探討漸進式熱預處理以及缺血預處理於大鼠心肌缺血再灌流損傷之保護作用

Abstract

缺血性心臟病是世界主要致死原因之一。當冠狀動脈遭到堵塞時，產生急性心肌梗塞，導致血管下游組織缺血、供氧減少而促使心臟組織走向死亡。對於這樣的清況，公認的治療方法為通過原發性冠狀動脈介入治療來恢復血流使其恢復含氧。然而，恢復含氧血流卻相反地會誘導心肌細胞死亡並增加梗死面積，從而降低其效益，這便是所謂的缺血/再灌流損傷。心肌可以藉由預處理、後處理和藥物預處理達到保護效果。全身性的漸進式熱預處理(PTP)可能具有產生抗氧化壓力的心臟保護作用，可有效減少細胞凋亡，並降低缺血/再灌流損傷的情形。在缺血預處理(IPC)的部分，長期缺血前給予短時間的缺血處理已被公認為有效預防缺血/再灌流損傷的心臟保護機制之一。熱預處理和缺血預處理這兩種不同的預處理類型都可以提供心血管保護，任何一種應用都可以在心肌I/R損傷後保持心臟微血管的功能和結構完整性。然而，在 PTP 中更有效的保護作用仍有待探索，例如三重漸進式熱預處理 (3PTP)，它能夠提供比單一熱預處理更有效的心血管保護，並可能維持缺血/再灌流損傷後心臟微血管的功能和結構完整性。與細胞凋亡、自噬、壞死等其他形式的細胞死亡不同，依賴鐵的程序性細胞死亡是一種新型的細胞死亡方式，也是包含心血管疾病、I/R 損傷等疾病研究的新指標。在本次研究中，我們應用了兩種不同類型的預處理，3PTP 和 IPC分別來保護心臟功能，旨在研究它們對心臟 I/R 中心肌結構、細胞凋亡、炎症和鐵凋亡的保護機制。在心肌缺血/再灌流損傷模型當中，大鼠通過閉塞冠狀動脈左前降支使其經歷60分鐘的缺血然後疏通血管再灌注240分鐘，並同時檢測血流動力學參數，包括心電圖、微循環、心率、左心室舒張末期壓、心室腔內壓力的心室血壓的最大上升速率 (+dp/dt) 和血壓最大下降速率 (-dp/dt)。心肌梗塞的大小通過 Evans blue-TTC 染色方式檢測。通過西方墨點法和組織免疫染色確定預處理誘導的生化保護機制。在我的結果中顯示心肌缺血/再灌流會抑制心臟的微循環，誘導心電圖 S-T 段升高，增加鐵依賴性細胞死亡模式發生、紅血球聚集、白細胞和巨噬細胞/單核細胞浸潤、梗塞面積增加、白血球生長因子和TUNEL檢測的陽性反應增加。而在接受預處理的保護方法後能產生抵抗心肌缺血/再灌流傷害的心臟保護效果，包含+dp/dt的數值回升，左心室舒張末期壓改善，紅血球、白血球浸潤現象和TUNEL檢測的陽性反應減低，心臟組織破碎化和梗塞面積降低，以及鐵依賴性細胞死亡指標蛋白減少。總言之，本研究確定了 IPC在 I/R 誘導的心臟損傷的情況下，能提供對抗鐵依賴性細胞死亡的心臟保護作用。以及，經過改良的 3PTP 可能通過 Bag3 介導的受損心臟結構和功能完整性的保護機制，對 I/R 損傷的心臟提供了防禦效果。因此可認為，三階段式熱預處理(3PTP)及缺血預處理(IPC)的治療方式能減輕心肌缺血/再灌流傷害下的左心室結構惡化和功能障礙。Ischemic heart disease is a leading cause of death worldwide. The occlusions of the coronary arteries lead to the ischemia that causes the decrease in oxygen supply and generates an acute myocardial infarction. Primary percutaneous coronary intervention has been recognized as the common treatment to recover blood flow and achieve reoxygenation. The reoxygenation paradoxically induces the death of cardiomyocytes and increases the infarct size, as known as ischemia/reperfusion (I/R) injury. Myocardial protection can be achieved by the methods of preconditioning, postconditioning and pharmacologic preconditioning. Among these methods, whole-body hyperthermia through progressive thermal preconditioning (PTP) has the protective effect against oxidative stress, which may have cardioprotective properties against I/R injury via diminishing apoptosis. According to previous evidence, ischemic preconditioning (IPC) has been recognized as one of the effective cardioprotective mechanisms to prevent I/R injury.PTP and IPC can provide cardiovascular protection, either application can preserve the function and the structural integrity of cardiac microvasculature after myocardial I/R injury. However, the more effective protective role in PTP remains to be explored, like triple progressive thermopreconditioning (3PTP), which can provide more effective cardiovascular protection than single thermal preconditioning. Differs from other forms of cell death such as apoptosis, autophagy, and necrosis, programmed cell death dependent on iron is a new type of cell death. It is also a new indicator for disease research, including cardiovascular disease after cardiac I/R injury. In this study, we applied two different types of preconditioning, 3PTP and IPC, to protect cardiac function, aiming to investigate their protective mechanisms against myocardial structure, apoptosis, inflammation, and ferroptosis in heart I/R.In the model of myocardial I/R injury, rats underwent 60 minutes of ischemia by the occlusion of left anterior descending coronary artery, and were followed by 240 minutes reperfusion. Hemodynamic parameters including the electrocardiogram, microcirculation, heart rate (HR), left ventricular end-diastolic pressure (LVEDP), maximal rate of rise in blood pressure in the ventricular chamber (+dp/dt), and maximal rate of decline in blood pressure in the ventricular chamber (-dp/dt) were determined. Myocardial infarct size was evaluated by the Evans blue-TTC method. Preconditioning-induced protective mechanisms were determined by Western blot and immunohistochemistry.Cardiac I/R depressed cardiac microcirculation, induced S-T segment elevation, increased infarct size, and increased the phenomena including iron-dependent cell death, erythrocyte accumulation, leukocytes infiltration, macrophage/monocyte infiltration, granulocyte colony-stimulating factor (G-CSF), and TUNEL positive cells. Preconditioning strategies evoked significant cardio-protections against I/R injury, which were characterized by the increase in +dp/dt value, the improvement of LVEDP, and the decrease in erythrocyte and leukocyte infiltration, TUNEL-positive cells, fragmentation and infarct area, and iron-dependent cell death marker. In summary, these findings identified the protective effect of IPC against iron-dependent cardiomyocyte death after cardiac I/R injury. Furthermore, the modified 3PTP provided cardioprotective effect against I/R injury and preserved the structural and functional integrity of damaged heart possibly by the Bag3-mediated mechanism, which co-chaperone with members of the heat-shock protein family of proteins to facilitate the removal of misfolded and degraded proteins. In conclusion, the strategies of 3PTP and IPC can alleviate the left ventricular structural deterioration and dysfunction caused by I/R injury.