core areas at 10, 24, and 36 h of reperfusion 56
Figure 4-9. Representative photographs depicted the expression of 4-HNE in the penumbra and ischemic core areas at 10, 24 and 36 h of reperfusion 57
Figure 4-10. Representative photographs depicted the expression of 8-OHdG in the penumbra and ischemic core areas at 10, 24 and 36 h of reperfusion. 58
Figure 4-11. Photographs depicted the distribution of TUNEL cells in the penumbra and ischemic core areas at 10, 24 and 36 h of reperfusion 59
Figure 4-12. Representative photographs depicted active caspase 3 and NeuN expression in the penumbra area at 10 h of reperfusion 61
Figure 4-13. Representative photographs depicted NeuN expression in the penumbra and ischemic core areas at 36 h of reperfusion 62

表目錄
Table 3-1. Primer sequences 32
Table 4-1. Comparison of physiological parameter among the groups 39
Table 4-2. The expression of oxidized hydroehtidine, ICAM-1, MPO and NF-κB immunoreactive (positive) cells (vessels) 48
Table 4-3. Physiological parameters 50
Table 4-4. The effects of ferulic acid on Mac-1, 4-HNE, 8-OHdG and TUNEL positive cells 63
Table 4-5. The effects of ferulic acid on active caspase 3 and NeuN-labeled cells 65
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