臺灣博碩士論文加值系統

丹皮酚對缺血─再灌流損傷大鼠腦梗塞效用及其機轉之研究
研究生 ：程錦宜
單位 ：中國醫學研究所
指導教授 ：謝慶良博士
中文摘要
根據傳統中醫典籍記載，牡丹皮具有涼血的作用，而它的主要成份丹皮酚被認為具有解熱、阻斷鈣離子通道和清除自由基的作用，因此本研究的目的是為了探討丹皮酚對腦梗塞的效用及其機轉。總共60隻雄性Spragrue-Dawley（SD）大鼠被研究，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分鐘，然後24小時的再灌流，建立一個缺血─再灌流腦損傷大鼠的腦梗塞動物模型。丹皮酚10 mg/kg，15 mg/kg和20 mg/kg分別於腦血流阻斷前20分鐘，以及丹皮酚20 mg/kg於腦血流阻斷後30分鐘從尾靜脈注射，以腦梗塞面積的比值和大鼠的神經缺損分級來評估丹皮酚對腦梗塞的效用。在整個實驗過程，大鼠的直腸溫度、平均動脈壓和心跳速率也被測量。另外，我們分別測量腦血流阻斷前、阻斷後90分鐘，以及再灌流後2小時和24小時時股動脈的lucigenin-chemiluminesence（CL）counts，以及再灌流24小時時腦梗塞區域的ED1染色陽性細胞，來探討丹皮酚對腦梗塞的作用機轉。結果顯示丹皮酚 15 mg/kg、20 mg/kg於腦血流阻斷前治療，和20 mg/kg腦血流阻斷後治療都能減少缺血─再灌流損傷大鼠的腦梗塞面積和神經缺損，以及這些大鼠於再灌流24小時時的直腸溫度較低。另外，丹皮酚20 mg/kg腦血流阻斷前治療能減少再灌流2小時時的lucigenin-CL counts，和再灌流後24小時後腦梗塞區域的ED1的陽性染色細胞，但對血糖則沒有影響。
結論是丹皮酚15 mg/kg和20 mg/kg從尾靜脈注射能減少這種腦梗塞和改善神經缺損。又丹皮酚能減少缺血─再灌流2小時Lucigenin-CL counts，和再灌流24小時時梗塞區的ED1染色陽性細胞，但對血糖沒有作用。另外，丹皮酚15 mg/kg、20 mg/kg再灌流24小時時有較低的體溫。因此我們推測丹皮酚能用來治療人類腦梗塞的急性期，而丹皮酚的這種作用至少部分是來自於它對氧化自由基產生的抑制或清除，以及抑制microglial cell的活化，而與血糖無關。至於丹皮酚組有較低的體溫可能來自抑制組織損傷的發炎反應有關。

Abstract
According to Traditional Chinese Medicine Writings, Moutan cortex can cool the blood. Paeonol is a component of Moutan cortex, is considered that has the action of antipyretic, blocking calcium ion channel and scavenging free radical. Therefore, the aim of the present study is to investigate effect and mechanisms of paeonol on cerebral infarct. A total 60 male Sprague-Dawley (SD) rats were studied, a model of ischemia-reperfusion injured cerebral infarct was established by occluding common carotid arteries and the right middle cerebral artery for 90 min, then reperfusion for 24 hrs. Paeonol 10 mg/kg, 15 mg/kg and 20 mg/kg were administered, respectively, from tail vein of the rat 20 min prior to the cerebral blood flow, and 20 mg/kg was administered 30 min after the cerebral blood flow was occluded. The cerebral infarct area ratio and neurological deficit score were used as an index to evaluate the effect of paeonol on cerebral infarct. Throughout the experimental procedure, the rectal temperature, mean arterial blood pressure and heart rate of the rat also were measured. In addition, we studied the effective mechanisms of paeonol, the lucigenin-Chemiluminesence (CL) counts were measured before, and 90 min after occluding the cerebral blood flow of bilateral common carotid arteries and right middle cerebral artery, and 2 hrs and 24 hrs after reperfusion, respectively. The ED1 staining in the cerebral infarct area and blood sugar levels also were measured after reperfusion 24 hrs. The results indicated that paeonol 15 mg/kg, 20 mg/kg pretreatment and 20 mg/kg posttreatment all decreased the cerebral infarct area ratio and neurological deficit score, and the rectal temperature was lower in the paeonol 15 mg/kg, 20 mg/kg pretreatment and 20 mg/kg posttreatment than these with paeonol 10 mg/kg pretreatment, or without paeonol treatment. In addition, paeonol 20 mg/kg pretreatment can decreased the lucigenin-CL counts during reperfusion 2hs, and also can decreased ED1 positive staining cells in the cerebral infarct area, but paeonol cannot change blood sugar levels.
In conclusion, paeonol 15 mg/kg, 20 mg/kg can decrease cerebral infarct area ratio and neurological deficit score in ischemia-reperfusion injured cerebral infarct rat. Paeonol can decreased lucigenin-CL counts at reperfusion 2hrs, and ED1 staining positive cell at reperfusion 24hrs, but cannot change blood sugar levels. In addition, the rectal temperature is lower in the paeonol 15 mg/kg, 20 mg/kg treated groups. Therefore, we suggest that paeonol can be used to treat acute cerebral infarct in humans, and its action, at least in part, result from the suppressive or scavenging effect of superoxide free radical generation, and inhibit activator of microglia cells, but not results from blood sugar. There are lower rectal temperature in the rats with paeonol 15 mg/kg, 20 mg/kg treatment, suggesting paeonol can inhibit inflammatory reaction of tissue damage.

