PERK信号通路介导的线粒体未折叠蛋白反应在低氧缺血性脑损伤中的作用

Role of PERK signaling pathway-mediated mitochondrial unfolded protein response in hypoxic-ischemic brain injury

摘要

目的 探讨蛋白激酶RNA样ER激酶（protein kinase RNA-like ER kinase，PERK）信号通路介导的线粒体未折叠蛋白反应（mitochondrial unfolded protein response，mtUPR）在缺氧缺血性脑损伤（hypoxic-ischemic brain injury，HIBI）中的作用。 方法 将大鼠随机分为假手术（Sham）组和5个HIBI亚组（HIBI后3、6、12、24、48 h）。用于蛋白质印迹检测PERK、转录激活因子4（activating transcription factor 4，ATF4）、热休克蛋白60（heat shock protein 60，HSP60）蛋白的时程表达。将大鼠随机分为Sham组、HIBI组、HIBI + PERK组和HIBI+载体（Vector）组，每组15只。HIBI+PERK组和HIBI+Vector组大鼠在HIBI手术前1 h，将基于腺病毒相关病毒（adeno-associated virus，AAV）的PERK过表达质粒或AAV载体注射到脑室内，用于特异性表达PERK。在HIBI后24 h进行FJC染色分析神经元变性和DHE染色、酶联免疫吸附试验分析氧化应激。将大鼠随机分为Sham组、HIBI组、HIBI+PERK激动剂（CCT020312）组，每组12只。在HIBI手术前1 h，向HIBI+CCT020312组大鼠脑室内注射CCT020312。在HIBI后3周进行开阔场地测试和莫里斯水迷宫测试。 结果 与Sham组相比，PERK、ATF4、HSP60在HIBI后3 h开始明显升高，在12 h达到高峰，然后逐渐下降，直到48 h（F=60.23、56.72、74.31，均P<0.001）。与HIBI组相比，HIBI+PERK组神经元变性的数量（100.2±3.1 vs. 582.4±15.7，P<0.001）、活性氧（reactive oxygen species，ROS）（42.4±2.9 vs. 17.7±2.1，P<0.01）、丙二醛（Malondialdehyde，MDA）（0.81±0.06 vs. 0.54±0.04，P<0.001）水平显著降低，和谷胱甘肽过氧化物酶（glutathione peroxidase，GSHPx）（112.4±3.6 vs. 177.5±6.6，P<0.05）、超氧化物歧化酶（superoxide Dismutase，SOD）活性（46.3±1.9 vs. 64.2±2.3，P<0.05）活性明显增加。与Sham组相比，HIBI组大鼠海马组织中PERK（1.00±0.03 vs. 1.66±0.08，P<0.01）、ATF4（1.00±0.04 vs. 1.53±0.06，P<0.05）、动力蛋白相关蛋白1（dynamin-related protein 1，Drp1）（1.00±0.02 vs. 1.98±0.07，P<0.01）、HSP60（1.00±0.03 vs. 1.37±0.04，P<0.05）蛋白表达均明显增加（P<0.05）。与HIBI组相比，HIBI+PERK组大鼠海马组织中PERK（1.66±0.08 vs. 2.95±0.17，P<0.01）、ATF4（1.53±0.06 vs. 3.42±0.22，P<0.01）、HSP60（1.37±0.04 vs. 2.03±0.09，P<0.05）蛋白表达均明显增加（F=46.72、30.63、20.64，P<0.001），和Drp1（1.98±0.07 vs. 1.04±0.05，P<0.05）蛋白表达明显降低（F=35.72，P<0.001）。HIBI+CCT020312组的平均逃避潜伏期和平台穿越次数均较HIBI组明显增加（F=246.84、113.62，P<0.001）。 结论 PERK减轻HIBI模型诱导的氧化应激和神经元凋亡，其机制可能涉及PERK/ATF4信号通路对mtUPR的调节。通过CCT020312给药具有神经保护作用。

