Thaliporphine衍生物對心臟缺血‐再灌流之保護與心血管作用評估

Abstract

背景：心肌梗塞是現在世界上導致死亡的重要原因之一；在過去，急性心肌梗塞之後最常做的治療即是進行冠狀動脈再灌流 (reperfusion)，然而，恢復原先缺血心肌之血流往往會造成所謂的缺血-再灌流損傷 (ischemia-reperfusion injury)。心肌缺血-再灌流損傷與急性發炎反應有關，其原本是有利於組織修復之起始及痂 (scar) 的形成，但是另一方面又會惡化心肌損傷的情形；而且通常再灌流所造成之傷害更甚於缺血所導致的。缺血-再灌流之病理成因十分複雜，像是氧化壓力之產生 (會導致DNA斷裂以及脂質和蛋白質的過氧化) 及發炎反應 (會使得補體與嗜中性白血球活化) 皆會導致再灌流損傷。如何減少再灌流損傷便成為現今治療上的重要課題。TM-1-1 DP是一種結構為phenolic aporphine生物鹼之衍生物，其為(+)-thaliporphine之新衍生物。之前的研究發現(+)-thaliporphine之衍生物TM-1及TM-1-1皆具有減少缺血-再灌流損傷之效果，但由於其中樞副作用太高，較不具臨床上之實用價值，本篇研究使用新合成之(+)-thaliporphine水溶性衍生物TM-1-1 DP，測試其是否亦具有心臟保護作用及其是否具有更好之安全性。 方法：本篇實驗使用已麻醉之公的SD大鼠，使其心肌遭受到一小時缺血-兩小時再灌流，並於再灌流或缺血前十分鐘投予各種劑量 (0.005-1 mg/kg) 之TM-1-1 DP或是只給生理食鹽水，來觀察大鼠之平均動脈血壓、心跳、心電圖以及心肌壞死區域有何變化；且抽血去測量血清中之CK-MB、LDH和NO是否會受到影響；另外，本篇實驗也於此模式中收取大鼠之缺血部位組織，進行西方點墨法來測試一些與缺血-再灌流有關的蛋白質，觀察其是否會受到TM-1-1 DP之影響而發生改變以探究其機轉；再者，對正常大鼠尾靜脈注射高劑量的TM-1-1 DP，測試其是否具有安全性。 結果：由實驗結果顯示，TM-1-1 DP在濃度為0.015到0.15 mg/kg之間能夠減少心肌壞死區域；而其濃度在0.05 mg/kg時展現了減少心肌壞死區域之最佳療效。而在有效劑量之下，TM-1-1 DP並不會對活體大鼠之平均動脈血壓及心跳造成影響；而且在注射了TM-1-1 DP有效劑量之六十倍藥物後，並不會造成實驗動物有任何死亡之情形出現；更甚者，TM-1-1 DP能夠減少細胞壞死指標CK-MB和LDH，且能夠增加eNOS、NO及其他有助於增進心肌細胞存活之分子 (如AKT和ERK等)，而且還可以降低一些發炎物質 (如TNFα) 和其他會導致細胞死亡蛋白之表現量。 結論：本篇研究發現，TM-1-1 DP能夠減少缺血-再灌流所導致之損傷，其機轉可能是藉由活化eNOS、NO、AKT、ERK而產生心臟保護之效果，並且能夠降低iNOS、TNFα、VCAM、P38、Caspase 3等蛋白之表現而使得心肌細胞死亡之情形減少，進而抑制心肌缺血-再灌流之損傷；然而，更多的心臟保護機制仍有待更深入之研究來釐清。

Background: Myocardial infarction is the major cause of death in the world. In the past, coronary artery reperfusion therapy has become an established management for acute myocardial infarction. However, restoration of blood flow to previously ischemic myocardium results in the so-called ischemia-reperfusion (I/R)-injury. Myocardial ischemia-reperfusion injury is associated with an acute inflammatory process that may be beneficial in initiating tissue repair and scar formation, but it is also known to extend myocardial injury. It is well known that injury from reperfusion could be more serious than ischemia. The pathogenesis of ischemia-reperfusion injury consists of many mechanisms. For example, generation of reactive oxygen (breaking DNA , peroxidizing lipid or protein) or inflammatory responses (complement or neutrophil activation) might cause reperfusion injury. Therapy to reduce reperfusion injury has become an important topic today. TM-1-1 DP, an aporphine alkaloid derivative, is a new molecular derivative of thaliporphine. Previous studies have demonstrated that TM-1 and TM-1-1 which are the derivatives of thaliporphine had beneficial effects for the treatment of ischemia-reperfusion injury. However, they may cause central side effects such as tremor when infused at 3 to 5 folds of therapeutic doses. This study used TM-1-1 DP which is a new water-soluble derivative of thaliporphine in order to investigate its protective effects of cardiovascular system and its safety. Method: The male anaesthetized Sprague-Dawley (SD) rats subjected to myocardial ischemia (60 min) and reperfusion (2 h) were treated with TM-1-1 DP (0.005-1 mg/kg) or with vehicle (saline only) at 10 min before ischemia or reperfusion. We observed their mean arterial blood pressure, heart rate, ECG and infract size during and after the periods of ischemia-reperfusion. The CK-MB, LDH and NO of the serum were measured and the western blot was also used in the present study to characterize the mechanism of TM-1-1 DP effect. Moreover, the normal rats were injected with TM-1-1 DP of high doses so as to test its security. Results: TM-1-1 DP at 0.015 to 0.15 mg/kg was found to reduce the infarct size. TM-1-1 DP at 0.05 mg/kg was found to possess maximal effects on reducing the infarct size. At the effective dose, TM-1-1 DP does not affect the mean arterial blood pressure and heart rate in vivo. No experimental animal died in the study when given 60 times of effective dose of TM-1-1 DP. In addition, TM-1-1 DP could reduce the CK-MB and LDH which are the indicators of the cell necrosis. Furthermore, TM-1-1 DP not only increased the contents of eNOS, NO and the cell survival proteins (AKT and ERK) but also decreased the expression of the inflammatory mediators (TNFα) and the cell death proteins. Conclusion: The results of this study suggest that TM-1-1 DP is beneficial for the treatment of reperfusion-induced myocardial damage. The cardioprotective mechanism is that TM-1-1 DP can activate the eNOS, NO, AKT and ERK as well as inhibit the iNOS, TNFα, VCAM, P38 and Caspase 3 in ischemia-reperfusion heart. However, the molecular mechanisms remain to be clarified in further studies.