目錄
第一章 前言
1
1-1.前言
1
1-2.腦梗塞動物模型探討
1
1-3.腦梗塞與自由基及發炎細胞的探討
1
1-4.活血化瘀藥對於缺血-再灌流損傷腦梗塞之研究
2
1-5.本研究的目的
2
第二章 文獻探討
4
2-1.傳統中醫有關中風的描述
4
2-2.中醫典籍有關牡丹皮的記載
5
2-3.丹皮酚的現代研究
5
2-4.缺血後自發性高溫
6
2-5.膠質細胞活化與增殖
6
2-6.白血球之黏附與聚集
8
2-7.自由基產生
10
2-8.延遲性神經損傷
10
2-9.自由基、缺血後炎症反應與腦梗塞關係探討
12
2-10.丹皮酚和中風的關係
13
第三章 材料與方法
14
3-1.動物
14
3-2.缺血─再灌流損傷大鼠腦梗塞模型之建立
14
3-3.藥物的製備、給藥劑量及方式
15
3-4.實驗分組
15
3-5.神經學狀態評估
16
3-6.腦切片梗塞面積染色分析
16
3-7.全血分析及血清生化檢查
17
3-8.缺血─再灌流損傷大鼠腦梗塞模型之建立
18
3-9.實驗分組
18
3-10.超氧陰離子的測定
18
3-11.免疫組織化學染色分析
19
3-12.血清生化檢查
21
3-13.統計分析
21
第四章 結果
22
4-1.缺血─再灌流損傷大鼠腦梗塞之動物模型
22
4-2.丹皮酚對.缺血─再灌流損傷大鼠腦梗塞之效用
22
4-3.丹皮酚對.缺血─再灌流損傷大鼠神經學狀態之效用
22
4-4.丹皮酚對.缺血─再灌流損傷大鼠直腸溫度的效用
23
4-5.丹皮酚對.缺血─再灌流損傷大鼠平均動脈壓和心跳速率的效用
23
4-6.丹皮酚對.缺血─再灌流損傷大鼠體重的效用
23
4-7.丹皮酚對.缺血─再灌流損傷大鼠週邊血球的效用
24
4-8.丹皮酚對.缺血─再灌流損傷大鼠肝、腎功能的效用
24
4-9.丹皮酚對缺血─再灌流損傷大鼠
24
4-10.丹皮酚對缺血─再灌流損傷大鼠血糖的效應
25
4-11.丹皮酚對缺血─再灌流損傷大鼠ED1染色陽性細胞的效應
25
第五章 討論(Discussion)
40
5-1.缺血─再灌流損傷大鼠腦梗塞之動物模型
40
5-2.丹皮酚能減小缺血性-再灌流損傷大鼠的腦梗塞面積
40
5-3.丹皮酚能改善缺血性-再灌流損傷大鼠的神經狀態
40
5-4.丹皮酚不會改變缺血性-再灌流損傷大鼠的平均動脈壓
40
5-5.丹皮酚對缺血性-再灌流損傷大鼠的體重沒有影響
41
5-6.丹皮酚對缺血性-再灌流損傷大鼠的肝功能
SGOT、SGPT和腎功能BUN、Creatinine沒有影響
41
5-7.丹皮酚對缺血性-再灌流損傷大鼠周邊血球沒有影響
41
5-8.丹皮酚減低缺血性-再灌流損傷大鼠的直腸溫度，可能來自它的抗發炎反應
41
5-9.丹皮酚對缺血性-再灌流損傷大鼠有抑制氧化自由基的產生或清除的作用
42
5-10.丹皮酚對缺血性-再灌流損傷大鼠的血糖沒有影響
43
5-11.丹皮酚能減少缺血性-再灌流損傷大鼠ED1染色陽性細胞的作用
43
第六章 結論 (Conclusion)
45
參考文獻
46
英文摘要
55
圖目錄
Figure 1 腦冠切片圖(Brain coronal sections)
26
Figure 2 梗塞面積(Infarct areas)
27
Figure 3 神經學缺陷評估(Neuro-deficit score assessment)
31
Figure 4 體重喪失評估(Weight loss assessment)
32
Figure 5 超氧陰離子之測量(Superoxide anion measurement)
35
Figure 6 血糖濃度之測量(Blood sugar measurement)
36
Figure 7 ED1 immunostaining-positive cells measurement
37
Figure 8 ED1之計算面積位置標示圖
38
Figure 9 ED1之免疫組織化學分析(Immunohistochemistry assay)
39
表目錄
Table 1.直腸溫度變化表(The change of rectal temperature)
28
Table 2.平均動脈壓變化表(The change of mean blood pressure)
29
Table 3.心跳速率變化表(The change of heart rate)
30
Table 4.周邊血球指標變化表
(Peripheral blood cells and differential values)
33
Table 5.肝腎功能測量表(The liver and renal functions)
34

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