文献检索，用中文搜 PubMed

BACKGROUND

Thrombolytic treatment with tissue plasminogen activator (tPA) is one of the approved pharmacological therapies for acute ischemic stroke. However, the use of tPA is limited due to hemorrhagic transformation (HT) and the narrow therapeutic time window. Previous studies demonstrated that asparagine endopeptidase (AEP), a widely expressed pH-dependent endo-lysosomal cysteine protease, can induce neuronal death during ischemia-reperfusion injury. But whether AEP is engaged in HT during ischemia-reperfusion injury is unclear. In the current study, we expanded the role of AEP on HT after delayed tPA administration.

METHODS

In order to investigate the effects of AEP on HT after delayed tPA administration following ischemic stroke, the middle cerebral artery occlusion/reperfusion (MCAO/R) was performed in wild-type (WT) and AEP knockout (KO) transgenic mice, followed by delayed administration of tPA (10 mg/kg, 3 h after occlusion). Additionally, we explored the potential of R13, a specific TrkB agonist with a strong inhibitory impact on AEP, to mitigate injury induced by tPA. 24 h after tPA administration, the following parameters were assessed: infarct volume, behavioral tests, hemorrhagic levels, Evans blue leakage, tight and adherens junction protein expression, blood-brain barrier (BBB) function, cerebral vascular structure, matrix metalloproteinases (MMPs), and BBB-regulated protein low-density lipoprotein receptor-related protein 1 (LRP-1) expression. To construct an in vitro model to examine the effects of AEP on ischemia-reperfusion injury after tPA treatment, human umbilical vein endothelial cells (HUVECs) were exposed to 4 h of oxygen-glucose deprivation (OGD), followed by treatment with tPA (500 ng/mL). 7,8-dihydroxyflavone (7,8-DHF), a natural TrkB agonist with an inhibitory effect on AEP, was applied before OGD.

RESULTS

Compared with tPA-treated WT mice, AEP KO mice treated with tPA showed improved infarct volume, neurological function, brain edema, brain hemoglobin levels, Evans blue leakage, vascular tight junctions, and basement membrane structure combined with reduced AEP expression and activity within the peri-infarct area. In addition, the mice treated with R13 exhibited protective effects on the BBB. Furthermore, we found that the expression of MMP2, MMP9, and LRP-1 in the brain was inhibited by both AEP knockout and R13 treatment. Moreover, HUVECs treated with 7,8-DHF showed improvements in tight and adherens junction proteins and suppressed levels of MMP2, MMP9, and LRP-1.

CONCLUSION

Our findings demonstrate that AEP exacerbates HT induced by delayed tPA treatment in acute ischemic stroke by activating LRP-1, MMP2, and MMP9, which disrupts BBB integrity. We further confirmed R13 as a preventive therapy to attenuate HT induced by delayed tPA treatment in acute ischemic stroke. The present study suggests AEP inhibition may serve as a promising strategy to enhance the safety of delayed tPA thrombolysis for ischemic stroke.

BACKGROUND

METHODS

RESULTS

CONCLUSION

背景

组织型纤溶酶原激活剂（tPA）溶栓治疗是急性缺血性卒中已获批的药物治疗方法之一。然而，由于出血性转化（HT）以及狭窄的治疗时间窗，tPA的应用受到限制。既往研究表明，天冬酰胺内肽酶（AEP）是一种广泛表达的pH依赖性溶酶体内半胱氨酸蛋白酶，可在缺血再灌注损伤期间诱导神经元死亡。但AEP是否参与缺血再灌注损伤期间的HT尚不清楚。在本研究中，我们拓展了AEP在延迟给予tPA后对HT的作用。

方法

为研究AEP对缺血性卒中延迟给予tPA后HT的影响，在野生型（WT）和AEP基因敲除（KO）转基因小鼠中进行大脑中动脉闭塞/再灌注（MCAO/R），随后延迟给予tPA（10 mg/kg，闭塞后3小时）。此外，我们探究了R13（一种对AEP有强烈抑制作用的特异性TrkB激动剂）减轻tPA诱导损伤的潜力。在给予tPA 24小时后，评估以下参数：梗死体积、行为测试、出血水平、伊文思蓝渗漏、紧密连接和黏附连接蛋白表达、血脑屏障（BBB）功能、脑血管结构、基质金属蛋白酶（MMPs）以及BBB调节蛋白低密度脂蛋白受体相关蛋白1（LRP-1）表达。为构建体外模型以研究AEP对tPA治疗后缺血再灌注损伤的影响，将人脐静脉内皮细胞（HUVECs）暴露于4小时氧糖剥夺（OGD），随后用tPA（500 ng/mL）处理。在OGD前应用7,8-二羟基黄酮（7,8-DHF），一种对AEP有抑制作用的天然TrkB激动剂。

结果

与tPA治疗的WT小鼠相比，tPA治疗的AEP KO小鼠梗死体积、神经功能、脑水肿、脑血红蛋白水平、伊文思蓝渗漏、血管紧密连接和基底膜结构均有改善，同时梗死周边区域AEP表达和活性降低。此外，用R13治疗的小鼠对BBB具有保护作用。再者，我们发现AEP基因敲除和R13治疗均抑制了大脑中MMP2、MMP9和LRP-1的表达。此外，用7,8-DHF处理的HUVECs紧密连接和黏附连接蛋白有所改善，MMP2、MMP9和LRP-1水平受到抑制。