8-Br-AMP於缺血-再灌流後腦損傷之保護作用

Abstract

摘要 由於中風的高致死率，在許多開發中及已開發國家高居十大死因第三名，且造成病患長久行動不便及家人負擔，然至今卻仍缺乏有效之治療方式。在本篇論文中，藉由將老鼠兩側總頸動脈及右側中大腦動脈結紮造成大腦局部缺血(ischemia)，一個半小時後鬆開使血液回流(reperfusion)，建立一個transient focal ischemia模式。8-Br-AMP是一個可通過細胞膜的adenine nucleotide analogue，在血液回流前5分鐘靜脈給予8-Br-AMP (10 mg/kg)，24小時後經由TTC染色法發現其減少缺血-再灌流後腦損傷體積。另外，在血液灌流4小時後發現給予8-Br-AMP老鼠大腦皮質中萃取出較少的Evans Blue，顯示 8-Br-AMP減少了缺血-再灌流後增加的血腦障壁通透性。接著以西方點墨法發現在缺血-再灌流後24小時，血腦障壁的tight junction protein ZO-1表現減少，而給予8-Br-AMP的老鼠則部分抑制所減少的ZO-1，而另一個tight junction protein：occludin即使在缺血-再灌流後或給予8-Br-AMP都沒有明顯的變化。同時我們也發現8-Br-AMP減少腦梗塞後所誘導的COX-2表現，然而由RT-PCR發現，缺血-再灌流後所誘導的IL-1β mRNA似乎不受8-Br-AMP影響。此外，8-Br-AMP增加Hsp60 Mrna及蛋白質表現，顯示8-Br-AMP可能促進粒線體功能；此外，8-Br-AMP不影響缺血-再灌流後AMPK磷酸化程度。在SH-SY5Y細胞中，以hypoxia-reoxygenation模擬ischemia- reperfusion，發現給予8-Br-AMP可減少細胞死亡。而若在細胞中給予MPP+ (mitochondria complex І抑制劑)造成神經毒性， 利用MTT reaction、流式細胞儀，得知8-Br-AMP可抑制因粒線體功能缺失所導致的細胞死亡。綜合以上結果推斷， 8-Br-AMP可能經由減少血腦障壁通透性及粒線體功能喪失，於缺血-再灌流之動物模式中達到神經保護作用。

Abstract Estimated mortality of stroke is about 25%, which makes it the third major cause of death in industrialized countries. Due to the high rate of severe permanent disability, stroke is a burden not only for the affected patients but their families. Therefore, it is important to develop effective neuroprotectant. The occlusion of middle cerebral artery (MCA) and bilateral common carotid arteries was made in male Sprague–Dawley rats, and reperfusion was performed 1.5 h after the occlusion. 8-Br-AMP (10 mg/kg) was administered 5 minutes before the onset of reperfusion. The infarct volume of 8-Br-AMP-treated rats decreased 24 hours after MCA occlusion in comparison with control. Damage to the blood–brain barrier was judged by the extravasation of Evans blue dye, and the content of Evans Blue decreased in the rats treated with 8-Br-AMP. Western blots showed that the blood–brain barrier associated protein ZO-1 decreased after ischemia, which was partially antagonized by 8-Br-AMP. However, there was no detectable change in another blood–brain barrier associated protein, occludin. COX-2 expression also increased in response to ischemia-reperfusion, which was attenuated by 8-Br-AMP. Compared with sham group, the mRNA level of IL-1β increased after MCAO, and 8-Br-AMP did not significantly affect the mRNA expression of IL-1β. In addition, it was found that 8-Br-AMP increased the protein and mRNA level of Hsp60, which is important for mitochondrial protein folding and function. However, 8-Br-AMP did not significantly affect the phosphorylation of energy-sensitive protein, AMPK. To simulate pathophysiological ischemia/reperfusion, SH-SY5Y cells were exposed to hypoxia 24 hours and then reoxygenation 24 hours. Pretreatment of 8-Br-AMP led to the increase of the viability of SH-SY5Y cells after hypoxia/reoxygenation. In addition, pretreatment of cells with 8-Br-AMP antagonized mitochondrial dysfunction induced by MPP+. These results suggest that infusion with 8-Br-AMP resulted in reduction of injury after focal cerebral ischemia, and the protection was possibly due to the decreased disruption of blood–brain barrier and mitochondrial dysfunction.