CDK5特异抑制肽联合低温对于缺血性中风的神经保护机制研究

Hypothermia is one of the effective neuroprotective therapies for stroke treatment. However, side effect of widely inhibition limits its clinical application. In our previous study, we found there is the synergic effect of the magnesium sulfate and hypothermia for treating ischemic stroke, which implied that some neuroprotective agents might be the remedy and promote the neuroprotection of hypothermia. Cyclin-dependent kinase 5 (CDK5)/p35 activity is important for the nervous system development, function and survival. Under pathological conditions, p35 is cleaved into p25 and induces neuropathology. Pathological CDK5/p25 activity contributes to the occurrence and deteriorating of kinds of neurodegenerative diseases and ischemia. We had demonstrated that CDK5 specific inhibitory peptide (TFP5) reduces the phenotypes of Alzheimer's disease by inhibiting the hyperactivity of CDK5/p25. In this study, we will investigate role of TFP5 for specifically blocking CDK5/p25 activity and protecting neuron apoptosis in ischemia stroke model. We will further study the combination effect of TFP5 and hypothermia on the neuroprotection in ischemia treatment. This study will provide pre-clinical evidences for combination of TFP5 and hypothermia as a novel strategy for the ischemic stroke treatment.

低温是一种临床证明有效的脑保护措施，但其脑保护机制的局限性限制了其临床疗效。我们发现，低温与硫酸镁在干预缺血性中风时具有协同作用。提示和神经保护剂联合应用可能弥补低温神经保护机制的缺陷。CDK5与其调节蛋白p35结合并活化，为神经细胞发挥正常功能所必须，而多种病理因素可使p35裂解成为p25，p25与CDK5结合使其活性过高失调，高活性的CDK5与多种神经退行性疾病和缺血性中风密切相关。我们发现，CDK5特异抑制肽（TFP5）能通过特异抑制CDK5/p25高活性改善阿尔兹海默病小鼠的预后，我们设想TFP5能通过相似的机制干预缺血缺氧性细胞凋亡，并与低温发挥一定程度协同作用。本课题拟在缺血性中风模型上，对TFP5的CDK5/p25特异抑制作用及其神经保护作用进行研究，并进一步探讨TFP5与低温的协同作用和内在机制。研究结果将为TFP5联合低温作为一种治疗缺血性中风的新策略提供实验依据。

低温多靶点、多层面的脑保护机制和神经保护剂单靶点、定向的脑保护机制在缺血性中风早期联用时有潜在的相辅相成的作用。TFP5是一种源于CDK5调节蛋白p35的CDK5特异抑制肽，前期研究表明TFP5能通过抑制CDK5的异常高活性改善阿尔兹海默病（AD）、帕金森病（AD）小鼠的预后。鉴于缺血性脑损伤病理过程中CDK5起到了类似AD和PD的病理作用，我们推测TFP5同样可能改善缺血性中风的预后。本研究复制了成年Sprague–Dawley大鼠大脑中动脉栓塞（MCAO）模型，观察了TFP5以及TFP5联合低温的脑保护作用，结果发现：1. 100mg/kg或小于这个剂量的TFP5对于大鼠各主要脏器无明显损伤；TFP5(30mg/kg)能够穿过血脑屏障，且3周后仍能在脑中发现；激光共聚焦显微镜显示，药物在细胞核及细胞质均有分布；2. TFP5(30mg/kg)腹腔注射能够显著减小MCAO大鼠早期的梗死体积，机制可能与其减轻兴奋性神经毒性、神经炎症、细胞凋亡、星形胶质细胞损伤和血脑屏障破坏有关；3. TFP5(30mg/kg)和低温早期干预缺血性中风的效用相当，低温基本不影响TFP5的神经保护机制，且当低温与TFP5联用时，动物的神经功能缺损趋向于更小。我们的研究结果提示，TFP5是一种潜在的缺血性中风早期治疗药物，而TFP5联合低温将可能更好的改善该病的预后。