Abstract

Objective To examine the role of mitochondrial unfolded protein response（mtUPR） mediated by the protein kinase RNA-like ER kinase（PERK） signaling pathway in hypoxic-ischemic brain injury（HIBI）. Methods Rats were randomly divided into the sham group and five HIBI groups（3，6，12，24 and 48 h after HIBI） for Western blot analysis of PERK，activating transcription factor 4 （ATF4），and heat shock protein 60（HSP60） protein expression over time. In another experiment，rats were randomly divided into the sham group，HIBI group，HIBI+PERK group，and HIBI+Vector group，with 15 rats in each group. Rats in the HIBI+PERK group and HIBI+Vector group were given intracerebroventricular injection of adeno-associated virus（AAV）-based PERK overexpression plasmid or empty AAV vector 1 h before HIBI to induce specific PERK expression. FJC staining was performed 24 h after HIBI to analyze neuronal degeneration，and oxidative stress was examined by DHE staining and enzyme-linked immunosorbent assay. In a third experiment，rats were randomly divided into the sham group，HIBI group and HIBI+CCT020312（PERK agonist） group，with 12 rats in each group. Rats in the HIBI+CCT020312 group were given intracerebroventricular injection of CCT020312 one hour before HIBI. Open field test and Morris water maze test were performed 3 weeks after HIBI. Results Compared with the sham group，the HIBI groups showed significantly increased PERK，ATF4，and HSP60 expression at 3 h post-HIBI，which peaked at 12 h and then gradually decreased until 48 h（F=60.23，56.72，74.31，all P<0.001）. The HIBI+PERK group had significantly lower number of degenerated neurons（100.2±3.1 vs. 582.4±15.7，P<0.001），reactive oxygen species（ROS） level （42.4±2.9 vs. 17.7±2.1，P<0.01），and malondialdehyde（MDA） level（0.81±0.06 vs. 0.54±0.04，P<0.001），but significantly higher glutathione peroxidase（GSH-Px） activity （112.4±3.6 vs. 177.5±6.6，P<0.05） and superoxide dismutase （SOD） activity （46.3±1.9 vs. 64.2±2.3，P<0.05），as compared with the HIBI group. PERK（1.00±0.03 vs. 1.66±0.08，P<0.01），ATF4 （1.00±0.04 vs. 1.53±0.06，P<0.05），dynamin-related protein 1 （Drp1；1.00±0.02 vs. 1.98±0.07，P<0.01），and HSP60 （1.00±0.03 vs. 1.37±0.04，P<0.05） protein expression in the hippocampal tissue were significantly higher in the HIBI group than in the sham group. Furthermore，the HIBI+PERK group demonstrated significantly higher PERK（1.66±0.08 vs. 2.95±0.17，P<0.01），ATF4（1.53±0.06 vs. 3.42±0.22，P<0.01），and HSP60 （1.37±0.04 vs. 2.03±0.09，P<0.05） protein expression（F=46.72，30.63，20.64，P<0.001） but significantly lower Drp1（1.98±0.07 vs. 1.04±0.05，P<0.05） protein expression in the hippocampal tissue than the HIBI group（F=35.72，P<0.001）. Mean escape latency and the number of platform crossings were significantly higher in the HIBI+CCT020312 group than in the HIBI group（F=246.84，113.62，P<0.001）. Conclusion PERK attenuates oxidative stress and neuronal apoptosis induced by HIBI，possibly through the regulation of mtUPR via the PERK/ATF4 signaling pathway. CCT020312 has neuroprotective effects in HIBI.

关键词

蛋白激酶RNA样ER激酶 / 线粒体未折叠蛋白反应 / 缺氧缺血性脑损伤 / 神经元

Key words

protein kinase RNA-like ER kinase / mitochondrial unfolded protein response / hypoxic ischemic brain injury / neuron

基金资助

湖北省卫生健康科研基金资助项目(2021B548K)