DeMars Kelly M, McCrea Austin O, Siwarski David M, Sanz Brian D, Yang Changjun, Candelario-Jalil Eduardo
Department of Neuroscience, McKnight Brain Institute, University of Florida, Gainesville, FL, United States.
Front Neurosci. 2018 Feb 20;12:89. doi: 10.3389/fnins.2018.00089. eCollection 2018.
Ischemic stroke occurs when a clot forms in the brain vasculature that starves downstream tissue of oxygen and nutrients resulting in cell death. The tissue immediately downstream of the blockage, the core, dies within minutes, but the surrounding tissue, the penumbra is potentially salvageable. Prostaglandin E binds to four different G-protein coupled membrane receptors EP1-EP4 mediating different and sometimes opposing responses. Pharmacological activation of the EP4 receptor has already been established as neuroprotective in stroke, but the mechanism(s) of protection are not well-characterized. In this study, we hypothesized that EP4 receptor activation reduces ischemic brain injury by reducing matrix metalloproteinase (MMP)-3/-9 production and blood-brain barrier (BBB) damage. Rats underwent transient ischemic stroke for 90 min. Animals received an intravenous injection of either the vehicle or L-902,688, a highly specific EP4 agonist, at the onset of reperfusion. Brain tissue was harvested at 24 h. We established a dose-response curve and used the optimal dose that resulted in the greatest infarct reduction to analyze BBB integrity compared to vehicle-treated rats. The presence of IgG, a blood protein, in the brain parenchyma is a marker of BBB damage, and L-902,688 (1 mg/kg; i.v.) dramatically reduced IgG extravasation ( < 0.05). Consistent with these data, we assessed zona occludens-1 and occludin, tight junction proteins integral to the maintenance of the BBB, and found reduced degradation with L-902,688 administration. With immunoblotting, qRT-PCR, and/or a fluorescence resonance energy transfer (FRET)-based activity assay, we next measured MMP-3/-9 since they are key effectors of BBB breakdown in stroke. In the cerebral cortex, not only was MMP-3 activity significantly decreased ( < 0.05), but L-902,688 treatment also reduced MMP-9 mRNA, protein, and enzymatic activity ( < 0.001). In addition, post-ischemic administration of the EP4 agonist significantly reduced pro-inflammatory cytokines IL-1β ( < 0.05) and IL-6 ( < 0.01) in the ischemic cerebral cortex. Most importantly, one injection of L-902,688 (1 mg/kg; i.v) at the onset of reperfusion significantly reduces neurological deficits up to 3 weeks later ( < 0.05). Our data show for the first time that pharmacological activation of EP4 with L-902,688 is neuroprotective in ischemic stroke by reducing MMP-3/-9 and BBB damage.
当大脑血管中形成血栓，使下游组织缺乏氧气和营养物质，导致细胞死亡时，就会发生缺血性中风。堵塞部位下游紧邻的组织即核心区域，会在几分钟内死亡，但周围组织即半暗带，有可能被挽救。前列腺素E与四种不同的G蛋白偶联膜受体EP1 - EP4结合，介导不同的、有时甚至是相反的反应。EP4受体的药理学激活已被证实对中风具有神经保护作用，但其保护机制尚未完全明确。在本研究中，我们假设EP4受体激活通过减少基质金属蛋白酶（MMP）-3/-9的产生和血脑屏障（BBB）损伤来减轻缺血性脑损伤。大鼠经历90分钟的短暂性缺血性中风。在再灌注开始时，动物接受静脉注射载体或L - 902,688（一种高度特异性的EP4激动剂）。在24小时时采集脑组织。我们建立了剂量反应曲线，并使用导致梗死面积最大减少的最佳剂量，与载体处理的大鼠相比，分析血脑屏障的完整性。脑实质中血液蛋白IgG的存在是血脑屏障损伤的标志物，L - 902,688（1mg/kg；静脉注射）显著减少了IgG外渗（<0.05）。与这些数据一致，我们评估了紧密连接蛋白闭合蛋白-1和闭合蛋白，它们是维持血脑屏障不可或缺的，发现L - 902,688给药后其降解减少。接下来，我们通过免疫印迹、定量逆转录-聚合酶链反应和/或基于荧光共振能量转移（FRET）的活性测定法来测量MMP - 3/-9，因为它们是中风中血脑屏障破坏的关键效应因子。在大脑皮层，不仅MMP - 3活性显著降低（<0.05），而且L - 902,688治疗还降低了MMP - 9的mRNA、蛋白和酶活性（<0.001）。此外，缺血后给予EP4激动剂显著降低了缺血性大脑皮层中促炎细胞因子白细胞介素-1β（<0.05）和白细胞介素-6（<0.01）。最重要的是，在再灌注开始时一次性注射L - 902,688（1mg/kg；静脉注射），在长达3周后显著降低了神经功能缺损（<0.05）。我们的数据首次表明，用L - 902,688对EP4进行药理学激活，通过减少MMP - 3/-9和血脑屏障损伤，对缺血性中风具有神经保护作用。
