- 學位論文
後制約訓練對肝臟缺血再灌流傷害的保護效應與機制
The protective effects and mechanisms of ischemic postconditioning on hepatic ischemia-reperfusion injury
摘要
缺血再灌流傷害(ischemia-reperfusion injury)常發生在器官移植、心肌梗塞、腦中風、創傷等情況，恢復血流(或稱再灌流)對於缺血的組織而言是必要的，然而再灌流往往帶來缺血以外的傷害，因此研究減少缺血再灌流傷害對於提升器官移植的成功率以及心肌梗塞或腦中風的預後是相當重要的。目前主要研究減少缺血再灌流傷害的方法有下列幾種：1. 藥物2.幹細胞療法3.制約訓練4.胞器治療(例如粒線體)。制約訓練(conditioning)是給予短暫的缺血再灌流處理，又可分為前制約訓練、後制約訓練以及遠端制約訓練，依照實行的時間點以及是否直接實行於缺血器官來做為區分，其保護效果與機制也略有不同。本研究中，吾人以肝臟缺血再灌流的動物模式，來探討後制約訓練(ischemic postconditioning, iPoC)對於肝臟缺血再灌流的保護效應與機制。首先，Wistar大鼠肝臟經過45分鐘缺血，立即給予重複三個循環的1分鐘的肝臟缺血和1分鐘的再灌流處理(iPoC組)，跟只有肝臟缺血的組別(IR組)比較，血液中ALT的升高值、肝臟細胞的凋亡以及4-hydroxy-2- nonenal (4-HNE，脂質過氧化產物)的增加情形都減少。缺血再灌流會破壞粒線體的膜電位(mitochondrial membrane potential)而導致cytochrome c 釋放至細胞質，引發肝細胞的凋亡。在iPoC的組別中，細胞質cytochrome c 的表現有明顯下降的情形，且粒線體膜電位的破壞也比較輕微。粒線體膜電位的改變是跟mitochondria permeability transition (MPT)有關。對肝臟缺血的大鼠給予NIM811 (MPT抑制劑)可以模擬iPoC的保護效應，減少cytochrome c的釋放；若給予atractyloside (MPT誘發劑)則會破壞iPoC的保護效應，表示iPoC是透過調控MPT來減少肝臟缺血再灌流傷害。 進而以陣列分析與定量聚合脢反應比較iPoC組跟IR組肝臟的microRNA的變化，發現iPoC會明顯增加肝臟中miR-183的表現增加以軟體搜尋發現參與細胞凋亡過程的一個重要蛋白質，Apaf-1，是miR-183的作用標的。Dual luciferase assay證實miR-183會透過結合至Apaf-1 的3’-UTR來抑制其表現。吾人以氧氣及葡萄糖剝奪模式(oxygen-glucose deprivation, OGD)，驗證低氧後制約訓練(hypoxic postconditioining, HPoC)會使大鼠肝臟細胞株-Clone 9 cells的miR-183表現增加、減少細胞凋亡以及Apaf-1的表現；給予miR-183模擬物可以模擬HPoC的保護效果，但給予miR-183抑制劑則會破壞HPoC的保護效果。同時，觀察到miR-183模擬物會減少凋亡訊息中Apaf-1下游的active caspase-3、cleaved caspase-9的表現，但不會影響上游cytochrome c 的表現。顯示miR-183是透過對於Apaf-1的抑制而減少下游caspase signaling，達到保護效果。接著，以C57BL/6小鼠的肝臟缺血再灌流的模式，與IR組比較，發現後制約訓練 (iPoC)引起miR-183的表現有明顯上升，且減少了ALT的上升、肝臟細胞的傷害(TUNEL assay和Suzuki’s classification評估)以及Apaf-1的表現。經由小鼠脾臟輸注miR-183模擬物可以模擬iPoC的保護效果，輸注miR-183抑制劑則破壞iPoC的保護效應。綜合上述，吾人發現後制約訓練可以透過:(1)調控MPT，減少ROS產生，以及(2)引起miR-183表現上升，抑制其目標mRNA，Apaf-1，來達到對肝臟缺血再灌流的保護效果。
並列摘要
Ischemia-reperfusion injury (IR injury) happens during organ transplantation, cardiac infarct, stroke, and trauma. Recovery of the blood, which called reperfusion, is necessary for the ischemic tissues. However, reperfusion causes extra injury other than ischemia. Therefore, investigation of attenuating IR injury is essential for the success rate of organ transplantation and the prognosis of cardiac infarct and stroke. Therapies for reducing IR injury are as follows: 1. Drugs, 2.Cell therapy, 3. Conditioning, 4. Organelle therapy. Conditioning is a maneuver received a brief period of ischemia and reperfusion, and it can be divided into preconditioning, postconditioning, and remote conditioning according to the executive time point and tissues. The protective mechanisms are different between these maneuvers. In this study, we set up an animal model of hepatic IR injury to investigate the protective effects and mechanisms of ischemia postconditioning (iPoC) on hepatic IR injury. First, we discovered that three cycles of 1 min reperfusion by releasing the clip across the left hepatic artery followed by 1 min of ischemia of liver by clamping the left hepatic artery performed on Wistar rats (iPoC group) reduced the elevation of serum ALT, apoptosis of hepatocytes, and 4-hydroxy-2-nonenal (4-HNE, a product of lipid peroxidation) compare to that of IR goup. The cytosolic cytochrome c expression significantly decreased, and the mitochondrial membrane potential was also preserved in iPoC group. Mitochondria permeability transition (MPT) was responsible for changes of mitochondrial membrane potential. The protective effects of iPoC was reduced by mPTP opener with ATR and mimicked by the inhibitor of mPTP opening with NIM811. Furthermore, the miRNAs profiles in the rats’ livers with or without iPoC after IR injury was analyzed by microarray. The target of miRNA was identified by luciferase assay. MicroRNA mimics and inhibitors were used in oxygen-glucose deprivation (OGD) injury in Clone 9 cells and partial liver IR injury in mice to test the function of miR-183 in the postconditioning. The expression of miR-183 was increased in the iPoC livers, and miR-183 repressed Apaf-1 mRNA expression. Hypoxic postconditioning (HPoC) and miR-183 mimics significantly decreased cell death after OGD, but miR-183 inhibitors eliminated the protection of HPoC. The increased expression of Apaf-1 and downstream capsase 3 / 9 activations after OGD were mitigated by HPoC or the addition of miR-183, while miR-183 inhibitor diminished the effect of HPoC on Apaf-1-capsapse signaling. Ischemic postconditoining (iPoC) and agomiR-183 decreased the elevation of serum ALT after liver IR in the mice, but antagomiR-183 mitigated the effect of iPoC. The results of H&E and TUNEL staining were compatible with biochemical assay. Besides, iPoC and agomiR-183 decreased the expression of Apaf-1 after IR injury in mice’s livers, while antagomiR-mediated prevention of miR-183 expression led to an increased protein expression of Apaf-1 in the postischemic livers. This study suggested that iPoC protected liver from IR injury through: (1). Modulating MPT and reducing the production of reactive oxygen species, (2). Up-regulating miR-183 to inhibit the expression of its target mRNA, Apaf-1.
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延伸閱讀
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