Metformin對心肌缺血再灌流之保護作用

The protective effect of metformin on cardiac ischemia reperfusion.

關如珺(Ru-Jiun Guan)

指導教授 ： 賴凌平

國立臺灣大學/醫學院/藥理學研究所/碩士(2008年)

https://doi.org/10.6342/NTU.2008.00458

摘要

中文摘要

背景：組織缺血後再灌流引起的損傷，在臨床上是很常見的，病人施行心血管手術或器官移植後、或於心肌梗塞、冠狀動脈阻塞及缺血性中風情況下，都會引起再灌流損傷，並影響病人的預後。許多臨床試驗都顯示，不管是第一型或第二型糖尿病，都容易發生缺血性心臟疾病及造成嚴重的後遺症，所以希望能研究抗糖尿病用藥對於心臟的保護作用。本篇實驗主要在研究metformin對於心臟冠狀動脈阻塞後再灌流所造成的損傷，是否具有保護的作用，並探討其可能的機轉。

方法：心臟取自成年雄性、重約250~350公克的Wistar品系大鼠，使用Langendorff離體心臟灌流模式，將灌流壓力固定於80mmHg，待平衡後結紮左前降支冠狀動脈，造成30分鐘的缺血，之後再灌流2小時，實驗分為不加藥組(CTL)及加藥組(MET)，加藥組為再灌流的前十分鐘(即缺血20分鐘時)到再灌流兩小時過程中使用含有不同濃度metformin(0.1mg/L, 1mg/L, 10mg/L)的灌流溶液，再灌流結束後利用triphenyl tetrazolium chloride(TTC)染色結果，觀察心肌梗死區域占心臟缺血區域的比率，並利用西方點墨法及KATP channel blocker，更進一步探討metformin的作用機轉。

結果：從TTC染色的結果發現，心肌梗死區域占心臟缺血區域的比率，CTL組為59.3±4.2%(n=8)，MET(1mg/L)組為30.6±2.6%(n=8)，加藥組顯著地減少心肌梗死範圍。利用LDH釋放測定觀察細胞壞死，MET組的心肌細胞壞死現象明顯較CTL組少，而西方墨點法則用以觀察metformin相關蛋白質表現量，發現MET組AMPK的磷酸化有顯著增加，total AMPK量則無改變，而利用cytosol蛋白質測量caspase-3 activity也沒有變化。最後在平衡時即給予兩種不同的KATP channel blocker，glibenclamide(10μM)及5-HD(100μM)，觀察心肌梗死區域占心臟缺血區域的比率，我們發現給予glibenclamide可以使metformin的保護作用消失，給予5-HD則無法影響metformin的保護作用。

結論：metformin對於心臟冠狀動脈阻塞後再灌流損傷具有保護作用，可能的機轉是透過AMPK活化及sarcolemmel KATP channels，減少心肌細胞壞死途徑，來降低心臟再灌流損傷引起的心肌梗死。

關鍵字

心肌缺血再灌流損傷 ； 心肌壞死 ； metformin ； AMPK ； KATP channels

並列摘要

Abstract

Background: Reperfusion injury during the recovery phase of tissue ischemia is important in many clinical situations such as cardiac transplantation, myocardial infarction and stroke. It influences the outcome of the patients. Patients with type 1 or type 2 diabetes are at risk for developing cardiovascular diseases including ischemic heart disease, acute myocardial infarction and postinfarct complications. Therefore, it is very interesting to know whether anti-diabetic medicines are also protective against ischemic heart disease. The aim of the present study was to investigate the protective effects of metformin on coronary artery occlusion-reperfusion induced myocardial injury.

Methods: We used mature male Wistar rat weighing 250~350g for the experiments. The hearts were subjected to Langendorff-perfusion with a coronary perfusion pressure of 80 mmHg. We occluded the left anterior descending artery for 30mins and then restored the coronary blood flow (reperfusion) for 2 hours. Animals were divided into control and metformin groups. In the metformin group, a solution containing metformin (0.1mg/L, 1mg/L, 10mg/L) was given from 10 minutes before reperfusion to the end of reperfusion. Triphenyl tetrazolium chloride (TTC) staining was used to show the area of infarction. Western blot and KATP channel blockers was also used in the present study.

Results: In TTC staining, the percentage area of infarction was significantly decreased in the metformin group when compared with the control group (CTL=59.3±4.2%, n=8; MET=30.6±2.6 %, n=8). We also measured the LDH release from the effluent of the perfused heart. The total LDH value was significantly lower in the metformin group. Western blot analysis revealed that phospho-AMPK was increased in the metformin group. Caspase-3 activity, total AMPK expression showed no significant differences between the two groups. In addition, the effects of two different KATP channel blockers glibenclamide(10μM) and 5-HD(100μM) on the protective effect of metformin was investigated. We found that glibenclamide significantly inhibited the protective effect of metformin while 5-HD did not.

Conclusion: Metformin significantly reduced the infarct size in myocardial ischemia/reperfusion injury. The smaller infarct size was attributed to a decrease of myocardial necrosis. The decreased reperfusion injury was related to phospho-AMPK and sarcolemmel KATP channels.

並列關鍵字

Myocardial ischemia/reperfusion injury ； Myocardium necrosis ； Metformin ； AMPK ； KATP channels

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