State Key Laboratory of Bioactive Substances and Functions of Natural Medicines, Beijing Key Laboratory of New Drug Mechanisms and Pharmacological Evaluation Study, Department of Pharmacology, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Neuroscience Center, Chinese Academy of Medical Sciences, Beijing, 100050, China.
Eur J Neurosci. 2014 Jun;39(12):2107-18. doi: 10.1111/ejn.12551. Epub 2014 Mar 20.
Ischemic stroke is currently treated with thrombolytic therapy with a drawback to induce hemorrhagic transformation (HT) if applied beyond its relatively narrow treatment time window. The present study was designed to examine the role of IMM-H004, a derivative of coumarin, in recombinant tissue plasminogen activator (tPA)-induced HT. Rats subjected to 6 h of thromboembolic occlusion or middle cerebral artery occlusion received tPA with or without IMM-H004. Delayed tPA intervention drastically increased the risk of HT and exaggerated the ischemic injury. To assess the effect of IMM-H004 on delayed treatment of tPA-induced toxicity after ischemia and reperfusion, various approaches were used, including a behavior test, TTC-staining, determination of cerebral hemorrhage, laser speckle imaging, Western blot, gelatin zymogram, immunohistochemistry and immunofluorescence staining. Experiments were also conducted in vitro in human brain microvascular endothelial cells (HBMECs) and PC12 cells to explore the mechanism for the role of IMM-H004. Combination therapy of tPA and IMM-H004 prevented the development of HT, and reduced the mortality rate, infarct volume and brain edema. IMM-H004 also exerted a protective role by decreasing matrix metalloproteinases, the co-localization of matrix metalloproteinase-2 with astrocytes and increasing occludin. Experiments in HBMECs and PC12 revealed an elevation in ATP level and a protein kinase A- and PI3K-dependent activation of Akt by IMM-H004 after tPA administration. These results suggest IMM-H004 as a promising adjuvant to alleviate the detrimental side effects of tPA in clinical therapy of ischemic stroke, and contribute to better understand the mechanism for the beneficial role of this novel remedy.
缺血性脑卒中目前采用溶栓疗法治疗，但如果在相对较窄的治疗时间窗之外应用，则会导致出血性转化（HT）。本研究旨在研究香豆素衍生物 IMM-H004 在重组组织型纤溶酶原激活剂（tPA）诱导的 HT 中的作用。接受 6 小时血栓栓塞闭塞或大脑中动脉闭塞的大鼠接受 tPA 联合或不联合 IMM-H004。延迟 tPA 干预极大地增加了 HT 的风险，并加重了缺血性损伤。为了评估 IMM-H004 对缺血再灌注后 tPA 诱导毒性的延迟治疗的影响，采用了各种方法，包括行为测试、TTC 染色、脑出血测定、激光散斑成像、Western blot、明胶酶谱、免疫组织化学和免疫荧光染色。还在人脑血管内皮细胞（HBMEC）和 PC12 细胞中进行了体外实验，以探讨 IMM-H004 作用的机制。tPA 和 IMM-H004 的联合治疗可预防 HT 的发生，降低死亡率、梗死体积和脑水肿。IMM-H004 通过降低基质金属蛋白酶、基质金属蛋白酶-2 与星形胶质细胞的共定位以及增加闭合蛋白，发挥保护作用。在 HBMEC 和 PC12 中的实验表明，在 tPA 给药后，IMM-H004 可升高 ATP 水平，并通过蛋白激酶 A 和 PI3K 依赖性激活 Akt。这些结果表明，IMM-H004 作为一种有前途的佐剂，可减轻 tPA 在缺血性脑卒中临床治疗中的有害副作用，并有助于更好地理解这种新型治疗方法的有益作用的机制。
