基于ERK1/2信号转导通路的针刺延长脑梗死溶栓时间窗的实验研究

Thrombolytic therapy is the most effective mean to treat the cerebral ischemia, but the very short time window of thrombolysis (3-4.5h) make it difficult to applicate to clinical treatment, so trying to find the method of extending the time window of thrombolysis, will provide more chances to treat more cerebral ischemic patients ..Our previous studies have found that acupuncture can extend the time window by inhibiting the activity of MMP-9 in the ischemic penumbra (IP) area. In the present study, scholars have determined that activation of ERK1 / 2 signaling pathway play a key role in regulating the MMP-9 activity. Based on those experiment results, and put forward hypotheses that acupuncture can extend the cerebral thrombolysis time window by activating ERK1 / 2 signaling pathway.In the study protocol, the cerebral ischemia model was established by modified autologous thromboembolic model, acupuncture was administrated after the model was successful, through observing neurological changes, analysising BBB permeability changes, determining the intracranial hemorrhage and cerebral edema changes before and after rt-PA thrombolysis therapy(at different time), to clarify the effect of acupuncture. Then from the molecular level to detect ERK1 / 2 signaling pathway related indicators mRNA and protein expression in the IP zone, determined activity of MMP-9, and clarify the relationship between these. Moreover, observing the Tight Junction structure and cytoskeleton from the cellular level, and demonstrating the project hypothesis in multi-levels .It may provide new theories and means to treat cerebral ischemic patients, and has the important instruction to guide clinical.

脑梗死超早期溶栓是治疗该病最有效手段，但该方法受到短暂的溶栓时间窗（3-4.5h）限制，临床实施比例很低，如能探寻到延长时间窗的方法，将会为患者赢得更多救治机会。.本课题组前期研究发现，针刺及时介入，具有延长溶栓时间窗的可行性，且该作用与降低IP区MMP-9活性密切相关。近期研究揭示，激活ERK1/2信号转导通路是下调MMP-9的关键途径。基于此，提出本项目假说：针刺可通过激活ERK1/2信号通路延长脑梗死溶栓时间窗。本研究采用大鼠自体血栓栓塞性模型，以针刺为干预手段，观察模型大鼠溶栓(分不同时间点)前后神经行为学、BBB通透性、颅内出血及脑水肿变化，以明确针刺效应。继则从分子水平检测IP区ERK1/2信号通路及炎症反应相关指标mRNA及蛋白表达、MMP-9活性的变化，并明确这些指标之间关系；用电镜从细胞水平观察BBB的TJ及细胞骨架等变化，多层次论证本项目假说，对临床具有重要指导意义。

背景：脑梗死超早期溶栓是治疗该病最有效手段，但溶栓时间窗（3-4.5h）有限制，导致临床实施比例很低，同时，超时间窗外溶栓会引发出血转化、脑水肿等并发症，如能探寻到延长时间窗的方法，将会为患者赢得更多救治机会。.内容：本研究采用大鼠自体血栓栓塞性模型，以“醒脑开窍”针刺法为干预手段，观察模型大鼠不同时间点溶栓后神经行为学、BBB通透性、颅内出血及脑水肿变化，以明确针刺效应，继则从分子水平检测IP区ERK1/2信号通路(Ras、b-Raf、MEK1/2、ERK1/2)、炎症反应相关指标(IL-1β、TNF-α)，以及MMP9表达的变化，并明确这些指标之间关系；采用透射电镜观察BBB的超微结构的变化，多层次论证针刺调控ERK1/2信号通路延长脑梗死溶栓时间窗的作用机制。.结果：1、早期针刺可以改善时间窗外单纯溶栓导致的BBB破坏，降低BBB的通透性，减轻溶栓后出血性转化以及水肿程度并发症，从而提高溶栓安全性；并且针刺能减小脑梗死体积，改善神经功能，增加了单纯溶栓的疗效，进一步明确了针刺可延长脑梗死溶栓安全时间窗至6h；2、早期针刺治疗可抑制脑梗死溶栓大鼠ERK1/2信号通路的激活，下调MMP9表达水平，降低脑梗死溶栓大鼠脑组织中炎症因子TNF-α、IL-β含量，抑制炎症反应；3、此外电镜观察发现早期针刺可以改善时间窗外单纯溶栓大鼠BBB超微结构的破坏，起到保护BBB的作用。.结论：针刺能够延长脑梗死溶栓安全时间窗至6h，其机制与抑制ERK1/2信号通路的激活有关。