The protective effect of metformin on cardiac ischemia reperfusion.

中文摘要景：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。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。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。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。Abstract ackground: 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.ethods: 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.esults: 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.onclusion: 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.目錄試委員審定書……………………………………….. i謝…………………………………………………….. ii寫表………………………………………………….. iii文摘要……………………………………………….. iv文摘要……………………………………………….. vi一章 緒論…………………………………………... 11-1：缺血再灌流細胞死亡情況…………………………………… 11-1：中華民國九十五年十大死因................................................... 21-2：心肌缺血再灌流損傷機轉....................................................... 41-3：缺血再灌流造成細胞內鈣離子過載………………………… 51-4：缺血再灌流造成自由基大量產生…………………………… 61-5：自由基造成之後續反應……………………………………… 71-6：MPTP影響缺血再灌流心肌細胞死亡方式………………… 91-7：缺血再灌流引起之心血管傷害............................................... 101-8：KATP channel構造組成.............................................................. 111-9：KATP channel開啟，鉀離子由細胞內往細胞外移動............. 121-10：KATP channel對於心臟缺血再灌流之保護作用................... 141-2：各種KCBs及KCOs的專一性及IC50……………………… 151-3：糖尿病導致coronary artery disease(CAD)的因素.................. 171-11：口服降血糖藥物的分類與作用機轉………………………. 181-4：各類口服降血糖藥物的優缺點……………………………… 191-12：Metformin的降血糖作用………………………………....... 211-13：AMPK的生理功能…………………………………………. 221-14：AMPK的三個次單元結構…………………………………. 241-15：AMP對AMPK的異位調控……………………………….. 241-5：能活化AMPK的狀況、壓力與藥物……………………...... 251-16：Metformin活化AMPK的分子傳導途徑………………….. 25究動機與目的………………………………………………………. 26二章 實驗材料與方法…………………………….. 27三章 實驗結果…………………………………….. 413-1：左前降支冠狀動脈結紮後造成灌流流速的下降比率……... 473-2：心臟缺血區域占整顆心臟百分比………………………....... 473-3：Metformin可以減少心臟缺血再灌流的心肌梗死範圍……. 483-1：Metformin對缺血再灌流過程最終心跳速率的影響………. 493-2：Metformin對缺血再灌流過程最終心臟灌流流速的影響…. 493-4：Metformin可以減少Genomic DNA fragmentation…………. 503-5：Metformin可以減少心臟缺血再灌流的心肌細胞壞死……. 513-6：Metformin對缺血再灌流後心肌細胞caspase-3 activity無顯著影響………………………………………………………… 23-7：Metformin可以促使缺血再灌流後AMPK磷酸化增加…… 533-8：AMPK活化可以減少心肌梗死範圍……………………....... 543-9：Glibenclamide可以抑制Metformin減少心臟缺血區域心肌梗死範圍的作用……………………………………………... 53-10：5-HD無法抑制Metformin減少心臟缺血區域心肌梗死範圍的作用……………………………………………………. 6四章 討論…………………………………………... 574-1：心臟缺血後給予不同長度的再灌流時間心肌壞死及凋亡比例的變化……………………………………………………. 94-2：AMPK的下游目標及影響的代謝作用…………………...... 624-3：缺血再灌流時AMPK活化對於心肌細胞代謝的影響…..... 64五章 結論與展望………………………………….. 69考文獻……………………………………………….. 7