探討 5-甲氧基色胺酸之血管保護療效與抗氧化能力

Abstract

研究背景 冠狀動脈疾病 (coronary artery disease, CAD) 主要是由冠狀動脈阻塞導致，儘管目前可透過經皮冠狀動脈介入術與冠狀動脈繞道手術進行治療，但所產生的心肌損傷依舊無法停止，且後續引發心臟功能失調的病理機制涉及多種層面，以致全球的住院率與致死率居高不下，因此需要新穎的治療策略。 研究目的 根據先導性研究，5-甲氧基色胺酸 (5-Methoxytryptophan, 5-MTP) 在氧化壓力上扮演調控角色，而產生活性氧物質的主要來源為粒線體。為了探討5-MTP的作用機制，以人類臍靜脈血管內皮細胞 (human umbilical vein endothelial cell, HUVEC) 氧化壓力平台測試粒線體的各項指標。本研究建立HUVEC氧化壓力平台，進而以此平台評估5-MTP於HUVEC之調控粒線體功能與血管保護效果。 實驗方法 以剝奪血清與生長因子並給予100 μM的過氧化氫 (hydrogen peroxide, H2O2)來建立HUVEC氧化壓力平台，而5-MTP的配置濃度則是依據先導性研究的心肌梗塞大鼠之5-MTP血中濃度，平均濃度為20 μg/mL，以此濃度為治療濃度。實驗分成以下4組： 1. Control (Ctl) 組：定期更換新鮮Complete-Endothelial Cell Growth medium 2 (ECGM2) 培養基。 2. Ctl + 5-MTP組：5-MTP預處理24小時後更換新鮮ECGM2培養基。 3. ROS組：剝奪血清與生長因子且同時添加100 μM的H2O2反應3或4小時。 4. ROS + 5-MTP組：5-MTP預處理24小時後進行剝奪血清與生長因子且同時添加100 μM的H2O2反應3或4小時。 為了評估粒線體的各項指標，透過mitobright、mtSOX與MT-1三種螢光染劑，分別檢測粒線體形變、超氧陰離子 (O2–) 與膜電位的變化。 結果與討論 實驗結果顯示，ROS組相比於Ctl組，其網狀的健康粒線體呈現統計顯著性的下降，而顆粒狀的受損粒線體則統計顯著性的上升，且伴隨O2–過度增加與粒線體膜電位異常。經由5-MTP預處理治療，能統計顯著性的減緩粒線體片段化、降低O2–含量，並減少膜電位異常。在西方墨點法分析中，參與5-MTP之生合成路徑的羥吲哚O-甲基轉移酶 (hydroxyindole O-methyltransferase, HIOMT) 呈現統計顯著性的上升。實驗結果指出ROS組與ROS + 5-MTP組皆能夠顯著提升HIOMT的表現量，但是這兩組之間無統計顯著性差異。在細胞存活率分析上，實驗結果指出ROS + 5-MTP組相較於ROS組，能統計顯著性的降低細胞死亡。粒線體的O2–與膜電位主要受電子傳遞鏈與能量代謝影響，而粒線體形變異常可能與dynamin-related protein 1過度活化而誘發顆粒狀型態的粒線體有關，推測5-MTP參與這些訊息路徑，往後若需釐清5-MTP作用於HUVEC之詳細機轉，可從以上途徑深入探討。 結論 從各項研究結果指出，5-MTP的治療機轉可藉由減緩粒線體的O2–含量，並減少膜電位異常與形變來達到氧化壓力下之血管保護效果。

Background Coronary artery disease (CAD) is caused by coronary artery occlusion. Although percutaneous coronary intervention and coronary artery bypass surgery are possible treatments, the myocardial damage cannot be reversed. The subsequent pathological mechanisms of cardiac dysfunction include multiple levels, leading to high rates of hospitalization and mortality worldwide. To effectively treat CAD, innovative therapies are required. Objectives 5-Methoxytryptophan (5-MTP) regulates oxidative stress, and the mitochondria are the primary sources of reactive oxygen species. A human umbilical vein endothelial cell (HUVEC) oxidative stress platform was used to evaluate multiple mitochondrial indicators and investigate the mechanisms of 5-MTP. We explored the influence of 5-MTP on mitochondrial function and the vasoprotective effect in a HUVEC oxidative stress platform. Methods To create a HUVEC oxidative stress platform, HUVECs were exposed to 100 μM H2O2 in a serum and growth factor deprivation (SGFD) medium. The concentration of 5-MTP was 20 μg/mL and was based on the mean plasma concentration of 5-MTP in myocardial infarction rats. Cells were divided into four groups: (1) cells in the control (Ctl) group were supplied with fresh endothelial cell growth medium 2 (ECGM2); (2) cells in the Ctl + 5-MTP group received 5-MTP treatment for 24 h in ECGM2; (3) cells in the ROS group were subjected to SGFD plus 100 μM H2O2 for 3 or 4 h; (4) cells in the ROS + 5-MTP group were exposed to 5-MTP therapy for 24 h before oxidative damage for 3 or 4 h. Three fluorescent dyes, mitobright, mtSOX, and MT-1, were used to measure changes in the morphology of mitochondria, superoxide (O2–), and membrane potential, respectively. Results and Discussion The results revealed that the healthy reticulated mitochondria decreased in number in the ROS group. However, the level of granular damaged mitochondria increased, the level of O2– increased excessively, and the aberrant mitochondrial membrane potential decreased. 5-MTP alleviated morphological abnormalities in mitochondria, the level of O2– and the mitochondrial membrane potential. According to a Western blot analysis, hydroxyindole O-methyltransferase (HIOMT), which is involved in the biosynthesis of 5-MTP, was affected by 5-MTP. The results demonstrated that HIOMT expression could be significantly increased by both the ROS and the ROS + 5-MTP groups. At the same time, no statistically significant difference was observed between the ROS and the ROS + 5-MTP groups. Regarding cell viability, the results revealed that the ROS + 5-MTP group could considerably reduce cell death. The electron transport chain and energy metabolism primarily affected the superoxide and mitochondrial membrane potential, and the abnormal mitochondrial morphology may be linked to the excessive activation of dynamin-related protein 1 to induce mitochondrial morphological changes. To fully comprehend the mechanism of 5-MTP in HUVECs, these pathways should be evaluated in future research. Conclusion According to our results, the therapeutic effects of 5-MTP involve suppressing superoxide production, abnormal membrane potential, and morphological alterations in the mitochondria to achieve vasoprotection in response to oxidative stress.