花生四烯酸造成心肌細胞死亡之機制探討

Abstract

花生四烯酸（Arachidonic acid；AA）對於細胞的生理功能有相當廣泛的影響。除可做為類二十碳酸（eicosanoids）之前趨物質外，AA亦可直接或者間接地藉由其下游代謝產物來影響細胞膜上的離子通道、活化PKC（protein kinase C）、抑制細胞內蛋白質的合成。細胞內平時有良好的調控機制可將AA維持在適當的濃度。然而，在細胞遭受缺血性傷害（ischemia）時，會活化PLC （phospholipase C）及PLA2（phospholipase A2），且此時ATP的生成減少，使酯化作用受阻，於是AA聚集而促使大量自由基產生，進而對細胞造成傷害。細胞質鈣離子的過度負荷 (calcium overload)是當心肌細胞受到缺血性傷害時會產生的重要現象。在缺血性傷害時，細胞質及粒線體的鈣離子會大量上升，並與細胞發生不可逆的病理病程有密切的關連。心肌細胞死亡可以分為兩類，凋亡（apoptosis）與壞死（necrosis）。在本篇研究中，我們給予AA（10 μM）來模擬細胞受到缺血性傷害時的部分狀況。在我們初代培養的幼鼠心肌細胞上，利用膜片箝制技術（Patch clamp technique）發現AA在心肌細胞上可活化一種非選擇性陽離子通道。而利用顯微螢光測定術（Microspectrofluorometry）和雷射共軛焦顯微鏡（Laser confocal scanning microscopy）可觀察到AA使細胞質與粒線體內鈣離子濃度及鈉離子增加的現象。細胞質內鈣離子及鈉離子增加的程度會隨著AA濃度而提升，亦即有濃度依性（dose dependent）的現象。此外，我們用螢光染劑TMRM來測量粒線體的膜電位變化，發現AA會造成粒線體膜電位去極化及粒線體形狀變形。目前已知粒線體膜電位去極化會造成粒線體雙層膜上之滲透性轉換孔洞（permeation transition pore）的開啟，進而釋放出細胞凋亡機制的上游訊息cytochrome c。cytochrome c釋出後會活化一連串的caspase cascade而造成細胞凋亡（apoptosis）。我們檢測心肌細胞經AA處理後造成細胞凋亡的機制（包括caspase-3活化、DNA斷裂、cytochrome c釋放及細胞核濃縮的比例增加）。另外，我們利用ethidium homodimer-1（EthD-1）發現AA也會造成細胞壞死。AA處理後會造成細胞膜的不完整，因此心肌細胞可以被EthD-1染到細胞核。AA會造成細胞內ATP含量顯著的下降。而3-aminobenzamide (3-AB), 1,5-dihydroxyisoquinoline (DHQ) 和pepstatin A可以防止經由AA造成的細胞壞死，暗示PARP的活化可能是負責凋亡到壞死的重要轉折點。此外，我們發現AA可造成細胞內活性氧化物的增加。從我們的結果發現AA可以活化一種非選擇性陽離子通道，且同時造成細胞凋亡與壞死的產生。

It has been known that arachidonic acid (AA) and other nonesterified fatty acids (FAs) could have harmful effects during cardiac ischemia. Cellular calcium overload is an important determinant of ischemic myocardial injury. Over recent years, mitochondrial calcium overload has also been thought to be critical in the pathogenesis of irreversible ischemic cell death. The death of cells can be classified into two categories: apoptosis and necrosis. In the present study, we applied AA (10 μM) to mimic the conditions of ischemic reperfusion injury. Using patch clamp technique, we showed that AA-induced Ca2+ entry is mediated via a non-selective cation channel. Using microfluorometry and time-lapse confocal recording of live cardiomyocytes, we showed that AA caused a marked increase in Na+ and Ca2+ levels in both the cytosol ([Na+]i , [Ca2+]i)and mitochondria ([Na+]m , [Ca2+]m). The magnitude of the rise in [Ca2+]i and [Na+]i induced by AA is dose-dependent. The mitochondrial membrane potential (△Ψm) was monitored with tetra-methyl rhodamine-methyl ester (TMRM). AA treatment collapsed △Ψm and induced the structural change of mitochondria. It has been known that △Ψm depolarization result in the opening of permeation transition pore (PTP), followed by cytochrome c release, and finally leading to programmed cell death pathway. Using immunofluorescence techniques, we have examined the AA-induced apoptotic machinery (including caspase activation, DNA fragmentation, cytochrome c release and nucleus condensation) in primary culture of rat cardiomyocytes. Additionally, we also found AA treatment cause a loss of membrane integrity, as indicated by ethidium homodimer-1 (EthD-1) uptake into cardiomyocytes. This result suggests that AA also induces necrotic cell death. AA also caused a marked decrease in cellular ATP levels. Necrosis could be prevented by 3-aminobenzamide (3-AB), 1,5-dihydroxyisoquinoline (DHQ) and pepstatin A, indicating that PARP activation was responsible for the apoptosis-to-necrosis switch. Furthermore, ROS augmentation can also be detected after AA treatment. Our findings suggest that AA activates a non-selective cation channel, and induces a mixture of both apoptotic and necrotic cell death in cardiomyocytes.