KS-C370G對心臟缺血-再灌流之保護與心血管作用評估

Abstract

在心肌缺血-再灌流的病理機制中，許多研究認為當冠狀動脈恢復血流使心肌重新獲得灌流時，反而會造成比缺血更嚴重之傷害。目前認為心肌再灌流傷害的機制中，大量產生的自由基是造成傷害的主要來源，可能透過直接攻擊細胞膜上的脂質與蛋白質或是DNA使其功能異常，或是間接活化發炎反應如活化補體與嗜中性白血球等作用，因此抗氧化方式的治療被認為能有效的降低心肌缺血-再灌流時的傷害。近幾年來，發現從蜂膠中萃取的成分CAPE (caffeic acid phenethyl ester) 具有優秀的抗氧化活性，且有許多研究皆已證明CAPE能於心肌缺血-再灌流時經由許多不同的機制提供心臟保護的作用。而於一系列CAPE的amide類合成衍生物中，發現其中的KS-C370G具有最強的抗氧化作用，因此選此化合物來與CAPE相比較以評估其心血管作用以及對抗心肌缺血-再灌流的保護作用，尤其著重於後者再灌流時的傷害。此外，選用No.369，其為KS-C370G結構修飾的衍生物，利用其不具有抗氧化活作用的特性與CAPE與KS-C370G相比較以釐清抗氧化是否為KS-C370G保護心臟所必需的活性。 於離體心臟的實驗中，結果顯示KS-C370G會使右心室收縮力降低；相對地，CAPE則會使左心房與右心房收縮力降低；而兩者皆不會影響右心房自發性心跳的速率。 於心肌缺血-再灌流的實驗中，利用模式一：結紮左冠狀動脈五分鐘-再灌流三十分鐘引發心律不整以評估抗心律不整的活性。結果顯示腹腔注射投與15 mg/kg的CAPE與KS-C370G相較於控制組於VT與VF發生的時間長度並無影響，因此兩者皆無抗心律不整的活性。 於心肌缺血-再灌流的實驗中，利用模式二：結紮左冠狀動脈四十五分鐘-再灌流兩小時造成細胞壞死以評估心臟保護的作用。結果顯示腹腔注射投與3 mg/kg 與15 mg/kg的CAPE與KS-C370G皆能隨著劑量有效降低缺血區域中心肌壞死的百分比，但投與藥物並不會降低CK-MB與LDH的活性。此外，腹腔注射投與15 mg/kg的No.369對於心肌壞死的程度並無保護作用。因此推論CAPE與KS-C370G的心臟保護可能主要來自於其本身的抗氧化能力。 很有趣地，結果顯示在投與藥物前十五分鐘腹腔注射3 mg/kg的NOS抑制劑L-NAME，會使CAPE與KS-C370減少心肌壞死的保護作用消失，因此推論NO可能參予在CAPE與KS-C370G的心肌保護作用中。但是在NO含量的分析中，結果發現兩者皆不會影響體內NO的含量。 於心肌缺血-再灌流的各個時間點紀錄的心跳速率與平均血壓，給予15 mg/kg的CAPE、KS-C370G與No.369皆不會影響心跳與血壓的表現，因此推論心臟保護作用並不是來自於降低心跳與血壓以減少心臟耗氧作功。 此外，更進一步去測定心肌malondialdehyde (MDA) 的含量與 myoperoxidase (MPO) 的活性分別代表脂質被過氧化與嗜中性白血球活化的情形，結果顯示腹腔注射投與15 mg/kg的CAPE與KS-C370G皆能使MDA含量明顯降低，但No.369無此作用，因此再次確認了兩者的保護作用的確來自於氧化壓力的降低；同樣地，腹腔注射投與15 mg/kg的CAPE與KS-C370G皆能使心肌MPO活性明顯降低，但No.369亦無此作用，因此推論兩者的心臟保護作用可能是透過本身的抗氧化作用間接地減少發炎反應的產生。 綜合以上結果，顯示KS-C370G與CAPE減少心肌壞死程度、降低心肌氧化壓力與減少嗜中性白血球活化等心臟保護作用可能主要來自於其本身的抗氧化活性，且主要進而透過NO的作用來達到保護的效果，但詳細的機轉與更多的證據還待未來去釐清與發掘。

