肝臟後制約訓練中對自噬的觀察

Abstract

前言:缺血後制約訓練(ischemic postconditioning, iPoC)是藉由改變缺血後的再灌流(reperfusion)程序而引發保護效應。不同以往以持續、不間斷的方式恢復灌流，缺血後制約訓練(ischemic postconditioning, iPoC)是以間歇、短暫的方式恢復灌流，藉此減少組織所產生的缺血再灌流傷害。本研究用小鼠模式，驗證缺血後制約訓練對肝臟缺血再灌流傷害的保護效果及對於細胞自噬的觀察。 材料與方法：實驗分為三個組別，control 組、IR 組及 iPoC 組。將雄性 C57BL/6小鼠(重約 20g-25g)的肝臟左外側葉及中葉以動脈夾阻斷血流 30 分鐘(缺血期)後，再移除動脈夾以恢復肝臟血液供應 60 分鐘(再灌流期)，此為缺血(IR)組。缺血後制約訓練(ischemic postconditioning, iPoC)係在恢復灌流前，給予肝臟 3 個短暫的部分缺血再灌流循環，每循環均包括 30 秒的缺血和 30 秒的再灌流。控制(comtrol)組，則是將小鼠的腹腔打開 30 分鐘，期間不做任何處理，接著將腹腔縫合並同樣給予 60 分鐘的再灌流時間。再灌流 60 分鐘後，收集血液樣本及取下肝臟組織做分析，包括評估血清中 ALT 值、H&E 染色、細胞凋亡評估(TUNEL assay)、細胞凋亡及自噬相關蛋白變化(Western blotting)，電子顯微鏡觀察肝臟細胞內細胞自噬的狀態。 結果:與 control 組相比，IR 組血清中ALT有顯著性的上升(51.8±4.59 U/L v.s 520.4±48.42 U/L，p<0.05)，而 iPoC 組血清中的 ALT 上升程度比 IR 組低(300.3±28.9 U/L v.s 20.4±48.42 U/L，p<0.05)。H&E染色中，與 control 組相比，IR 組的細胞呈現大量的空泡狀(1.25±0.32 score v.s 2.99±0.45 score，p<0.05)及壞死(1.27±0.16 score v.s 3.09±0.14 score，p<0.05)；在 iPoC 組與 IR 組比較，空泡狀(1.86±0.21 score v.s 2.99±0.45 score，p<0.05)及壞死(2.36±0.16 score v.s 3.09±0.14 score，p<0.05)的情況有相對減少。在細胞凋亡分析中，與 control 組相比，IR 組的 TUNEL-positive的細胞所占比例較高(2.6±2.04 % v.s 73.5±0.38 %，p<0.05)，但在經過 iPoC 治療後與 IR 組相比，TUNEL-positive 的細胞則有顯著下降(54.4±1.52 % v.s 73.5±0.38 %，p<0.05)。再灌流 60 分鐘後，與 control 組相比，IR 組細胞質中的 cytochrom c 表現有顯著上升(p<0.05)，經過 iPoC 治療後(iPoC 組)則有顯著下降(p<0.05)，在 cleaved caspase 3 部分，與 control 組比較，IR組有顯著的上升(p<0.05)在經過 iPoC 治療後(iPoC 組)與IR組相較，沒有顯著差異(1.32±0.23 v.s 1.56±0.22，p=0.46)。再灌流60分鐘後，自噬體(autophagosome)蛋白之變化，與 control 組相比，IR 組 LC3-II表現有顯著性的上升(1.0±0.12 v.s 1.59±0.15，p<0.05)，在經過iPoC治療後(iPoC組)與IR組相比，沒有顯著差異(1.53±0.2 v.s 1.59±0.15，p=0.59 )，在自噬溶小體(autolysosome)的部分，與 control 組相比，IR 組 p62 表現量有顯著的下降(1.0±0.09 v.s 0.71±0.08，p<0.05)，但 iPoC 組的 p62 表現量與 IR 組相比，則有顯著的提高(1.38±0.26 v.s 0.71±0.08，p<0.05)。 結論:後制約訓練可以減少肝臟缺血再灌流所造成的傷害，並會減少自噬溶小體的形成，影響自噬作用的進行。

Introduction: Ischemic postconditioning (iPoC), a repetitive, brief ischemia-reperfusion maneuver performed at or before the initiation of tissue reperfusion, has been shown to mitigate reperfusion injury in heart and brain. The aim of this study is to investigate the effects of iPoC on liver ischemia-reperfusion injury. Methods: Partial liver ischemia-reperfusion injury was induced by clamping the left lateral lobes and median lobes of the liver for 30 minutes on male C57BL/6 mouse (20g-25g). Three cycles of 30 seconds of reperfusion followed by 30 seconds of ischemia was performed just before reperfusion began in iPoC group. Blood and liver samples were harvested at 60 minutes after reperfusion for assessments which include serum ALT, H&E staining, TUNEL staining, western blot, and electron microscopy (EM) study. The results were compared between the control, IR, and iPoC groups. Results: Our data shows that iPoC could reduce the elevation of serum ALT level after reperfusion 60 minutes (300.3±28.9 U/L v.s 520.4±48.42 U/L, p<0.05), and decrease the percentage of apoptotic hepatocytes(54.4±1.52 % v.s 73.5±0.38 %，p<0.05). The H&E staining showed that ischemic postconditioning could reduce necrosis (2.36±0.16 score v.s 3.09±0.14 score, p<0.05) and vacuolization(1.86±0.21 score v.s 2.99±0.45 score, p<0.05). Western blot showed increase cytochrome c, expression after ischemia reperfusion injury of liver, and decrease the expression of cytochrome c after ischemic postconditioning. Expression levels of cleaved caspase3 and LC3-II were increased after IR injury but there were no difference between IR and iPoC group. Expression levels of p62 were decreased after IR injury but increased after ischemic postconditioning. Conclusion: This study shows that ischemic postconditioning can attenuate cell deaths and influence autophagy after reperfusion injury of liver.