内质网应激在微囊藻毒素致大鼠认知损伤中的作用及调控机制

Contamination of freshwater systems by cyanobacterial blooms is a worldwide problem, causing serious water pollution and public health hazard to humans and livestock by virtue of their ability to produce cyanotoxins. Microcystins (MCs) are the most dangerous group of cyanotoxins and more than 80 variants are known, many of which are potent hepatotoxins, with microcystin-LR (MCLR) being one of the most abundant and toxic variants in blooms. Liver is the prime target organ affected by MCLR. Recently, neurotoxic potential of MCLR has gotten more and more attention. Despite previous studies suggesting that MCLR could execute hyperphosphorylation of tau and spatial memory retention deficits in rats,the molecular and cellular mechanisms underlying these impairments remain unclear. Recent evidence indicated that the activation of endoplasmic reticulum stress (ERS) be involved in the related pathological findings to cognitive impairment, and ERS may play a significant role in the pathogenesis of MCLR toxicity in murine liver. However, though activation of ERS was observed in the brain of rats in our previous study, the role and regulation of ERS in neurotoxic effect of MCLR were still poorly understood. Glycogen synthase kinase (GSK-3), originally identified as regulator of glycogen metabolism, is now known to be important in neuronal plasticity, gene expression and cell survival. GSK-3β is specifically expressed in the brain. Our previous study demonstrated that GSK-3β involved deficits in learning and memory induced by MCLR acute exposure. Until now, the possible molecular link between GSK-3β and the cognitive impairment induced by MCLR is still missing. To achieve this, we explore the cognitive dysfunction induced by MCLR, the signal transduction mechanisms mediated by ERS and the role of GSK-3β in the cognitive toxicity of MCLR through behavioral test, mophological observation, in vivo electrophysiological recording, immunoradiometric assay, phosphoproteome analysis and molecular biological technique. Our results will elucidate the molecular mechanisms in neurotoxic effect of MCLR from a new perspective and provide defined targets for new treatment strategies, prevention or ameliorating the hazards of MCs.

世界范围内蓝藻水华的频频发生给饮水安全和人畜健康造成了严重的威胁。微囊藻毒素（MCs）是危害最大的蓝藻毒素，其中以MCLR的毒性最强。肝脏是MCLR毒性效应的主要靶器官。近年来，MCs的神经毒性受到越来越多的关注。国内外学者和我们的前期工作显示MCLR能够诱发认知损伤。内质网应激（ERS）与认知障碍的病理改变密切相关，研究证实ERS活化参与了MCLR诱导的肝脏损伤，我们的前期研究发现MCLR能够激活大鼠脑内的ERS，但ERS在MCLR认知毒性效应中的作用及其调节机制，迄今知之甚少。本项目运用行为学、形态学、在体场电位记录、放射免疫分析、磷酸化蛋白质组学及其它分子生物学技术观察MCLR导致的认知损害及由ERS介导的信号级联，重点关注糖原合成酶激酶-3β（GSK-3β）磷酸化调节在MCLR认知损伤效应中的作用。本课题将从新的视觉阐明MCLR认知毒性的分子机制，为防治MCs的毒性提供新靶点。

世界范围内蓝藻水华的频频发生给饮水安全和人畜健康造成了严重的威胁。微囊藻毒素（MCs）是危害最大的蓝藻毒素，其中以MC-LR的毒性最强。本项目采用Morris水迷宫和离体场电位记录评价大鼠空间学习记忆能力，从整体上发现，MC-LR能够损伤大鼠认知功能，显著增加内质网应激标志分子的表达，而内质网应激的抑制剂TUDCA能够减弱MC-LR导致的损害。同时，MC-LR处理组大鼠内质网丧失经典的紧密平行排列结构，内质网肿胀、断裂，排列不规则，而TUDCA处理能够减轻MC-LR导致的内质网超微结构的损伤。以原代培养的海马神经元为模型，采用蛋白质组学技术发现，MC-LR处理后，45种蛋白质的表达发生变化，利用生物信息学确定蛋白质分类与功能，主要涉及到钙离子信号转导及凋亡、突触传递及生长、应激相关信号、细胞骨架蛋白、代谢相关蛋白、转录调节与蛋白质合成及蛋白磷酸酶活性相关蛋白。其中，钙调神经磷酸酶（CaN）和核因子活化T细胞胞浆蛋白3（NFATc3）的表达增加，Western blot 和酶活检测的结果表明MC-LR能够活化CaN，促使NFATc3从胞浆转位到细胞核，免疫荧光双标和细胞活力检测表明，MCLR诱发的细胞凋亡与坏死与Ca活化，诱导cyt c从线粒体释放到胞浆内有关。细胞内钙信号紊乱是内质网应激的重要表现之一，采用荧光数字钙影像技术发现，MC-LR能够诱导海马神经元细胞内钙离子浓度升高，其钙离子的来源为内质网钙库。内质网膜上IP3R的拮抗剂（2-APB，xestospongin C）和PLC的抑制剂（U73122）能够抑制MCLR对胞内钙的动员。钙升高能够活化凋亡信号通路，而BAPTA/AM, 2-APB和U73122能够显著缓解MCLR诱导的细胞活力降低和增加的凋亡性和坏死性细胞死亡。蓝藻水华主要危害的是水生态系统中的鱼类，因此我们以斑马鱼为研究对象，观察了内质网应激效应在MC-LR诱发的胚胎发育毒性中的介导作用。研究结果表明，斑马鱼胚胎暴露于MCLR后，可以浓度依赖性地增加斑马鱼仔鱼的畸形率，降低心率和体长，其效应能被内质网应激的阻断剂TUDCA（20 μM）逆转。吖啶橙染色结果显示，MCLR能够诱导斑马鱼仔鱼心包囊区细胞凋亡，其效应也能被TUDCA阻断。RT-PCR的结果表明，MCLR能够上调内质网相关基因的表达，同时增加细胞凋亡通路上关键分子的基因与蛋白的表达，其效