摘要:心臟冠狀動脈栓塞(ischemia),當血栓溶解後,血液再流入,稱為再灌流(reperfusion),此時心肌同時產生的大量的含氧自由基(ROS)及含氮自由基(RNS),會造成心肌缺血-再灌流之心肌損傷(I/R injury),導致心肌細胞死亡及心臟衰竭。之前在神經細胞及神經膠細胞的研究,已知外加glutamate、RNS (NO•及ONOO-)、或家用殺蟲劑(Methylisothiazolinone),可引發細胞內Zn2+釋放,GSHi depletion,12-lipoxygenase (12-LOX)活化,造成細胞內ROS 大量產生,導致神經細胞及神經膠細胞之凋亡。已知粒線體對細胞死亡之重要性,但在神經細胞及神經膠細胞的研究所使用的是: (a). low power 20X objective CCD imaging,因此無法分辨細胞質或粒腺體內Zn2+storage sites。(b).使用的是對Zn2+及O2-•相當不敏感的Newport Green 及HE,無法偵測粒腺體內微量Zn2+及O2-•的變化。(c). 除了RNS,在任何種類之細胞,截至目前為止,從未有論文研究其它種類之氧化壓力(I/R、ROS),是否亦導致細胞內Zn2+釋放,而造成細胞之死亡。本研究計畫(第一年),心肌細胞以time-lapse confocal microscopy、用相當敏感之O2-.及Zn2+染劑,同時觀察當細胞處理缺血-再灌流(I/R)、ROS、RNS,細胞質及粒線體內Zn2+/O2-.動態變化,這是在任何種類的細胞,從未的研究過的。由我們preliminary data: Fig. 1. 發現,心肌在缺血-再灌流處理下,細胞質及粒腺體內之Zn2+大量之上升,TPEN (Zn2+-chelator)具有強大保護效果。令人驚訝的是,雖然baicalein 對I/R 引起的O2-‧及Zn2+上升完全無效,但baicalein 及luteolin (12-LOX 抑制劑,亦屬flavones)可完全抑制由I/R、ROS、RNS 引起之心肌凋亡(Figs. 1E, F, 3); 因O2-‧上升無法抑制,代表baicalein 及luteolin 之心肌保護效果並非是antioxidants 之作用,因此它們所抑制之target protein (e.g. 12-LOX),乃位於Zn2+引起之凋亡路徑之下游訊號(見模式圖)。這是心肌在I/R 處理下,從未被發現而令人興奮之結果。但是我們進一步發現心肌在knockdown of 12-LOX mRNA >70% 後(Fig. 4A),對ROS、RNS 之處理完全無保護效果(Fig. 4Ba-b)。由於神經細胞的研究,將baicalein 視為是12-LOX 抑制劑,但未作knockdown12-LOX 之實驗,因此處理RNS 引起之神經細胞內Zn2+大量上升,導致活化凋亡路徑下游之12-LOX,可能作了錯誤之推斷。因此baicalein 及luteolin 對oxidative stress (氧化壓力)所造成之強大保護效果(Fig. 3),有待本計劃進一步釐清。因此未來三年計畫如能深入探討baicalein、luteolin (屬flavones是plants及 vegetables天然成分)保護及改善缺血-再灌流所導致之細胞凋亡之分子機制,將對缺血-再灌流造成之心肌損傷有相當重要性。第一年計劃,我們將深入研究心肌細胞處理I/R、ROS、RNS 之後,引起細胞質及粒線體Zn2+大量釋放,導致心肌細胞凋亡之分子機制。探討TPEN 及flavones (baicalein 及luteolin)對心肌氧化壓力引起之Zn2+大量釋放後,保護心肌細胞之分子機制。第二年計劃:(i). 探討ERK 活化是否位於Zn2+引起心肌死亡之下游訊號?(ii). 將已知之flavones 可能之target proteins,加以knockdown (12-LOX、12/15-LOX、NFкB、HIF1α) 或過度表現HIF1α,深入研究上述蛋白質,對心肌細胞存活或導致凋亡可能之分子機制。(iii).探討MT-IIa (細胞內Zn2+-binding protein) 加以knockdown 或過度表現,研究free-Zn2+ levels 上升或下降對心肌細胞促進死亡或存活之分子機制。第三年計劃:(i). 如將baicalein 及luteolin 已知的四種可能target proteins,加以knockdown 或過度表現後,仍無法有類似flavones 及TPEN 之強大保護效果,則將進行DNA microarray,找出真正flavone-sensitive target protein,以對抗心肌缺血-再灌流所造成之心肌死亡之參考。(ii). 將以成鼠動物模式,在I/R 處理後,觀察心肌細胞是否與第一年之Zn2+i-inducedapoptosis 分子機制類似?比較它們的異同。並將探討TPEN、baicalein、luteolin 可能保護心臟及改善心臟I/R 所導致之心肌細胞凋亡。如能順利執行本三年計劃,將對心臟缺血-再灌流造成之心肌損傷有相當重要性。
Abstract: Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are both suggested to beinvolved in cardiac ischemia-reperfusion (I/R) injury. However, it is not known whetherROS-/RNS-induced intracellular Zn2+(Zn2+i) release is involved in the I/R-induced cardiacinjury. High-affinity Zn2+- and O2-•-selective fluorescent probes are used to simultaneouslymonitor dynamic changes in cytosolic and mitochondrial Zn2+ levels ([Zn2+]cyt and [Zn2+]m)and in the [O2-•]cyt in primary cultured neonatal ventricular myocytes. Our preliminaryresults (Figs. 1-4) showed that, when myocytes