36-13對心肌細胞在氧化壓力下的保護作用

Abstract

背景：缺血性心臟病是國人常見的心臟疾病之一；冠狀動脈急性阻塞後，再灌流恢復血流供應雖是必要的治療措施，但是再灌流後所產生的大量自由基，卻可能對心臟造成更嚴重的傷害。本篇實驗研究類黃酮化合物36-13，對氧化壓力下的心肌細胞損傷是否有保護效果，並研究其可能機轉。 方法及結果：本實驗利用50μM H2O2對新生大鼠心肌細胞及H9C2胚胎大鼠心肌細胞株造成氧化壓力。先利用MTT assay觀察36-13對心肌細胞的保護效果，並比較36-13和抗氧化劑NAC的有效保護濃度，再比較兩者對細胞內ROS生成量的影響；接著利用西方墨點法，觀察在氧化壓力下不同時間點時不同蛋白質（eNOS、AMPK、Akt、ERK）活化的情形，然後給予各蛋白的抑制劑，以了解各蛋白對細胞存活率的影響。最後再用西方墨點法更進一步探討36-13的作用機轉。從MTT assay的結果發現，3μM 36-13和50μM NAC可以顯著提升氧化壓力下的細胞存活率；不過觀察胞內ROS的結果顯示，36-13無法和NAC一樣顯著減少H2O2引起的胞內ROS生成。西方墨點法定量蛋白活性的結果發現，和未給藥組相比，36-13可促進氧化壓力下peNOS、pAMPK、pAkt的表現；接著給予Protein inhibitors後進行MTT assay的結果顯示，抑制peNOS和pAMPK會顯著降低36-13對心肌細胞的保護效果。後續西方墨點法的結果，顯示36-13引起的eNOS活化，是受到AMPK所調控。 結論：36-13對氧化壓力下的心肌細胞具有保護效果，其作用機轉並非透過抗氧化作用或是減少細胞內ROS生成，而可能是透過活化AMPK調控eNOS活性去對抗氧化壓力，進而達到細胞保護的效果。

Background: Ischemic heart disease is one of the most common heart diseases worldwide. Although restore blood flow after acute coronary artery occlusion is inevitable, reperfusion injury might cause more severe damage to the heart. This study examines whether the flavonoid-like compound, 36-13, has cardioprotective effects under oxidative stress, and investigates the possible underlying mechanism. Methods and Results: This study used 50μM H2O2 to induce oxidative stress of NRVM and H9C2 cell line. We first used MTT assay to observe the cardioprotective effect of 36-13, compared the effective concentration with antioxidant NAC, and then compared their influence on the intracellular ROS. Next, the activation of different proteins ( eNOS, AMPK, Akt, and ERK ) at different time points under oxidative stress were shown by western blot. Then we pretreated different protein inhibitors to investigate the importance of different protein on cell viability, and followed by western blot to investigate detail mechanism. The result of MTT assay shows that 3μM 36-13 and 50μM NAC can enhance the cell viability significantly under oxidative stress, but 36-13 can’t reduce intracellular ROS induced by H2O2 as NAC. Western blot shows that 36-13 can enhance expressions of peNOS, pAMPK, and pAkt under oxidative stress. The following MTT assay reveals that inhibition of eNOS and AMPK phosphorylation can attenuate the protective effect of 36-13 significantly. The result of further western blot study indicates that the activation of eNOS caused by 36-13 was regulated by AMPK. Conclusion: 36-13 reveals protective effects to cardiomyocytes under oxidative stress, not through antioxidant or reduces intracellular ROS generation, but activates AMPK to regulate eNOS.