2011-08-012024-05-14https://scholars.lib.ntu.edu.tw/handle/123456789/658998摘要：背景：心肺停止併心肺復甦急救為一特殊且嚴重的全身性缺血/再灌流傷害之例子。在所有的器官組織中，心臟及腦部為最不能耐受此傷害之器官，往往造成病患循環衰竭及復甦後永久性的神經缺損及障礙。反應性氧化物為缺血暨再灌流傷害中一個重要的病理因子，抗氧化治療理論上可減少反應性氧化物所造成的傷害並改善功能及預後。吾人先前的研究指出前花青素為多酚類中一有效的抗氧化成分，可清除反應性氧化物並減少心肌細胞缺血暨再灌流傷害所造成的死亡。因此吾人在此一系列研究中將此成果由細胞層級研究推展至動物層級，並在第二、三年進一步探索其於神經保護之作用以及機轉，以期用於治療復甦急救後心肌以及腦部之缺血暨再灌流傷害，真正改善此類病患之預後。方法：吾人將分三年以：(1)大鼠窒息導致心肺停止/心肺復甦急救之模型，(2)海馬迴腦切片缺血暨再灌流傷害模型，(3)神經培養細胞之缺血暨再灌流傷害模型三種模型進行研究。首先將用兩種procyanidin B2 治療模式：(1) 慢性預防；(2) 急性治療，以存活率、心臟功能及神經功能恢復程度來比較兩種治療之成效。其次吾人將探討前花青素治療對全身系統及局部器官(心臟及腦)ROS 產生之影響，並釐清其ROS 廓清與保護作用的關係。進一步，吾人將利用海馬迴腦切片與神經培養細胞兩模型，以螢光顯微鏡即時、連續地監測細胞中superoxide, H2O2/hydroxyl radical 與nitric oxide (NO)的變化，深入探究黃芩素治療中對神經ROS 及NO 產生的影響。另外，吾人亦將測定下游細胞凋亡之指標 (DNA fragmentation、TUNEL staining 及caspase 活性)，釐清前花青素對神經之保護是否亦透過抗凋亡來達成。最後，吾人將離清Akt-eNOS-NO 途徑在前花青素誘發細胞內訊息傳遞及下游抗凋亡保護機轉所扮演的角色，並與心肌細胞及動物心臟實驗之結果比較，以找出潛在共通的保護機轉，除了抗氧化保護外，NO 是否扮演重要的訊息傳導角色，可資應用於其他細胞之缺血暨再灌流傷害之保護。預期結果：動物、組織及細胞三個層次缺血/再灌流傷害模型的建立，將提供轉譯醫學研究的平台，三者間將互補所需，提供不同層級的存活、功能及巨觀與微觀生理指標。而前花青素對動物心臟及神經系統的保護，將印證吾人於心臟細胞研究之觀察結果，並提供前花青素治療神經保護機轉研究的一個重大方向。至於Akt-eNOS-NO 途徑與各種下游細胞凋亡指標的因果關係研究，將可勾勒出前花青素訊息傳導保護機轉的路徑。臨床應用：本系列實驗提供了前花青素於保護復甦後心臟及全腦傷害的可能機轉，除抗氧化效果外，亦釐清Akt-eNOS-NO 途徑在此間所可能扮演的角色。同時，兩者保護機轉中各環節的比較，將可找出共通的保護機制，提供作為發展藥物治療的潛在標的。<br> Abstract: Backgrounds: Cardiac arrest and cardiopulmonary resuscitation (CPR) is a specific model ofglobal ischemia/reperfusion (I/R) injury. Among all organ systems, heart and brain are twomost important organs that greatly confer prognostic implication to survival. While oxidativestress is significantly increased during I/R, antioxidant therapy is a reasonable therapeuticintervention that may reduce oxidant injury and improve the prognosis of these patients. Ourprevious study suggests herbal antioxidant procyanidin B2 exhibits great potentials inscavenging the reactive oxygen species (ROS) and protects cardiomyocytes from I/R injury.Herein we try to translate the result to an animal model of cardiac arrest and CPR, and furthertest its protective effect in neuronal I/R injury using both animal and neuronal model of I/R.Methods: Three models of I/R injury will be used for investigation. (1) Rat asphyxial cardiacarrest and CPR model, (2) Hippocampus slice I/R injury model, (3) Cultured neuronal cell I/Rinjury model. First, the protective effects of different procyanidin B2 treatment will becompared (1) Chronic prevention and (2) Acute treatment. The ROS profiles of the 2protocols along I/R will also be correlated with the protective effect. Next, we wouldcontinuously monitor the dihydroethidine (for superoxide), 2’,7’-dichlorofluorescin diacetate(for H2O2/hydroxyl radical) and 4,5-diaminofluorescein diacetate fluorescence (for NO) inhippocampus slice and cells undergoing I/R to investigate the causal relationships with thedownstream apoptotic indicators such as caspase activation, Bax/Bcl-2, and DNA laddering.These will be verified using specific inhibitors with monitoring of the downstream indicators.Expected Results: The establishment of the three I/R models at cell, tissue and animal levelsoffers the platform for translational physiological research, based on which the survival,functional, and pathophysiological aspects of the study can be approached. First, the resultobtained in cardiomyocyte level will be translated to the whole heart, and then extended toanother important organ, brain. Next, the ROS and NO response in neuron and hippocampuswill bring insight to mechanistic studies of procyanidin B2 in both heart and brain. Most of all,the correlations of the Akt-eNOS-NO and the downstream apoptotic indicators can outline theprotective signaling pathways that procyanidin B2 exerts.Clinical Implications: This series of study translates the cell-level study to animal level. Italso explores the possible mechanisms involved in procyanidin’s protection against I/R injuryof the neurological system. Understanding of the protective mechanisms in bothcardiomyocyte and neurons can potentially figure out the common protective signalingpathway that is applicable to other type of cells, based on which potential targets forpharmacological intervention can be developed and improvement of the overall prognosis inpatients resuscitated from cardiac arrest improved.