Thrombolytic therapy with recombinant tissue plasminogen activator (rtPA) in ischaemic stroke has been associated with neurotoxicity, blood brain barrier (BBB) disruption and intra-cerebral hemorrhage. To examine rtPA cellular toxicity we investigated the effects of rtPA on cell viability in neuronal, astrocyte and brain endothelial cell (bEnd.3) cultures with and without prior exposure to oxygen-glucose deprivation (OGD). In addition, the neuroprotective peptide poly-arginine-18 (R18D; 18-mer of D-arginine) was examined for its ability to reduce rtPA toxicity. Studies demonstrated that a 4- or 24-h exposure of rtPA was toxic, affecting neuronal cell viability at ≥ 2 µM, and astrocyte and bEnd.3 cells viability at ≥ 5 μM. In addition, a 4-h exposure to rtPA after a period of OGD (OGD/rtPA) exacerbated toxicity, affecting neuronal, astrocyte and bEnd.3 cell viability at rtPA concentrations as low as 0.1 µM. Treatment of cells with low concentrations of R18D (0.5 and 1 µM) reduced the toxic effects of rtPA and OGD/rtPA, while on some occasions a higher 2 µM R18D concentrations exacerbated neuronal and bEnd.3 cell toxicity in OGD/rtPA exposed cultures. In exploratory studies we also demonstrated that OGD activates matrix metalloproteinase-9 (MMP-9) release into the supernatant of astrocyte and bEnd.3 cell cultures, but not neuronal cultures, and that OGD/rtPA increases MMP-9 activation. Furthermore, R18D decreased MMP-9 activation in OGD/rtPA treated astrocyte and bEnd.3 cell cultures. In summary, the findings show that rtPA can be toxic to neural cells and that OGD exacerbates toxicity, while R18D has the capacity to reduce rtPA neural cellular toxicity and reduce MMP-9 activation in astrocytes and bEnd.3. Poly-arginine-18 peptides, which are being developed as neuroprotective therapeutics for ischaemic stroke, therefore have the additional potential of reducing cytotoxic effects associated with rtPA thrombolysis in the treatment of ischaemic stroke.

中文翻译：

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在培养的神经细胞中评估重组组织纤溶酶原激活剂（rtPA）的毒性，并随后用聚精氨酸肽R18D处理。

在缺血性卒中中使用重组组织纤溶酶原激活剂（rtPA）进行溶栓治疗与神经毒性，血脑屏障（BBB）破坏和脑内出血有关。为了检查rtPA细胞毒性，我们研究了rtPA对有或没有事先暴露于氧-葡萄糖剥夺（OGD）的神经元，星形胶质细胞和脑内皮细胞（bEnd.3）培养物中细胞活力的影响。另外，检查了神经保护肽聚精氨酸-18（R18D； D-精氨酸的18聚体）降低rtPA毒性的能力。研究表明，rtPA暴露4或24小时具有毒性，在≥2 µM时会影响神经元细胞活力，而在≥5μM时会影响星形胶质细胞和bEnd.3细胞的活力。此外，经过一段时间的OGD（OGD / rtPA）暴露于rtPA 4小时会加剧毒性，影响神经元，rtPA浓度低至0.1 µM时星形胶质细胞和bEnd.3细胞活力。用低浓度的R18D（0.5和1 µM）处理细胞会降低rtPA和OGD / rtPA的毒性作用，而在某些情况下，较高的2 µM R18D浓度会在暴露于OGD / rtPA的培养物中加剧神经元和bEnd.3细胞毒性。在探索性研究中，我们还证明OGD激活基质金属蛋白酶9（MMP-9）释放到星形胶质细胞和bEnd.3细胞培养物的上清液中，而不是神经元培养物，并且OGD / rtPA增加MMP-9的激活作用。此外，R18D降低了OGD / rtPA处理的星形胶质细胞和bEnd.3细胞培养物中MMP-9的活化。总之，研究结果表明rtPA可能对神经细胞有毒，而OGD会加剧毒性，R18D具有降低rtPA神经细胞毒性和减少星形胶质细胞和bEnd中MMP-9活化的能力。3。聚精氨酸-18肽已被开发为缺血性中风的神经保护疗法，因此在治疗缺血性中风方面具有降低与rtPA溶栓相关的细胞毒性作用的潜力。