were exposed to the ischemia-reperfusion(I/R) or DTDP (an oxidant), a marked increase in the [O2-•]cyt, [Zn2+]cyt, and [Zn2+]m was seen(Figs. 1-2). TPEN (a Zn2+ chelator) and GSH both markedly inhibited the Zn2+i release andprotected against oxidative stress- or 20 μM Zn2+-induced caspase-3-dependent myocyteapoptosis (Figs. 1, 2, 3). Interestingly, baicalein and luteolin, two potent flavones[lipoxygenase (LOX) inhibitors], had little effect on the increase in the [O2-•]cyt and [Zn2+]cyt(Fig. 1B), while these flavones markedly prevented the I/R-, oxidant-, and Zn2+i-inducedcaspase-3-dependent apoptosis (Fig. 3), indicating that activation of the flavone-sensitivetarget protein occurs downstream of the O2-•-/Zn2+i-increase-induced apoptosis (seeHypothesis). However, knockdown of 12-LOX by RNA interference had no protective effectagainst the Zn2+irelease-/flavone-sensitive myocyte apoptosis.Our preliminary results therefore show that (i). the Zn2+i increase and the activation of anflavone-sensitive, but LOX-independent, apoptotic pathway are involved in oxidativestress-induced myocyte death. (ii). The flavones are potentially promising targets fortreatment of ischemic cardiovascular diseases.Since there is no molecular information in any cell type that:(i). whether the I/R and ROS-induced Zn2+i release is involved in the cell death.(ii). the molecular mechanism of marked protective effect of baicalein and luteolinagainst oxidative stress.In this three years’ project, the following questions may be answered.The first year (primary cultured neonatal rat cardiomyocytes):Investigation of oxidative stress-induced mitochondrial Zn2+ influx pathways and themolecular mechanism of TPEN-/flavone-sensitive cardiomyocyte apoptosis.The second year (primary cultured neonatal rat cardiomyocytes):(i). Investigation of possible role of ERK activation on the Zni2+ increase-inducedmyocyte apoptosis.(ii). Over-expression or knockdown of four possible flavone targets: to investigatepossible role of Zn2+-sensitive myocyte apoptosis induced by oxidative stress. The fourpotential flavones’ target proteins are: 12-lipoxygenase (12-LOX), 12/15-LOX, nuclearfactor κB (NFκB), hypoxia-inducible factor-1 (HIF1α).(iii). Over-expression or knockdown of MT-IIa: to investigate molecular mechanism ofchange in free-Zni2+ levels on the oxidative stress-induced myocyte apoptosis.The third year (animal study):(i). If the four known flavones’ targets are not protective against oxidative stress-inducedmyocyte death, DNA microarray will be performed to find out the flavone-target forcardiac protection.(ii).Ischemia-reperfusion in adult rat hearts: to investigate molecular mechanism offlavones-/TPEN-sensitive Zn2+increase resulting in adult cardiomyocyte death.