- 學位論文
Metformin 及 NTU1150 對新生大鼠心肌細胞的保護作用
The Protective Effects of the Metformin and NTU1150 on Neonatal Rat Ventricular Myocytes
摘要
背景: 降低心肌梗塞的受損範圍，是決定臨床治療結果的重要因子。因此，許多研究已朝向探索心肌缺血及再灌流傷害的分子機轉，並且尋找可減少心肌缺血及再灌流損傷的藥物。Ｍetformin 是治療糖尿病的常用藥物。其治療糖尿病的作用機轉，是透過誘發AMPK磷酸化， 而達到調整醣類、脂質、蛋白質的代謝。 由於metformin能活化AMPK，已有研究指出metformin 具有減少心肌缺血再灌流所造成的損傷的保護能力。本實驗目的為評估一合成藥物NTU-1150是否對心肌缺血再灌流所造成的損傷具有保護作用，並探討其可能的機轉，並與metformin之作用相比較。 實驗方法：心肌細胞取自第二天或第三天新生大鼠心臟。將心室心肌細胞培養於攝氏37度，5% CO2，20% O2的培養箱中。待取下的心肌細胞固定及穩定後，才進行以下的實驗。將細胞依加入不同的試劑，予以分組。各組細胞在20% O2的培養箱12小時後，移至氮氣充填之密閉低氧艙中6小時，再放回培養箱中12小時。使用 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium (MTT) 進行細胞活性測試，並利用西方墨點法，進行蛋白質表現分析。 實驗結果：NTU-1150 1 μM及metformin 30 μM對缺氧再灌流之新生大鼠心肌細胞的存活率有相似之增加效果。同時， NTU-1150及metformin也提高了pAMPK,pAKT, pERK, GLUT4 及pmTOR的表現量，也明顯降低caspase 3的活性。使用AMPK inhibitor (Compound C) 會減少pAMPK 及GLUT4的表現量，並且會增加caspase 3活性 及減少細胞存活率。而AKT inhibitor (LY294002) 則會降低pAMPK, pAKT, pERK, GLUT4的表現量，並提高caspase 3的活性及減少細胞存活率。 結論: NTU-11501 μM及metformin 30 μM對缺血再灌流新生大鼠心肌細胞存活率有相似之增強作用。而兩種化合物對AMPK及AKT磷酸化之增強作用可能與其促進GLUT4表現，及細胞存活率增加有關。
並列摘要
Background: Myocardial infarction is a major cardiovascular disease. Restoration of blood flow has become an established strategy for treatment of acute myocardial infarction. Restoration of blood flow to the ischemic myocardium can result in the ischemia-reperfusion injury. The pathogenic mechanisms of ischemia/reperfusion (I/R) injury include generation of ROS and the inflammatory response. Therapy to reduce reperfusion injury has become an important topic. In this study, we aimed to evaluate the cardioprotective efficacy of NTU-1150, a synthetic compound of caffeic acid amide derivative, on neonatal rat ventricular myocytes (NRVMs) in hypoxia/reoxygenation (H/R) condition. Metformin, a diabetic agent known to have cardioprotective in I/R rat myocardium, was also investigated and compared with the actions of NTU-1150. Material and Methods: NRVMs were obtained and studied with a designed H/R protocol. The NRVMs viability in H/R condition accompanied with the presence of vehicle, NTU-1150, or metformin, were checked by 3-(4,5-dimethylthiazol-2-yl) diphenyltetrazolium (MTT) assay. The corresponding protein expressions were analyzed by western blot. The molecular mechanisms related to the protective effects of NTU-1150 and metofrmin on NRVMs viability in H/R condition were tested with or without the presence of LY294002 (AKT inhibitor), or Compound C (AMPK inhibitor). Results: Both NTU-1150 at concentrations from 0.1 to 3 μM and metformin at 30 μM increased cell viability of NRVMs in H/R condition. This protective effect of both agents was associated with an increase in phosphorylation of AMPK, AKT, mTOR, ERK and expression of GLUT4. This protective effect by 1 μM NTU-1150 was comparable to that by 30 μM metformin. The protective effect of both agents was accompanied by a decrease of caspase 3 activity. Addition of LY294002 (15 μM) inhibited both NTU-1150 and metformin-stimulated AKT phosphorylation. The inhibition of AKT phosphorylation resulted in an inhibition of ERK and mTOR phosphorylation. On the other hand, the addition of compound C resulted in an inhibition of both NTU-1150 and metformin-induced AMPK phosphorylation. Compared to the inhibition of AKT phosphorylation by LY-294002, the inhibition of AMPK phosphorylation was not associated with any change of ERK activity but resulted in a more prominent inhibition of GLUT4 expression and cell viability. Conclusion: In this study, NTU-1150 1 μM and metformin 30 μM exerted similar protective effect on viability of NRVMs in H/R condition. Both the stimulation of AMPK phosphorylation and AKT phosphorylation contribute to the increase of GLUT4 expression and cell viability induced by NTU-1150 and metformin. Activation of AMPK contributes more to the cardioprotective activity of both agents.