In cardiac pathology, it is well known that injury from reperfusion could be more serious than ischemia, and generation of reactive oxygen species (ROS) might be the main cause of reperfusion injury. In the reperfusion period, ROS can exacerbate the injury from ischemia directly by breaking DNA and peroxidating lipid or protein, or indirectly by inducing inflammatory responses like complement or neutrophil activation. Therefore, antioxidant therapy is thought to be an effective method to reduce ischemia-reperfusion injury. In these years, the caffeic-acid compounds derived from the propolis of honeybee hives, CAPE (Caffeic acid phenethyl ester), is reported to have remarkable antioxidant effect, and it has been proved that CAPE can protect heart from ischemia-reperfusion injury by various mechanisms. In the series of synthetic CAPE derivatives, KS-C370G has the strongest antioxidant activity in DPPH-radical scavengering assay. Therefore, we chose this compound and evaluated its cardiovascular effects and cardioprotective effects against ischemia-reperusion injury, especially the latter. Furthermore, an analogue of KS-C370G “No.369”, with no radical-scavenging activity, was used to test whether the antioxidant effect was involved in the cardioprotective pathway. In isolated rat RV, contractile force was reduced by KS-C370G, In contrast, contractile force of the left and right atria were reduced by CAPE. Heart rate was not significantly alterd by both compounds. Regional myocardial ischemia-reperfusion model-1 was established by ligating the left main coronary artery of rat hearts forc5 min followed by reperfusion for 30 min in vivo to induce arrhythmia. CAPE and KS-C370G (both 15 mg/kg i.p.) has no effects on reducing VT and VF duration. These results suggested that both compounds had no anti-arrhythmic activity. Model-2 was established by ligating left main coronary artery for 45 min followed by reperfusion for 2 hour in vivo to evaluate the cardioprotective effects. KS-C370G 3 mg/kg and 15 mg/kg i.p. significantly reduced infarct size (IS) dose-dependently, but neither reduced the level of CK-MB and LDH. These results were similar to those of CAPE. Moreover, No.369 15 mg/kg i.p. had no cardioprotective effect in IS. These results suggested that the cardioprotective effects of CAPE and KS-C370G were mainly due to their antioxidant activity. Interestingly, when NOS inhibitor L-NAME (3 mg/kg i.p.) was added 15 min before drug injection, the cardioprotective effects of CAPE and KS-C370G (both 15 mg/kg i.p.) in IS were eliminated. This suggested that NO was an important factor responsible for cardioprotection. However, in the NO content assay, neither CAPE nor KS-C370G significantly increased NO content. In hemodynamic parameters in the time course of ischemia- reperfusion, CAPE, KS-C370G and No.369 (all 15 mg/kg i.p.) had no effects on HR and BP. This suggested that the cardioprotective effects were not due to reduced oxygen comsumption by decreasing heart work. Furthermore, malondialdehyde (MDA) content assay and myoperoxidase (MPO) activity assay were performed to evaluate the effects of compounds on lipid peroxidation and neutrophil acitivation recpectively. CAPE and KS-C370G (both 15 mg/kg i.p.) significantly reduced MDA content, but not No.369 (15 mg/kg i.p.). This indicated that CAPE and KS-C370G treatment reduced the oxidative stress. On the other hand, CAPE and KS-C370G also reduced MPO activity, but not No.369. This suggested that the cardioprotective effects of CAPE and KS-C370G was partly attributed to reducing inflammatory reponse by their antioxidant effects indirectly. In summary, KS-C370G, like CAPE, might exert cardioprotective activity mainly through its antioxidant ability resulting in reduced cardiomyocyte necrosis, lipid peroxidation and neutrophil activation. Nitric oxide might contribute to its protective effects. However, the mechanisms remain to be clarified in further studies.