巨噬细胞移动抑制因子MIF介导缺血预适应心肌保护作用的机制研究

Myocardial ischemic injury is the leading cause of cardiac morbidity and mortality worldwide. In the last decades, though significant progress in myocardial protection have been achieved, many patients with a heart attack having optimal restoration of blood flow still develop heart failure and 38% will die within one year, indicating the significance of ischemic myocardial protection. Ischemic preconditioning (IPC) by which brief and intermittent periods of coronary artery blockage precede a sustained myocardial ischemia, results in significant cardioprotection with marked infarct size reduction and preservation of contractile function. However, clinical translation of such powerful approach is hampered due to concerns of direct ischemia exerting on the heart and other organs, or the stroke risk due to dropping off of calcific or atherosclerotic plaques by repetitive clamping the aorta during open heart surgeries in aged patients. Thus, seeking an approach or identifying a novel target by which can mimic IPC protection while avoiding unfavourable side effects is always a challenge to the modern cardiology. Macrophage migration inhibitory factor (MIF) is a pleiotrophic cytokine. Recent studies reported that MIF released from a briefly ischemic myocardium exerted cardioprotection by reducing infarct size and preserving contractile function. Following such interesting findings, our recent pilot study using genetic modified mouse strain with MIF gene deficiency (MIFKO) in a Langendorff heart perfusion model has found that the powerful IPC protection in MIFKO mice was diminished with a larger infarct size than wild type (WT) control mice. This important finding indicates that myocardium released MIF may be a critical cytokine mediating IPC protection. In this project, we will further our preliminary findings to investigate the underlying mechanisms and explore the therapeutic potential. First, we will use in vitro heart perfusion model and the unique MIFKO mouse strain to study the influence of MIF on ischemic related signaling pathways; Second, we will using in vivo IPC model to document the key role of MIF in mediating IPC protection by determining the extent of inflammation, myocyte apoptosis, infarct size and cardiac function, and also the long-term influence on cardiac remodeling in both MIFKO and WT mice. Third, taking advantages of the MIFKO strain, we will investigate the role of MIF in promoting energy metabolism, inhibiting JNK pathways and anti-oxidation during ischemic injury. Fourth, most importantly, we will test the possibility of supply exogenous MIF in WT and MIFO mice to enhance or restore IPC protection and to mimic the IPC protection in vivo. Results from this proposal will advance our knowledge and understanding of the role of MIF in mediating IPC protection, and will provide critical information for designing a novel therapeutic strategy that target MIF to better protect ischemic hearts.

缺血预适应即在持续心肌缺血之前施予短暂、间歇冠脉血流阻断可以显著降低心梗面积，保护心脏功能。然而，由于直接施加缺血于心脏及其它脏器的弊端，阻碍了这一心肌保护方法在临床的应用。研究发现，由短暂缺血心脏产生的多功能的细胞因子，MIF，能够保护缺血的心肌。最近利用MIF基因敲除（MIFKO）小鼠的离体灌流模型，我们发现与对照小鼠相比，IPC引发的心梗面积减小的保护作用在MIFKO小鼠明显减弱，此结果说明，MIF可能是一重要的细胞因子，介导心肌IPC的保护。本课题拟采用基因干预小鼠品系建立离体及在体心肌缺血模型，通过检测炎性反应，细胞凋亡及心肌损害程度，心功能等指标证明MIF介导心肌IPC的保护；利用基因干预小鼠的优势，研究相关的分子机制，探讨MIF在促进能量代谢，抑制JNK信号通路及抗氧化方面的作用。同时进一步探索药理途径提高MIF水平所产生的模型IPC的保护，为缺血心肌的保护提供新的视角。

目的：缺血预适应（Ischemic preconditioning，IPC）是一种强有力的内源性保护机制，可以显著降低心梗面积，保护心脏功能。由短暂缺血心脏释放的多功能的细胞因子MIF，能够保护缺血的心肌。本研究假设MIF在IPC诱导的心肌保护中起关键作用，并介导IPC的心肌保护。方法：在离体和在体的条件下，持续缺血/再灌注之前给雄性野生型（WT）和MIF基因敲除（MIFKO）小鼠实施3个循环5 min缺血5 min再灌注的IPC干预，通过检测心梗面积、心肌细胞凋亡、炎性反应、心功能及远期对心肌重塑的影响，明确MIF介导的IPC保护的在体效果；通过检测RISK信号途径相关蛋白（p-ERK1/2、p-p70S6K、p-Akt、p-GSK-3β），p-AMPK，GLUT 4及线粒体呼吸链活性氧（Reactive oxygen species，ROS）的表达，分别研究MIF对RISK信号通路、促进能量代谢及抗氧化作用的影响。结果：研究结果显示，WT小鼠中IPC能够明显激活RISK信号途径相关蛋白、促进AMPK的磷酸化及GLUT4的胞膜转移，并能够显著抑制心肌ROS的产生，从而减少WT小鼠心肌梗死面积及炎性细胞浸润、抑制心肌细胞凋亡、改善心功能，从进而有效减轻WT心肌缺血再灌注损伤；而MIFKO小鼠接受IPC干预后不能进一步激活RISK途径相关蛋白，AMPK及GLUT4的激活也没有进一步改变，抗氧化作用减弱，因此不能有效减轻MIFKO心肌缺血再灌注损伤。活体转染AAV9-MIF提高MIF水平有效减轻心梗面积和炎性细胞浸润程度，从而达到模拟IPC保护的效果。结论：本研究证实了MIF通过RISK的激活，AMPK的活化以及ROS的抑制参与并介导了IPC的保护，药理途径补充提高心肌MIF水平可模拟IPC介导的心肌保护，并能进一步提高心脏对预知缺血损伤的耐受性。这为广大接受心脏手术的患者提供更好的心肌保护措施，为保护缺血性心肌损伤，提出了新的研究目标和治疗方向。