合成之咖啡酸衍生物36-12對心肌細胞在氧化壓力下保護作用之探討

Abstract

背景: 缺血性心臟病是常見的心血管疾病之一。當心肌缺血時，及早恢復血流供應在治療上是十分迫切的。然而，伴隨而來的再灌流損傷卻可能對心臟造成進一步的傷害，且至今仍無公認的治療準則問世。研究顯示，咖啡酸及其衍生物在心臟保護及抗氧化上皆有顯著的效果。本論文欲探討咖啡酸衍生物，compound 36-12，對於心肌細胞在氧化壓力下的保護效果，及其作用機制。 方法及結果: 本實驗使用50 μM的H2O2對H9c2細胞株所造成之氧化壓力，模擬再灌流對於心臟之傷害。首先，使用MTT試驗評估compound 36-12之保護能力，並與抗氧化劑NAC之效果做對照。同時，藉由螢光探針測量細胞中ROS的含量，以評估藥物的抗氧化效果。此外，也進一步使用蛋白抑制劑阻斷心臟保護相關蛋白之活性，以探討其作用機轉。最後，使用西方墨點法觀察氧化壓力下這些蛋白的活化情形，以及compound 36-12對其造成之影響。實驗結果發現在氧化壓力下，3 μM的compound 36-12即可產生顯著的心臟保護、及抗氧化效果，其程度和50 μM的NAC類似。透過蛋白抑制劑的使用，發現在抑制STAT3的情況下，compound 36-12的保護效果幾乎完全消失。至於其抗氧化作用雖被部分抑制，但仍能顯著降低ROS的生成量。而抑制JAK2之活性，則可提升氧化壓力下的細胞存活率，並降低ROS的生成量。藉由西方墨點法，發現compound 36-12不僅可顯著增加STAT3及AMPK之磷酸化、減少ERK磷酸化，也明顯降低了氧化壓力初期eNOS及JAK2的磷酸化程度。 結論: 在氧化壓力下，compound 36-12可大幅降低心肌細胞中ROS的生成量，並顯著提升了細胞存活率。此效果可能和對STAT3磷酸化之促進，以及對氧化壓力引起之JAK2磷酸化的抑制有關。而其對於STAT3磷酸化之促進效果，應非藉由JAK2而達成。

Background: Ischemic heart disease is one of the most prevalent cardiovascular problems in the world. Once ischemia occurs, restoration of blood flow as soon as possible is necessary for minimizing the damage to the least. However, reperfusion injury may even lead to more horrible outcome to the heart. As far, there is still no guideline for treatment of reperfusion injury. Caffeic acid is recognized as a good antioxidant and proved beneficial for cardioprotection by previous works. In this study, we try to figure out whether a synthetic caffeic acid derivative, compound 36-12, has cardioprotective effects under oxidative stress, and investigate the underlying mechanisms as well. Methods and Results: In this study, 50 μM H2O2 was used to induce oxidative stress in H9c2 cell line to mimic myocardial reperfusion injury. Firstly, we examined the protective effect of compound 36-12 by MTT assay, and compared it with NAC, a well-known antioxidant. Secondly, we measured intracellular ROS level by fluorescent probes to observe antioxidant activity of the drug. Besides, we also used different protein inhibitors to block signaling pathways which make contribution to cardioprotection, and then observed whether the protective effects of compound 36-12 was influenced by doing so. Lastly, activation extents of these proteins at different time points under oxidative stress were shown by Western blotting. Results showed that 3 μM compound 36-12 not only enhanced cell viability but also reduced intracellular ROS level under oxidative stress significantly. This protective effect was comparable to 50 μM NAC. Then, we found that the protective effect of compound 36-12 was totally abolished by STAT3 inhibition, and its ROS scavenging activity was attenuated as well. On the other hand, JAK2 inhibition could enhance cell viability and lower intracellular ROS level under oxidative stress. The results by Western blotting showed that compound 36-12 provoked STAT3 and AMPK phosphorylation obviously. At the same time, however, it also diminished the phosphorylation extent of ERK, as well as H2O2-induced phosphorylation of eNOS and JAK2. Conclusion: Compound 36-12 shows marvelously cardioprotective and ROS scavenging effects under oxidative stress, which have something to do with activation of STAT3 and inhibition of JAK2. Besides, the phosphorylation of STAT3 induced by compound 36-12, is through a JAK2-independent mechanism.