GRANZYME-B IS INVOLVED IN MEDIATING POST-ISCHEMIC NEURONAL DEATH DURING FOCAL CEREBRAL ISCHEMIA IN RAT MODEL
GRANZYME-B IS INVOLVED IN MEDIATING POST-ISCHEMIC NEURONAL DEATH DURING FOCAL CEREBRAL ISCHEMIA IN RAT MODEL
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DOI：
10.1016/j.neuroscience.2009.10.067
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发表时间：
2010-02-17
期刊：
影响因子：
3.3
通讯作者：
Babu, P. Prakash
中科院分区：
文献类型：
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作者：
Chaitanya, G. V.;Schwaninger, M.;Babu, P. Prakash
尽管外周免疫细胞可侵袭脑梗塞组织并引起免疫损伤，但细胞毒性T淋巴细胞(CTL)及其释放的毒素在介导神经元死亡中的作用尚不清楚。颗粒酶-b(Grazyme-b，Gra-b)是发现于CTL和自然杀伤细胞胞浆颗粒中的一种丝氨酸蛋白酶，通过激活多个caspase和启动caspase-in依赖的途径导致靶细胞死亡，在诱导靶细胞死亡中发挥重要作用。为了确定CTL和Gra-b是否参与脑缺血后的细胞死亡，我们研究了CD8(+)CTL和Gra-b在短暂性大脑中动脉闭塞(TMCAO)后脑梗塞和假手术动物中的作用。我们观察到在再灌流后1小时内，CTL在缺血区内有明显的细胞毒性T细胞浸润。缺血区Gra-b水平从1h至3d显著升高，且与趋化因子(IP-10/CXCL10、IL-2)和肿瘤坏死因子-α水平升高相关。免疫共沉淀实验表明，Gra-b在缺血样本中与Bid、PARP和caspase-3相互作用。Gra-b和TUNEL的免疫荧光分析表明，Gra-b在凋亡和坏死细胞中都存在。三重免疫染色进一步证实Gra-b阳性变性细胞为神经元。TMCAO后Gra-b和CTL在退行性神经元附近的空间分布、Gra-b水平的升高、TUNEL阳性神经元的定位以及与其他促凋亡蛋白的相互作用表明Gra-b和CTL在tMCAO后脑缺血后神经元死亡中发挥重要作用。基于上述发现，我们支持我们的假设，即激活的CTL分泌的Gra-b可能通过介导神经元死亡而参与加重缺血后的损伤。(C)2010年IBRO。爱思唯尔有限公司出版。保留所有权利。
Although peripheral immune cells infiltrate ischemic infarct tissue and elicit immune injury, the role of Cytotoxic T Lymphocytes (CTLs) and the toxins they release in mediating neuronal death is not well understood. Granzyme-b (Gra-b), a serine protease found in the cytoplasmic granules of CTLs and natural killer cells, plays an important role in inducing target cell death by activating several caspases and by initiating caspase-in dependent pathways that contribute to target cell death. To determine if CTLs and Gra-b are involved in post-ischemic cerebral cell death; we investigated the role of CD8(+) CTLs and Gra-b in ischemic rat brain infarct after transient middle cerebral artery occlusion (tMCAO) and in sham-operated animals. We observed that CTLs infiltrate the ischemic infarct within 1 h of reperfusion. There was a significant increase in Gra-b levels in the ischemic region starting from 1 h until 3 day which correlated with increased levels of chemokines (IP-10/CXCL10, IL-2) and TNF-alpha. Co-immunoprecipitation experiments show that Gra-b interacts with Bid, PARP, and caspase-3 in ischemic samples. Immunofluorescence analysis of Gra-b and TUNEL showed that Gra-b is present both in apoptotic and necrotic cells. Triple immunostaining further confirmed that the Gra-b positive degenerating cells were neurons. CTLs in close spatial proximity to degenerating neurons, increased levels of Gra-b, localization in neurons positive for TUNEL, and interaction with other pro-apoptotic proteins indicate that Gra-b and CTLs play a significant role in neuronal death following cerebral ischemia in the rat brain after tMCAO. Based on the above findings we support our hypothesis that Gra-b secreted from activated CTLs might be involved in aggravating post-ischemic damage by mediating neuronal death. (C) 2010 IBRO. Published by Elsevier Ltd. All rights reserved.